Bio

Academic Appointments


Research & Scholarship

Current Research and Scholarly Interests


My laboratory is involved in studying several aspects of adrenergic receptor biology. Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system and play a critical role in the regulation of cardiovascular function. Specific projects include:

1- RECEPTOR STRUCTURE: We are interested in understanding the three dimensional structure of adrenergic receptors and learning about the conformational changes that mediate signal transduction. We are taking several experimental approaches including mutagenesis, biochemical, and biophysical studies.

2-INTRACELLULAR TRAFFICKING OF ADRENERGIC RECEPTORS: The function of receptors can be modulated by changes in receptor structure (phosphorylation) and by changes in subcellular localization. We are using immunocytochemical approaches to study the targeting of receptors to specific subcellular domains and agonist mediated redistribution of receptors. Our goal is to determine the functional significance of differences in targeting and trafficking that we have observed in several adrenergic receptors, and to identify cellular proteins that mediate receptor trafficking.

3-PHYSIOLOGIC RELEVANCE OF ADRENERGIC RECEPTOR SUBTYPE DIVERSITY: Multiple closely related subtypes of adrenergic receptors have been identified through cloning studies. We are using targeted gene modification in mice to study the physiologic role of these closely related subtypes. We have disrupted the genes for five adrenergic receptors (alpha 2a, alpha 2b, alpha 2c, beta 1, and beta2) and are investigating the consequence of these disruptions on neural and cardiovascular physiology.

Teaching

2013-14 Courses


Publications

Journal Articles


  • The Dynamic Process of beta(2)-Adrenergic Receptor Activation CELL Nygaard, R., Zou, Y., Dror, R. O., Mildorf, T. J., Arlow, D. H., Manglik, A., Pan, A. C., Liu, C. W., Fung, J. J., Bokoch, M. P., Thian, F. S., Kobilka, T. S., Shaw, D. E., Mueller, L., Prosser, R. S., Kobilka, B. K. 2013; 152 (3): 532-542

    Abstract

    G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ?(2)-adrenergic receptor (?(2)AR), a prototypical GPCR. We labeled ?(2)AR with (13)CH(3)?-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ?(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ?(2)AR's ability to engage multiple signaling and regulatory proteins.

    View details for DOI 10.1016/j.cell.2013.01.008

    View details for Web of Science ID 000314362800022

    View details for PubMedID 23374348

  • Identification of GPCR-Interacting Cytosolic Proteins Using HDL Particles and Mass Spectrometry-Based Proteomic Approach PLOS ONE Chung, K. Y., Day, P. W., Velez-Ruiz, G., Sunahara, R. K., Kobilka, B. K. 2013; 8 (1)

    Abstract

    G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the ?(2)-adrenergic receptor (?(2)AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified ?(2)AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated ?(2)AR pull-down, 242 proteins in the inverse agonist-activated ?(2)AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the ?(2)AR-interacting proteins isolated was confirmed by Western blot; three known ?(2)AR-interacting proteins (Gs?, NHERF-2, and Grb2) and 3 newly identified known ?(2)AR-interacting proteins (AMPK?, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of ?(2)AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.

    View details for DOI 10.1371/journal.pone.0054942

    View details for Web of Science ID 000315210400043

    View details for PubMedID 23372797

  • High-resolution crystal structure of human protease-activated receptor 1 NATURE Zhang, C., Srinivasan, Y., Arlow, D. H., Fung, J. J., Palmer, D., Zheng, Y., Green, H. F., Pandey, A., Dror, R. O., Shaw, D. E., Weis, W. I., Coughlin, S. R., Kobilka, B. K. 2012; 492 (7429): 387-?

    Abstract

    Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2 Å resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family.

    View details for DOI 10.1038/nature11701

    View details for Web of Science ID 000312488200047

    View details for PubMedID 23222541

  • Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor JOURNAL OF BIOLOGICAL CHEMISTRY Chung, K. Y., Kim, T. H., Manglik, A., Alvares, R., Kobilka, B. K., Prosser, R. S. 2012; 287 (43): 36305-36311

    Abstract

    The G protein-coupled ?(2)-adrenoreceptor (?(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and ?-arrestin. As such, the energy landscape of ?(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of ?(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in ?(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of ?(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

    View details for DOI 10.1074/jbc.M112.406371

    View details for Web of Science ID 000310364000051

    View details for PubMedID 22893704

  • N-Terminal T4 Lysozyme Fusion Facilitates Crystallization of a G Protein Coupled Receptor PLOS ONE Zou, Y., Weis, W. I., Kobilka, B. K. 2012; 7 (10)

    Abstract

    A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the ?(2) adrenergic receptor (?(2)AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.

    View details for DOI 10.1371/journal.pone.0046039

    View details for Web of Science ID 000309580800006

    View details for PubMedID 23056231

  • A new era of GPCR structural and chemical biology NATURE CHEMICAL BIOLOGY Granier, S., Kobilka, B. 2012; 8 (8): 670-673

    Abstract

    G protein-coupled receptors (GPCRs) are versatile molecular machines that regulate the majority of physiological responses to chemically diverse hormones and neurotransmitters. Recent breakthroughs in structural studies have advanced our understanding of GPCR signaling, particularly the selectivity of ligand recognition and receptor activation of G proteins.

    View details for DOI 10.1038/nchembio.1025

    View details for Web of Science ID 000306527600002

    View details for PubMedID 22810761

  • Crystal structure of the mu-opioid receptor bound to a morphinan antagonist NATURE Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Mathiesen, J. M., Sunahara, R. K., Pardo, L., Weis, W. I., Kobilka, B. K., Granier, S. 2012; 485 (7398): 321-U170

    Abstract

    Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8?Å crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

    View details for DOI 10.1038/nature10954

    View details for Web of Science ID 000304099100032

    View details for PubMedID 22437502

  • Structure of the delta-opioid receptor bound to naltrindole NATURE Granier, S., Manglik, A., Kruse, A. C., Kobilka, T. S., Thian, F. S., Weis, W. I., Kobilka, B. K. 2012; 485 (7398): 400-U171

    Abstract

    The opioid receptor family comprises three members, the µ-, ?- and ?-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The ?-opioid receptor (?-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the µ-OR and ?-OR have recently been solved. Here we report the crystal structure of the mouse ?-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the µ-OR and ?-OR, the ?-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the ?-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

    View details for DOI 10.1038/nature11111

    View details for Web of Science ID 000304099100049

    View details for PubMedID 22596164

  • Structure and dynamics of the M3 muscarinic acetylcholine receptor NATURE Kruse, A. C., Hu, J., Pan, A. C., Arlow, D. H., Rosenbaum, D. M., Rosemond, E., Green, H. F., Liu, T., Chae, P. S., Dror, R. O., Shaw, D. E., Weis, W. I., Wess, J., Kobilka, B. K. 2012; 482 (7386): 552-556

    Abstract

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G(q/11)-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G(i/o)-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

    View details for DOI 10.1038/nature10867

    View details for Web of Science ID 000300770500056

    View details for PubMedID 22358844

  • Crystal structure of the beta(2) adrenergic receptor-Gs protein complex NATURE Rasmussen, S. G., DeVree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., Kobilka, T. S., Thian, F. S., Chae, P. S., Pardon, E., Calinski, D., Mathiesen, J. M., Shah, S. T., Lyons, J. A., Caffrey, M., Gellman, S. H., Steyaert, J., Skiniotis, G., Weis, W. I., Sunahara, R. K., Kobilka, B. K. 2011; 477 (7366): 549-U311

    Abstract

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The ?(2) adrenergic receptor (?(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric ?(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the ?(2)AR and Gs involve the amino- and carboxy-terminal ?-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the ?(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an ?-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the ?-helical domain of G?s relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

    View details for DOI 10.1038/nature10361

    View details for Web of Science ID 000295320900031

    View details for PubMedID 21772288

  • Conformational changes in the G protein Gs induced by the beta(2) adrenergic receptor NATURE Chung, K. Y., Rasmussen, S. G., Liu, T., Li, S., DeVree, B. T., Chae, P. S., Calinski, D., Kobilka, B. K., Woods, V. L., Sunahara, R. K. 2011; 477 (7366): 611-U143

    Abstract

    G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein ?-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human ?(2) adrenergic receptor (?(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the ?(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the ?(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the ?-subunit of Gs and consequently alters the 'P-loop' that binds the ?-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and ?-phosphate coordination are key determinants of GDP (and GTP) binding affinity.

    View details for DOI 10.1038/nature10488

    View details for Web of Science ID 000295320900044

    View details for PubMedID 21956331

  • Structural insights into adrenergic receptor function and pharmacology TRENDS IN PHARMACOLOGICAL SCIENCES Kobilka, B. K. 2011; 32 (4): 213-218

    Abstract

    It has been over 50years since Sir James Black developed the first beta adrenergic receptor (?AR) blocker to treat heart disease. At that time, the concept of cell surface receptors was relatively new and not widely accepted, and most of the tools currently used to characterize plasma membrane receptors had not been developed. There has been remarkable progress in receptor biology since then, including the development of radioligand binding assays, the biochemical characterization of receptors as discrete membrane proteins, and the cloning of the first G-protein-coupled receptors (GPCRs), which led to the identification of other members of the large family of GPCRs. More recently, progress in GPCR structural biology has led to insights into the three-dimensional structures of ?ARs in both active and inactive states. Despite all of this progress, the process of developing a drug for a particular GPCR target has become more complex, time-consuming and expensive.

    View details for DOI 10.1016/j.tips.2011.02.005

    View details for Web of Science ID 000290189200007

    View details for PubMedID 21414670

  • Structure and function of an irreversible agonist-beta(2) adrenoceptor complex NATURE Rosenbaum, D. M., Zhang, C., Lyons, J. A., Holl, R., Aragao, D., Arlow, D. H., Rasmussen, S. G., Choi, H., DeVree, B. T., Sunahara, R. K., Chae, P. S., Gellman, S. H., Dror, R. O., Shaw, D. E., Weis, W. I., Caffrey, M., Gmeiner, P., Kobilka, B. K. 2011; 469 (7329): 236-240

    Abstract

    G-protein-coupled receptors (GPCRs) are eukaryotic integral membrane proteins that modulate biological function by initiating cellular signalling in response to chemically diverse agonists. Despite recent progress in the structural biology of GPCRs, the molecular basis for agonist binding and allosteric modulation of these proteins is poorly understood. Structural knowledge of agonist-bound states is essential for deciphering the mechanism of receptor activation, and for structure-guided design and optimization of ligands. However, the crystallization of agonist-bound GPCRs has been hampered by modest affinities and rapid off-rates of available agonists. Using the inactive structure of the human ?(2) adrenergic receptor (?(2)AR) as a guide, we designed a ?(2)AR agonist that can be covalently tethered to a specific site on the receptor through a disulphide bond. The covalent ?(2)AR-agonist complex forms efficiently, and is capable of activating a heterotrimeric G protein. We crystallized a covalent agonist-bound ?(2)AR-T4L fusion protein in lipid bilayers through the use of the lipidic mesophase method, and determined its structure at 3.5?Å resolution. A comparison to the inactive structure and an antibody-stabilized active structure (companion paper) shows how binding events at both the extracellular and intracellular surfaces are required to stabilize an active conformation of the receptor. The structures are in agreement with long-timescale (up to 30??s) molecular dynamics simulations showing that an agonist-bound active conformation spontaneously relaxes to an inactive-like conformation in the absence of a G protein or stabilizing antibody.

    View details for DOI 10.1038/nature09665

    View details for Web of Science ID 000286143400043

    View details for PubMedID 21228876

  • Structure of a nanobody-stabilized active state of the beta(2) adrenoceptor NATURE Rasmussen, S. G., Choi, H., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., DeVree, B. T., Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., Schnapp, A., Konetzki, I., Sunahara, R. K., Gellman, S. H., Pautsch, A., Steyaert, J., Weis, W. I., Kobilka, B. K. 2011; 469 (7329): 175-180

    Abstract

    G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human ?(2) adrenergic receptor (?(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive ?(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11?Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.

    View details for DOI 10.1038/nature09648

    View details for Web of Science ID 000286143400030

    View details for PubMedID 21228869

  • Tandem Facial Amphiphiles for Membrane Protein Stabilization JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chae, P. S., Gotfryd, K., Pacyna, J., Miercke, L. J., Rasmussen, S. G., Robbins, R. A., Rana, R. R., Loland, C. J., Kobilka, B., Stroud, R., Byrne, B., Gether, U., Gellman, S. H. 2010; 132 (47): 16750-16752

    Abstract

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles.

    View details for DOI 10.1021/ja1072959

    View details for Web of Science ID 000284972400009

    View details for PubMedID 21049926

  • A Device for Separated and Reversible Co-Culture of Cardiomyocytes BIOTECHNOLOGY PROGRESS Chen, M. Q., Whittington, R. H., Day, P. W., Kobilka, B. K., Giovangrandi, L., Kovacs, G. T. 2010; 26 (4): 1164-1171

    Abstract

    A novel technique is introduced for patterning and controllably merging two cultures of adherent cells on a microelectrode array (MEA) by separation with a removable physical barrier. The device was first demonstrated by separating two cardiomyocyte populations, which upon merging synchronized electrical activity. Next, two applications of this co-culture device are presented that demonstrate its flexibility as well as outline different metrics to analyze co-cultures. In a differential assay, the device contained two distinct cell cultures of neonatal wild-type and beta-adrenergic receptor (beta-AR) knockout cardiomyocytes and simultaneously exposed them with the beta-AR agonist isoproterenol. The beat rate and action potential amplitude from each cell type displayed different characteristic responses in both unmerged and merged states. This technique can be used to study the role of beta-receptor signaling and how the corresponding cellular response can be modulated by neighboring cells. In the second application, action potential propagation between modeled host and graft cell cultures was shown through the analysis of conduction velocity across the MEA. A co-culture of murine cardiomyocytes (host) and murine skeletal myoblasts (graft) demonstrated functional integration at the boundary, as shown by the progression of synchronous electrical activity propagating from the host into the graft cell populations. However, conduction velocity significantly decreased as the depolarization waves reached the graft region due to a mismatch of inherent cell properties that influence conduction.

    View details for DOI 10.1002/btpr.431

    View details for Web of Science ID 000281045100030

    View details for PubMedID 20730771

  • Regulation of G-Protein Coupled Receptor Traffic by an Evolutionary Conserved Hydrophobic Signal TRAFFIC Angelotti, T., Daunt, D., Shcherbakova, O. G., Kobilka, B., Hurt, C. M. 2010; 11 (4): 560-578

    Abstract

    Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

    View details for DOI 10.1111/j.1600-0854.2010.01033.x

    View details for Web of Science ID 000275530700012

    View details for PubMedID 20059747

  • Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor NATURE Bokoch, M. P., Zou, Y., Rasmussen, S. G., Liu, C. W., Nygaard, R., Rosenbaum, D. M., Fung, J. J., Choi, H., Thian, F. S., Kobilka, T. S., Puglisi, J. D., Weis, W. I., Pardo, L., Prosser, R. S., Mueller, L., Kobilka, B. K. 2010; 463 (7277): 108-U121

    Abstract

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

    View details for DOI 10.1038/nature08650

    View details for Web of Science ID 000273344900040

    View details for PubMedID 20054398

  • Ligand-regulated oligomerization of beta(2)-adrenoceptors in a model lipid bilayer EMBO JOURNAL Fung, J. J., Deupi, X., Pardo, L., Yao, X. J., Velez-Ruiz, G. A., DeVree, B. T., Sunahara, R. K., Kobilka, B. K. 2009; 28 (21): 3315-3328

    Abstract

    The beta(2)-adrenoceptor (beta(2)AR) was one of the first Family A G protein-coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified beta(2)AR site-specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the beta(2)AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into beta(2)AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists.

    View details for DOI 10.1038/emboj.2009.267

    View details for Web of Science ID 000271891400005

    View details for PubMedID 19763081

  • The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex. Proceedings of the National Academy of Sciences of the United States of America Yao, X. J., Vélez Ruiz, G., Whorton, M. R., Rasmussen, S. G., DeVree, B. T., Deupi, X., Sunahara, R. K., Kobilka, B. 2009; 106 (23): 9501-9506

    Abstract

    G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs, drugs classified as inverse agonists can suppress basal activity. There is a growing body of evidence that basal activity is physiologically relevant, and the ability of a drug to inhibit basal activity may influence its therapeutic properties. However, the molecular mechanism for basal activation and inhibition of basal activity by inverse agonists is poorly understood and difficult to study, because the basally active state is short-lived and represents a minor fraction of receptor conformations. Here, we investigate basal activation of the G protein Gs by the beta(2) adrenergic receptor (beta(2)AR) by using purified receptor reconstituted into recombinant HDL particles with a stoichiometric excess of Gs. The beta(2)AR is site-specifically labeled with a small, environmentally sensitive fluorophore enabling direct monitoring of agonist- and Gs-induced conformational changes. In the absence of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy.

    View details for DOI 10.1073/pnas.0811437106

    View details for PubMedID 19470481

  • The structure and function of G-protein-coupled receptors NATURE Rosenbaum, D. M., Rasmussen, S. G., Kobilka, B. K. 2009; 459 (7245): 356-363

    Abstract

    G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein structure and biology. Great progress has been made over the past three decades in understanding diverse GPCRs, from pharmacology to functional characterization in vivo. Recent high-resolution structural studies have provided insights into the molecular mechanisms of GPCR activation and constitutive activity.

    View details for DOI 10.1038/nature08144

    View details for Web of Science ID 000266243700033

    View details for PubMedID 19458711

  • Structural insights into G-protein-coupled receptor activation CURRENT OPINION IN STRUCTURAL BIOLOGY Weis, W. I., Kobilka, B. K. 2008; 18 (6): 734-740

    Abstract

    G-protein-coupled receptors (GPCRs) are the largest family of eukaryotic plasma membrane receptors, and are responsible for the majority of cellular responses to external signals. GPCRs share a common architecture comprising seven transmembrane (TM) helices. Binding of an activating ligand enables the receptor to catalyze the exchange of GTP for GDP in a heterotrimeric G protein. GPCRs are in a conformational equilibrium between inactive and activating states. Crystallographic and spectroscopic studies of the visual pigment rhodopsin and two beta-adrenergic receptors have defined some of the conformational changes associated with activation.

    View details for DOI 10.1016/j.sbi.2008.09.010

    View details for Web of Science ID 000262064300014

    View details for PubMedID 18957321

  • New G-protein-coupled receptor crystal structures: insights and limitations TRENDS IN PHARMACOLOGICAL SCIENCES Kobilka, B., Schertler, G. F. 2008; 29 (2): 79-83

    Abstract

    G-protein-coupled receptors (GPCRs) constitute a large family of structurally similar proteins that respond to a chemically diverse array of physiological and environmental stimulants. Until recently, high-resolution structural information was limited to rhodopsin, a naturally abundant GPCR that is highly specialized for the detection of light. Non-rhodopsin GPCRs for diffusible hormones and neurotransmitters have proven more resistant to crystallography approaches, possibly because of their inherent structural flexibility and the need for recombinant expression. Recently, crystal structures of the human beta(2) adrenoceptor have been obtained using two different approaches to stabilize receptor protein and increase polar surface area. These structures, together with the existing structures of rhodopsin, represent an important first step in understanding how GPCRs work at a molecular level. Much more high-resolution information is needed for this important family of membrane proteins, however: for example, the structures of GPCRs from different families, structures bound to ligands having different efficacies, and structures of GPCRs in complex with G proteins and other signaling molecules. Methods to characterize the dynamic aspects of the GPCR architecture at high resolution will also be important.

    View details for DOI 10.1016/j.tips.2007.11.009

    View details for Web of Science ID 000253599000005

    View details for PubMedID 18194818

  • Signaling from beta(1)- and beta(2)-adrenergic receptors is defined by differential interactions with PDE4 EMBO JOURNAL Richter, W., Day, P., Agrawal, R., Bruss, M. D., Granier, S., Wang, Y. L., Rasmussen, S. G., Horner, K., Wang, P., Lei, T., Patterson, A. J., Kobilka, B., Conti, M. 2008; 27 (2): 384-393

    Abstract

    Beta1- and beta2-adrenergic receptors (betaARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by beta1AR but not beta2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that beta1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a beta2AR/beta-arrestin/PDE complex reported previously. The beta1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the beta2AR is a prerequisite for the recruitment of a complex consisting of beta-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of beta1- and beta2-adrenoceptor signaling.

    View details for DOI 10.1038/sj.emboj.7601968

    View details for Web of Science ID 000253408600009

    View details for PubMedID 18188154

  • GPCR engineering yields high-resolution structural insights into beta(2)-adrenergic receptor function SCIENCE Rosenbaum, D. M., Cherezov, V., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H., Yao, X., Weis, W. I., Stevens, R. C., Kobilka, B. K. 2007; 318 (5854): 1266-1273

    Abstract

    The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.

    View details for DOI 10.1126/science.1150609

    View details for Web of Science ID 000251086600034

    View details for PubMedID 17962519

  • High-resolution crystal structure of an engineered human beta(2)-adrenergic G protein-coupled receptor SCIENCE Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H., Kuhn, P., Weis, W. I., Kobilka, B. K., Stevens, R. C. 2007; 318 (5854): 1258-1265

    Abstract

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.

    View details for DOI 10.1126/science.1150577

    View details for Web of Science ID 000251086600033

    View details for PubMedID 17962520

  • Crystal structure of the human beta(2) adrenergic G-protein-coupled receptor NATURE Rasmussen, S. G., Choi, H., Rosenbaum, D. M., Kobilka, T. S., Thian, F. S., Edwards, P. C., Burghammer, M., Ratnala, V. R., Sanishvili, R., Fischetti, R. F., Schertler, G. F., Weis, W. I., Kobilka, B. K. 2007; 450 (7168): 383-U4

    Abstract

    Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 A/3.7 A resolution. The cytoplasmic ends of the beta2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the beta2AR are not seen. The beta2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the beta2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

    View details for DOI 10.1038/nature06325

    View details for Web of Science ID 000250918600046

    View details for PubMedID 17952055

  • A monoclonal antibody for G protein-coupled receptor crystallography NATURE METHODS Day, P. W., Rasmussen, S. G., Parnot, C., Fung, J. J., Masood, A., Kobilka, T. S., Yao, X., Choi, H., Weis, W. I., Rohrer, D. K., Kobilka, B. K. 2007; 4 (11): 927-929

    Abstract

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural information. We describe the generation of a monoclonal antibody that recognizes the third intracellular loop (IL3) of the native human beta(2) adrenergic (beta(2)AR) receptor; this antibody was critical for acquiring diffraction-quality crystals.

    View details for DOI 10.1038/NMETH1112

    View details for Web of Science ID 000250575700017

    View details for PubMedID 17952087

  • N-ethylmaleimide-sensitive factor regulates beta(2) adrenoceptor trafficking and signaling in cardiomyocytes MOLECULAR PHARMACOLOGY Wang, Y., Lauffer, B., von Zastrow, M., Kobilka, B. K., Xiang, Y. 2007; 72 (2): 429-439

    Abstract

    Recycling of G protein-coupled receptors determines the functional resensitization of receptors and is implicated in switching beta2 adrenoceptor (beta2AR) G protein specificity in cardiomyocytes. The human beta2AR carboxyl end binds to the N-ethylmaleimide-sensitive factor (NSF), an ATPase integral to membrane trafficking machinery. It is interesting that the human beta2AR (hbeta2AR) carboxyl end pulled down NSF from mouse heart lysates, whereas the murine one did not. Despite this difference, both beta2ARs exhibited substantial agonist-induced internalization, recycling, and Gi coupling in cardiomyocytes. The hbeta2AR, however, displayed faster rates of agonist-induced internalization and recycling compared with the murine beta2AR (mbeta2AR) and a more profound Gi component in its contraction response. Replacing the mbeta2AR proline (-1) with a leucine generated a gain-of-function mutation, mbeta2AR-P417L, with a rescued ability to bind NSF, faster internalization and recycling than the mbeta2AR, and a significant enhancement in Gi signaling, which mimics the hbeta2AR. Selective disruption of the mbeta2AR-P417L binding to NSF inhibited the receptor coupling to Gi. Mean-while, inhibiting NSF with N-ethylmaleimide blocked the mbeta2AR recycling after agonist-induced endocytosis. Expressing the NSF-E329Q mutant lacking ATPase activity inhibited the mbeta2AR coupling to Gi in cardiomyocytes. Our results revealed a dual regulation on hbeta2AR trafficking and signaling by NSF through direct binding to cargo receptor and its ATPase activity and uncovered an unprecedented role for the receptor binding to NSF in regulating G protein specificity that has diverged between mouse and human beta2ARs.

    View details for DOI 10.1124/mol.107.037747

    View details for Web of Science ID 000248282900023

    View details for PubMedID 17510209

  • Conformational complexity of G-protein-coupled receptors TRENDS IN PHARMACOLOGICAL SCIENCES Kobilka, B. K., Deupi, X. 2007; 28 (8): 397-406

    Abstract

    G-protein-coupled receptors (GPCRs) are remarkably versatile signaling molecules. Members of this large family of membrane proteins respond to structurally diverse ligands and mediate most transmembrane signal transduction in response to hormones and neurotransmitters, and in response to the senses of sight, smell and taste. Individual GPCRs can signal through several G-protein subtypes and through G-protein-independent pathways, often in a ligand-specific manner. This functional plasticity can be attributed to structural flexibility of GPCRs and the ability of ligands to induce or to stabilize ligand-specific conformations. Here, we review what has been learned about the dynamic nature of the structure and mechanism of GPCR activation, primarily focusing on spectroscopic studies of purified human beta2 adrenergic receptor.

    View details for DOI 10.1016/j.tips.2007.06.003

    View details for Web of Science ID 000248898900007

    View details for PubMedID 17629961

  • Structure and conformational changes in the C-terminal domain of the beta(2)-adrenoceptor - Insights from fluorescence resonance energy transfer studies JOURNAL OF BIOLOGICAL CHEMISTRY Granier, S., Kim, S., Shafer, A. M., Ratnala, V. R., Fung, J. J., Zare, R. N., Kobilka, B. 2007; 282 (18): 13895-13905

    Abstract

    The C terminus of the beta(2)-adrenoceptor (AR) interacts with G protein-coupled receptor kinases and arrestins in an agonist-dependent manner, suggesting that conformational changes induced by ligands in the transmembrane domains are transmitted to the C terminus. We used fluorescence resonance energy transfer (FRET) to examine ligand-induced structural changes in the distance between two positions on the beta(2)-AR C terminus and cysteine 265 (Cys-265) at the cytoplasmic end of transmembrane domain 6. The donor fluorophore FlAsH (Fluorescein Arsenical Helix binder) was attached to a CCPGCC motif introduced at position 351-356 in the proximal C terminus or at the distal C terminus. An acceptor fluorophore, Alexa Fluor 568, was attached to Cys-265. FRET analyses revealed that the average distances between Cys-265 and the proximal and distal FlAsH sites were 57 and 62A(,) respectively. These relatively large distances suggest that the C terminus is in an extended, relatively unstructured conformation. Nevertheless, we observed ligand-specific changes in FRET. All ligands induced an increase in FRET between the proximal C-terminal FlAsH site and Cys-265. Ligands that have been shown to induce arrestin-dependent ERK activation, including the catecholamine agonists and the inverse agonist ICI118551, led to a decrease in FRET between the distal FlAsH site and Cys-265, whereas other ligands had no effect or induced a small increase in FRET. Taken together the results provide new insight into the structure of the C terminus of the beta(2)-AR as well as ligand-induced conformational changes that may be relevant to arrestin-dependent regulation and signaling.

    View details for DOI 10.1074/jbc.M611904200

    View details for Web of Science ID 000246060300079

    View details for PubMedID 17347144

  • A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Whorton, M. R., Bokoch, M. P., Rasmussen, S. G., Huang, B., Zare, R. N., Kobilka, B., Sunahara, R. K. 2007; 104 (18): 7682-7687

    Abstract

    G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein (rHDL) phospholipid bilayer particle together with the stimulatory heterotrimeric G protein, Gs. Single-molecule fluorescence imaging and FRET analysis demonstrate that a single beta2AR is incorporated per rHDL particle. The monomeric beta2AR efficiently activates Gs and displays GTP-sensitive allosteric ligand-binding properties. These data suggest that a monomeric receptor in a lipid bilayer is the minimal functional unit necessary for signaling, and that the cooperativity of agonist binding is due to G protein association with a receptor monomer and not receptor oligomerization.

    View details for DOI 10.1073/pnas.0611448104

    View details for Web of Science ID 000246239400068

    View details for PubMedID 17452637

  • G protein coupled receptor structure and activation BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Kobilka, B. K. 2007; 1768 (4): 794-807

    Abstract

    G protein coupled receptors (GPCRs) are remarkably versatile signaling molecules. The members of this large family of membrane proteins are activated by a spectrum of structurally diverse ligands, and have been shown to modulate the activity of different signaling pathways in a ligand specific manner. In this manuscript I will review what is known about the structure and mechanism of activation of GPCRs focusing primarily on two model systems, rhodopsin and the beta(2) adrenoceptor.

    View details for DOI 10.1016/j.bbamem.2006.10.021

    View details for Web of Science ID 000245972500005

    View details for PubMedID 17188232

  • Organization of beta-adrenoceptor signaling compartments by sympathetic innervation of cardiac myocytes JOURNAL OF CELL BIOLOGY Shcherbakova, O. G., Hurt, C. M., Xiang, Y., Dell'Acqua, M. L., Zhang, Q., Tsien, R. W., Kobilka, B. K. 2007; 176 (4): 521-533

    Abstract

    The sympathetic nervous system regulates cardiac function through the activation of adrenergic receptors (ARs). beta(1) and beta(2)ARs are the primary sympathetic receptors in the heart and play different roles in regulating cardiac contractile function and remodeling in response to injury. In this study, we examine the targeting and trafficking of beta(1) and beta(2)ARs at cardiac sympathetic synapses in vitro. Sympathetic neurons form functional synapses with neonatal cardiac myocytes in culture. The myocyte membrane develops into specialized zones that surround contacting axons and contain accumulations of the scaffold proteins SAP97 and AKAP79/150 but are deficient in caveolin-3. The beta(1)ARs are enriched within these zones, whereas beta(2)ARs are excluded from them after stimulation of neuronal activity. The results indicate that specialized signaling domains are organized in cardiac myocytes at sites of contact with sympathetic neurons and that these domains are likely to play a role in the subtype-specific regulation of cardiac function by beta(1) and beta(2)ARs in vivo.

    View details for DOI 10.1083/jcb.200604167

    View details for Web of Science ID 000244348300013

    View details for PubMedID 17296797

  • Counting low-copy number proteins in a single cell SCIENCE Huang, B., Wu, H., Bhaya, D., Grossman, A., Granier, S., Kobilka, B. K., Zare, R. N. 2007; 315 (5808): 81-84

    Abstract

    We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta2 adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells (Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.

    View details for DOI 10.1126/science.1133992

    View details for Web of Science ID 000243259100039

    View details for PubMedID 17204646

  • Activation of G Protein Coupled Receptors MECHANISMS AND PATHWAYS OF HETEROTRIMERIC G PROTEIN SIGNALING Deupi, X., Kobilka, B. 2007; 74: 137-166

    Abstract

    G protein-coupled receptors (GPCRs) mediate responses to hormones and neurotransmitters, as well as the senses of sight, smell, and taste. These remarkably versatile signaling molecules respond to structurally diverse ligands. Many GPCRs couple to multiple G protein subtypes, and several have been shown to activate G protein-independent signaling pathways. Drugs acting on GPCRs exhibit efficacy profiles that may differ for different signaling cascades. The functional plasticity exhibited by GPCRs can be attributed to structural flexibility and the existence of multiple ligand-specific conformational states. This chapter will review our current understanding of the mechanism by which agonists bind and activate GPCRs.

    View details for DOI 10.1016/S0065-3231(07)74004-4

    View details for Web of Science ID 000250106300004

    View details for PubMedID 17854657

  • PDZ-domain arrays for identifying components of GPCR signaling complexes TRENDS IN PHARMACOLOGICAL SCIENCES Day, P., Kobilka, B. 2006; 27 (10): 509-511

    Abstract

    Many G-protein-coupled receptors (GPCRs) modulate the activity of multiple effectors. Yet, despite this apparent promiscuity, signaling in the context of differentiated cells is often highly specific. This specificity is attributable to the formation of cell-type-specific signaling complexes that are held together by scaffolding proteins, many of which contain one or more PDZ domains. Identifying the set of potential interactions among GPCRs, other signaling molecules and these scaffolding proteins is essential for understanding physiological signaling processes. A recent article describes an elegantly simple PDZ-domain array that can identify potential interacting partners of GPCRs and other signaling molecules.

    View details for DOI 10.1016/j.tips.2006.08.003

    View details for Web of Science ID 000241258000001

    View details for PubMedID 16904197

  • Differential targeting and function of alpha(2A) and alpha(2C) adrenergic receptor subtypes in cultured sympathetic neurons NEUROPHARMACOLOGY Brum, P. C., Hurt, C. M., Shcherbakova, O. G., Kobilka, B., Angelotti, T. 2006; 51 (3): 397-413

    Abstract

    Previous research suggested that alpha2A and alpha2C adrenergic receptor (AR) subtypes have overlapping but unique physiological roles in neuronal signaling; however, the basis for these dissimilarities is not completely known. To better understand the observed functional differences between these autoreceptors, we investigated targeting and signaling of endogenously expressed alpha2A and alpha2CARs in cultured sympathetic ganglion neurons (SGN). At Days 1 and 4, alpha2A and alpha2CARs could be readily detected in SGN from wild-type mice. By Day 8, alpha2A ARs were targeted to cell body, as well as axonal and dendritic sites, whereas alpha2C ARs were primarily localized to an intracellular vesicular pool within the cell body and proximal dendritic projections. Expression of synaptic vesicle marker protein SV2 did not differ at Day 8 nor co-localize with either subtype. By Day 16, however, alpha2C ARs had relocated to somatodendritic and axonal sites and, unlike alpha2A ARs, co-localized with SV2 at synaptic contact sites. Consistent with a functional role for alpha2 ARs, we also observed that dexmedetomidine stimulation of cultured SGN more efficiently inhibited depolarization-induced calcium entry into older, compared to younger, cultures. These results provide direct evidence of distinct developmental patterns of endogenous alpha2A and alpha2C AR targeting and function in a native cell system and that maturation of SGN in culture leads to alterations in neuronal properties required for proper targeting. More importantly, the co-localization at Day 16 of alpha2C ARs at sites of synaptic contact may partially explain the differential modulation of neurotransmitter release and responsiveness to action potential frequency observed between alpha2A and alpha2C ARs in SGN.

    View details for DOI 10.1016/j.neuropharm.2006.03.032

    View details for Web of Science ID 000240811900001

    View details for PubMedID 16750543

  • Coupling ligand structure to specific conformational switches in the beta(2)-adrenoceptor NATURE CHEMICAL BIOLOGY Yao, X., Parnot, C., Deupi, X., Ratnala, V. R., Swaminath, G., Farrens, D., Kobilka, B. 2006; 2 (8): 417-422

    Abstract

    G protein-coupled receptors (GPCRs) regulate a wide variety of physiological functions in response to structurally diverse ligands ranging from cations and small organic molecules to peptides and glycoproteins. For many GPCRs, structurally related ligands can have diverse efficacy profiles. To investigate the process of ligand binding and activation, we used fluorescence spectroscopy to study the ability of ligands having different efficacies to induce a specific conformational change in the human beta2-adrenoceptor (beta2-AR). The 'ionic lock' is a molecular switch found in rhodopsin-family GPCRs that has been proposed to link the cytoplasmic ends of transmembrane domains 3 and 6 in the inactive state. We found that most partial agonists were as effective as full agonists in disrupting the ionic lock. Our results show that disruption of this important molecular switch is necessary, but not sufficient, for full activation of the beta2-AR.

    View details for DOI 10.1038/nchembio801

    View details for Web of Science ID 000239146800010

    View details for PubMedID 16799554

  • Phospholipid biotinylation of polydimethylsiloxane (PDMS) for protein immobilization LAB ON A CHIP Huang, B., Wu, H. K., Kim, S., Kobilka, B. K., Zare, R. N. 2006; 6 (3): 369-373

    Abstract

    Polydimethylsiloxane (PDMS) surfaces can be functionalized with biotin groups by adding biotinylated phospholipids to the PDMS prepolymer before curing. The addition of beta-D-dodecyl-N-maltoside (DDM) in the solution blocks non-specific protein binding on these functionalized PDMS surfaces. We characterize the surface by measuring fluorescently labeled streptavidin binding. Single molecule tracking shows that the phospholipids are not covalently linked to PDMS polymer chains, but the surface functionalization is not removed by washing. We demonstrate the immobilization of biotinylated antibodies and lectins through biotin-avidin interactions.

    View details for DOI 10.1039/b515840k

    View details for Web of Science ID 000235993800005

    View details for PubMedID 16511619

  • Effect of targeted deletions of beta(1)- and beta(2)-adrenergic-receptor subtypes on heart rate variability AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Ecker, P. M., Lin, C. C., Powers, J., Kobilka, B. K., Dubin, A. M., Bernstein, D. 2006; 290 (1): H192-H199

    Abstract

    Beta-adrenergic receptors (beta-ARs) play a major role in regulating heart rate (HR) and contractility in the intact cardiovascular system. Three subtypes (beta1, beta2, and beta3) are expressed in heart tissue, and the role of each subtype in regulating cardiac function has previously been determined by using both pharmacological and gene-targeting approaches. However, previous studies have only examined the role of beta-ARs in the macrolevel regulation of HR. We employed three knockout (KO) mouse lines, beta1-KO, beta2-KO, and beta1/beta2 double KO (DL-KO), to examine the role that beta-AR subtypes play in HR variability (HRV) and in the sympathetic and parasympathetic inputs into HR control. Fast Fourier transformation (FFT) in frequency domain methods of ECG spectral analysis was used to resolve HRV into high- and low-frequency (HF and LF) powers. Resting HR (in beats/min) was decreased in beta1-KO [488 (SD 27)] and DL-KO [495 (SD 12)] mice compared with wild-type [WT; 638 (SD 30)] or beta2-KO [656 (SD 51)] (P < 0.0005) mice. Mice lacking beta1-ARs (beta1-KO and DL-KO) had increased HRV (as illustrated by the standard deviation of normal R-R intervals) and increased normalized HF and LF powers compared with mice with intact beta1-ARs (WT and beta2-KO). These results demonstrate the differential role of beta-AR subtypes in regulating autonomic signaling.

    View details for DOI 10.1152/ajpheart.00032.2005

    View details for Web of Science ID 000234148200023

    View details for PubMedID 16113068

  • Differential cardioprotective/cardiotoxic effects mediated by ss-adrenergic receptor subtypes AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Bernstein, D., Fajardo, G., Zhao, M. M., Urashima, T., Powers, J., Berry, G., Kobilka, B. K. 2005; 289 (6): H2441-H2449

    Abstract

    Recent data suggest that beta-adrenergic receptor subtypes couple differentially to signaling pathways regulating cardiac function vs. cardiac remodeling. To dissect the roles of beta1- vs. beta2-receptors in the pathogenesis of cardiomyopathy, doxorubicin was administered to beta1, beta2, and beta1/beta2 knockout (-/-) and wild-type mice. Expression and activation of MAPKs were measured. Wild-type and beta1-/- mice showed no acute cardiovascular effects, whereas beta2-/- mice all died within 30 min. The additional deletion of the beta1-receptor (beta1/beta2-/-) totally rescued this toxicity. beta2-/- mice developed decreased contractile function, hypotension, QTc prolongation, and ST segment changes and a 20-fold increase in p38 MAPK activity not seen in the other genotypes. The MAPK inhibitor SB-203580 rescued beta2-/- mice from this acute toxicity. The enhanced toxicity in beta2-/- mice was also recapitulated in wild-type mice with the beta2-selective antagonist ICI-118,551, although the rescue effect of the beta1-deletion was not recapitulated using the beta1-selective antagonist metoprolol or the nonselective beta-antagonist propranolol. These data suggest that beta2-adrenergic receptors play a cardioprotective role in the pathogenesis of cardiomyopathy, whereas beta1-adrenergic receptors mediate at least some of the acute cardiotoxicity of anthracyclines. Differential activation of MAPK isoforms, previously shown in vitro to regulate beta-agonist as well as doxorubicin cardiotoxicity, appears to play a role in mediating the differential effects of these beta-adrenergic receptor subtypes in vivo.

    View details for DOI 10.1152/ajpheart.00005.2005

    View details for Web of Science ID 000233176600023

    View details for PubMedID 16040722

  • Using synthetic lipids to stabilize purified beta(2) adrenoceptor in detergent micelles ANALYTICAL BIOCHEMISTRY Yao, Z. P., Kobilka, B. 2005; 343 (2): 344-346

    View details for DOI 10.1016/j.ab.2005.05.002

    View details for Web of Science ID 000231163900022

    View details for PubMedID 16005425

  • Probing the beta(2) adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists JOURNAL OF BIOLOGICAL CHEMISTRY Swaminath, G., Deupi, X., LEE, T. W., Zhu, W., Thian, F. S., Kobilka, T. S., Kobilka, B. 2005; 280 (23): 22165-22171

    Abstract

    The beta(2) adrenergic receptor (beta(2)AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. The beta(2)AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a molecular probe to identify mechanistic differences between beta(2)AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different site on the beta(2)AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a GPCR is a multistep process that can be dissected into its component parts using agonist fragments.

    View details for Web of Science ID 000229557900061

    View details for PubMedID 15817484

  • Mass spectrometric analysis of agonist effects on posttranslational modifications of the beta-2 adrenoceptor in mammalian cells BIOCHEMISTRY Trester-Zedlitz, M., Burlingame, A., Kobilka, B., von Zastrow, M. 2005; 44 (16): 6133-6143

    Abstract

    Posttranslational modifications (PTMs) of the beta-2 adrenoceptor (B2AR) play a fundamental role in receptor regulation by agonists. We have examined the effects of several agonists on net levels of B2AR palmitoylation and phosphorylation using epitope tagging in stably transfected human embryonal kidney (HEK) 293 cells, immunoaffinity purification, and mass spectrometry combined with the method of stable isotope labeling by amino acids in cell culture (SILAC). Palmitoylation of Cys341 was confirmed and did not change detectably after 30 min exposure of cells to saturating concentrations of dopamine, epinephrine, or isoproterenol. However, all of these agonists produced a marked increase in net phosphorylation. Phosphorylation of the third cytoplasmic loop was increased to a similar degree by all three agonists, whereas differences between agonists were observed in net phosphorylation of the carboxyl-terminal cytoplasmic domain (isoproterenol approximately epinephrine > dopamine). Interestingly, agonist-induced phosphorylation of the carboxyl-terminal cytoplasmic domain was observed exclusively in a proximal portion (between residues 339-369). None of the agonists produced detectable phosphorylation in a distal portion of the cytoplasmic tail, which contains all sites of agonist-induced phosphorylation identified previously by in vitro reconstitution. These results provide insight to agonist-dependent regulation of the B2AR in intact cells, suggest the existence of significant differences in regulatory phosphorylation events occurring between in vitro and in vivo conditions, and outline a general analytical approach to investigate regulated PTM of receptors in mammalian cells.

    View details for DOI 10.1021/bi0475469

    View details for Web of Science ID 000228678900018

    View details for PubMedID 15835901

  • Phosphodiesterase 4D is required for beta(2) adrenoceptor subtype-specific signaling in cardiac myocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xiang, Y., Naro, F., Zoudilova, M., Jin, S. L., Conti, M., Kobilka, B. 2005; 102 (3): 909-914

    Abstract

    beta adrenoceptor (betaAR) signaling is finely regulated to mediate the sympathetic nervous system control of cardiovascular function. In neonatal cardiac myocytes, beta1AR activates the conventional Gs/cAMP pathway, whereas beta2AR sequentially activates both the Gs and Gi pathways to regulate the myocyte contraction rate. Here, we show that phosphodiesterase 4D (PDE4D) selectively impacts signaling by beta2AR in neonatal cardiac myocytes, while having little or no effect on beta1AR signaling. Although beta2AR activation leads to an increase in cAMP production, the cAMP generated does not have access to the protein kinase A-dependent signaling pathways by which the beta1AR regulates the contraction rate. However, this restricted access is lost in the presence of PDE4 inhibitors or after ablation of PDE4D. These results not only suggest that PDE4D is an integral component of the beta2AR signaling complex, but also underscore the critical role of subcellular cAMP regulation in the complex control of receptor signaling. They also illustrate a mechanism for fine-tuned betaAR subtype signaling specificity and intensity in the cardiac system.

    View details for DOI 10.1073/pnas.0405263102

    View details for Web of Science ID 000226436000069

    View details for PubMedID 15644445

  • Toward understanding GPCR dimers NATURE STRUCTURAL & MOLECULAR BIOLOGY Parnot, C., Kobilka, B. 2004; 11 (8): 691-692

    View details for DOI 10.1038/nsmb0804-691

    View details for Web of Science ID 000222930600007

    View details for PubMedID 15280880

  • Agonist binding: A multistep process MOLECULAR PHARMACOLOGY Kobilka, B. 2004; 65 (5): 1060-1062

    View details for Web of Science ID 000220951600002

    View details for PubMedID 15102933

  • Protecting the myocardium: A role for the beta 2 adrenergic receptor in the heart CRITICAL CARE MEDICINE Patterson, A. J., Zhu, W. Z., Chow, A., Agrawal, R., Kosek, J., Xiao, R. P., Kobilka, B. 2004; 32 (4): 1041-1048

    Abstract

    The sympathetic nervous system enhances cardiac muscle function by activating beta adrenergic receptors (betaARs). Recent studies suggest that chronic betaAR stimulation is detrimental, however, and that it may play a role in the clinical deterioration of patients with congestive heart failure. To examine the impact of chronic beta1AR and beta2AR subtype stimulation individually, we studied the cardiovascular effects of catecholamine infusions in betaAR subtype knockout mice (beta1KO, beta2KO).Prospective, randomized, experimental study.Animal research laboratory.beta1KO and beta2KO mice and wild-type controls.The animals were subjected to 2 wks of continuous infusion of the betaAR agonist isoproterenol. Analyses of cardiac function and structure were performed during and 3 days after completion of the infusions. Functional studies included graded exercise treadmill testing, in vivo assessments of left ventricular function using Mikro-Tip catheter transducers, right ventricular pressure measurements, and analyses of organ weight to body weight ratios. Structural studies included heart weight measurements, assessments of myocyte ultrastructure using electron microscopy, and in situ terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling staining to quantitate myocyte apoptosis.We found that isoproterenol-treated beta2KO mice experienced greater mortality rates (p =.001, chi-square test using Fisher's exact method) and increased myocyte apoptosis at 3- and 7-day time points (p =.04 and p =.0007, respectively, two-way analysis of variance).The results of this study suggest that in vivo beta2AR activation is antiapoptotic and contributes to myocardial protection.

    View details for DOI 10.1097/01.CCM.0000120049.43113.90

    View details for Web of Science ID 000220872700021

    View details for PubMedID 15071399

  • Plasmon-waveguide resonance studies of ligand binding to the human beta(2)-adrenergic receptor BIOCHEMISTRY Devanathan, S., Yao, Z. P., Salamon, Z., Kobilka, B., Tollin, G. 2004; 43 (11): 3280-3288

    Abstract

    Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that can be used to probe the molecular interactions occurring within anisotropic proteolipid membranes in real time without requiring molecular labeling. This method directly monitors mass density, conformation, and molecular orientation changes occurring in such systems and allows determination of protein-ligand binding constants and binding kinetics. In the present study, PWR has been used to monitor the incorporation of the human beta(2)-adrenergic receptor into a solid-supported egg phosphatidylcholine lipid bilayer and to follow the binding of full agonists (isoproterenol, epinephrine), a partial agonist (dobutamine), an antagonist (alprenolol), and an inverse agonist (ICI-118,551) to the receptor. The combination of differences in binding kinetics and the PWR spectral changes point to the occurrence of multiple conformations that are characteristic of the type of ligand, reflecting differences in the receptor structural states produced by the binding process. These results provide new evidence for the conformational heterogeneity of the liganded states formed by the beta(2)-adrenergic receptor.

    View details for DOI 10.1021/bi035825a

    View details for Web of Science ID 000220276700030

    View details for PubMedID 15023079

  • Sequential binding of agonists to the beta(2) adrenoceptor - Kinetic evidence for intermediate conformational states JOURNAL OF BIOLOGICAL CHEMISTRY Swaminath, G., Xiang, Y., LEE, T. W., Steenhuis, J., Parnot, C., Kobilka, B. K. 2004; 279 (1): 686-691

    Abstract

    The beta2 adrenoreceptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes in purified beta2AR. Here we show that upon catecholamine binding, beta2ARs undergo transitions to two kinetically distinguishable conformational states. Using a panel of chemically related catechol derivatives, we identified the specific chemical groups on the agonist responsible for the rapid and slow conformational changes in the receptor. The conformational changes observed in our biophysical assay were correlated with biologic responses in cellular assays. Dopamine, which induces only a rapid conformational change, is efficient at activating Gs but not receptor internalization. In contrast, norepinephrine and epinephrine, which induce both rapid and slow conformational changes, are efficient at activating Gs and receptor internalization. These results support a mechanistic model for GPCR activation where contacts between the receptor and structural determinants of the agonist stabilize a succession of conformational states with distinct cellular functions.

    View details for DOI 10.1074/jbc.M310888200

    View details for Web of Science ID 000187555300082

    View details for PubMedID 14559905

  • The PDZ-binding motif of the beta(2)-adrenoceptor is essential for physiologic signaling and trafficking in cardiac myocytes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xiang, Y., Kobilka, B. 2003; 100 (19): 10776-10781

    Abstract

    beta1- and beta2-adrenergic receptors (AR) regulate cardiac myocyte function through distinct signaling pathways. In addition to regulating cardiac rate and contractility, beta1AR and beta2AR may play different roles in the pathogenesis of heart failure. Studies on neonatal cardiac myocytes from beta1AR and beta2AR knockout mice suggest that subtype-specific signaling is determined by subtype-specific membrane targeting and trafficking. Stimulation of beta2ARs has a biphasic effect on contraction rate, with an initial increase followed by a sustained Gi-dependent decrease. Recent studies show that a PDZ domain-binding motif at the carboxyl terminus of human beta2AR interacts with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, a PDZ-domain-containing protein. The human beta2AR carboxyl terminus also binds to N-ethylmaleimide-sensitive factor, which does not contain a PDZ domain. We found that mutation of the three carboxyl-terminal amino acids in the mouse beta2AR (beta2AR-AAA) disrupts recycling of the receptor after agonist-induced internalization in cardiac myocytes. Nevertheless, stimulation of the beta2AR-AAA produced a greater contraction rate increase than that of the wild-type beta2AR. This enhanced stimulation of contraction rate can be attributed in part to the failure of the beta2AR-AAA to couple to Gi. We also observed that coupling of endogenous, wild-type beta2AR to Gi in beta1AR knockout myocytes is inhibited by treatment with a membrane-permeable peptide representing the beta2AR carboxyl terminus. These studies demonstrate that association of the carboxyl terminus of the beta2AR with ezrin-binding protein 50/sodium-hydrogen exchanger regulatory factor, N-ethylmaleimide-sensitive factor, or some related proteins dictates physiologic signaling specificity and trafficking in cardiac myocytes.

    View details for DOI 10.1073/pnas.1831718100

    View details for Web of Science ID 000185415300038

    View details for PubMedID 12954981

  • Myocyte adrenoceptor signaling pathways SCIENCE Xiang, Y., Kobilka, B. K. 2003; 300 (5625): 1530-1532

    Abstract

    Adrenoceptors (ARs), members of the G protein-coupled receptor superfamily, form the interface between the sympathetic nervous system and the cardiovascular system, with integral roles in the rapid regulation of myocardial function. However, in heart failure, chronic catecholamine stimulation of adrenoceptors has been linked to pathologic cardiac remodeling, including myocyte apoptosis and hypertrophy. In cardiac myocytes, activation of AR subtypes results in coupling to different G proteins and induction of specific signaling pathways, which is partly regulated by the subtype-specific distribution of receptors in plasma membrane compartments containing distinct complexes of signaling molecules. The Connections Maps of the Adrenergic and Myocyte Adrenergic Signaling Pathways bring into focus the specific signaling pathways of individual AR subtypes and their relevant functions in vivo.

    View details for Web of Science ID 000183333100040

    View details for PubMedID 12791980

  • Identification of an allosteric binding site for ZN(2+) on the beta(2) adrenergic receptor JOURNAL OF BIOLOGICAL CHEMISTRY Swaminath, G., LEE, T. W., Kobilka, B. 2003; 278 (1): 352-356

    Abstract

    The activity of G protein-coupled receptors (GPCRs) can be modulated by a diverse spectrum of drugs ranging from full agonists to partial agonists, antagonists, and inverse agonists. The vast majority of these ligands compete with native ligands for binding to orthosteric binding sites. Allosteric ligands have also been described for a number of GPCRs. However, little is known about the mechanism by which these ligands modulate the affinity of receptors for orthosteric ligands. We have previously reported that Zn(II) acts as a positive allosteric modulator of the beta(2)-adrenergic receptor (beta(2)AR). To identify the Zn(2+) binding site responsible for the enhancement of agonist affinity in the beta(2)AR, we mutated histidines located in hydrophilic sequences bridging the seven transmembrane domains. Mutation of His-269 abolished the effect of Zn(2+) on agonist affinity. Mutations of other histidines had no effect on agonist affinity. Further mutagenesis of residues adjacent to His-269 demonstrated that Cys-265 and Glu-225 are also required to achieve the full allosteric effect of Zn(2+) on agonist binding. Our results suggest that bridging of the cytoplasmic extensions of TM5 and TM6 by Zn(2+) facilitates agonist binding. These results are in agreement with recent biophysical studies demonstrating that agonist binding leads to movement of TM6 relative to TM5.

    View details for DOI 10.1074/jbc.M206424200

    View details for Web of Science ID 000180255700047

    View details for PubMedID 12409304

  • Efficient adenylyl cyclase activation by a beta(2)-adrenoceptor-G(i)alpha(2) fusion protein BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Seifert, R., Wenzel-Seifert, K., Arthur, J. M., Jose, P. O., Kobilka, B. K. 2002; 298 (5): 824-828

    Abstract

    The G-protein G(i)alpha can activate adenylyl cyclase (AC), but the relevance of this AC activation is unknown. We used receptor-G protein co-expression and receptor-G protein fusion proteins to investigate G(i)alpha(2) regulation of AC in Sf9 cells. G(i)alpha(2) was fused to the beta(2)-adrenoceptor (beta(2)AR), a preferentially G(s)-coupled receptor, or the formyl peptide receptor (FPR), a G(i)-coupled receptor. The FPR co-expressed with, or fused to, G(i)alpha(2), reduced AC activity. In contrast, the beta(2)AR fused to G(i)alpha(2) was a highly efficient AC activator, while the beta(2)AR co-expressed with G(i)alpha(2) was not. Agonist efficiently stimulated incorporation of [alpha-32P]GTP azidoanilide into beta(2)AR-G(i)alpha(2). We explain AC activation by beta(2)AR-G(i)alpha(2) by a model in which there is interaction of the beta(2)AR and AC, preventing tethered G(i)alpha(2) from interacting with the inhibitory G(i)alpha site of AC. The postulated beta(2)AR/AC interaction brings G(i)alpha(2) into close proximity of the G(s)alpha site of AC, enabling G(i)alpha(2) to activate AC.

    View details for Web of Science ID 000179374500030

    View details for PubMedID 12419329

  • Abnormal cardiac function associated with sympathetic nervous system hyperactivity in mice AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Brum, P. C., Kosek, J., Patterson, A., Bernstein, D., Kobilka, B. 2002; 283 (5): H1838-H1845

    Abstract

    alpha(2A)-Adrenergic receptors (ARs) in the midbrain regulate sympathetic nervous system activity, and both alpha(2A)-ARs and alpha(2C)-ARs regulate catecholamine release from sympathetic nerve terminals in cardiac tissue. Disruption of both alpha(2A)- and alpha(2C)-ARs in mice leads to chronically elevated sympathetic tone and decreased cardiac function by 4 mo of age. These knockout mice have increased mortality, reduced exercise capacity, decreased peak oxygen uptake, and decreased cardiac contractility relative to wild-type controls. Moreover, we observed significant abnormalities in the ultrastructure of cardiac myocytes from alpha(2A)/alpha(2C)-AR knockout mice by electron microscopy. Our results demonstrate that chronic elevation of sympathetic tone can lead to abnormal cardiac function in the absence of prior myocardial injury or genetically induced alterations in myocardial structural or functional proteins. These mice provide a physiologically relevant animal model for investigating the role of the sympathetic nervous system in the development and progression of heart failure.

    View details for DOI 10.1152/ajpheart.01063.2001

    View details for Web of Science ID 000178625800012

    View details for PubMedID 12384461

  • Functional immobilization of a ligand-activated G-protein-coupled receptor CHEMBIOCHEM Neumann, L., Wohland, T., Whelan, R. J., Zare, R. N., Kobilka, B. K. 2002; 3 (10): 993-998

    Abstract

    G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. They are the largest family of receptors in the human genome and constitute the largest class of targets for drug discovery. To facilitate studies of GPCR activation and interactions with other proteins, we developed a simple method to immobilize a functional, detergent-solubilized GPCR on gold and glass surfaces. The beta(2) adrenergic receptor (beta(2)AR), a prototypical GPCR, was purified and labeled with a reporter fluorophore at a conformationally sensitive site. The detergent-soluble fluorescent beta(2)AR was immobilized through its amino-terminal FLAG epitope on a surface layered with biotinylated bovine serum albumin, avidin, and biotinylated M1 antibody. Agonist activation of the beta(2)AR was monitored in real time by fluorescence microscopy. This approach will make it possible to study conformational dynamics of single immobilized receptors and to generate arrays of functional GPCRs for novel high-throughput screening strategies.

    View details for Web of Science ID 000178555900009

    View details for PubMedID 12362365

  • Heterozygous alpha(2A)-adrenergic receptor mice unveil unique therapeutic benefits of partial agonists PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tan, C. M., Wilson, M. H., MacMillan, L. B., Kobilka, B. K., Limbird, L. E. 2002; 99 (19): 12471-12476

    Abstract

    Genetic manipulation of the alpha(2A)-adrenergic receptor (alpha(2A)-AR) in mice has revealed the role of this subtype in numerous responses, including agonist-induced hypotension and sedation. Unexpectedly, alpha(2)-agonist treatment of mice heterozygous for the alpha(2A)-AR (alpha(2A)-AR(+/-)) lowers blood pressure without sedation, indicating that more than 50% of alpha(2A)-AR must be activated to evoke sedation. We postulated that partial activation of alpha(2A)-AR in wild-type alpha(2A)-AR(+/+) animals could be achieved with partial agonists, agents with variable ability to couple receptor occupancy to effector activation, and might elicit one versus another pharmacological response. In vitro assays reveal that moxonidine is a partial agonist at alpha(2A)-AR. Although moxonidine was developed to preferentially interact with imidazoline binding sites, it requires the alpha(2A)-AR to lower blood pressure because we observe no hypotensive response to moxonidine in alpha(2A)-AR-null (alpha(2A)-AR(-/-)) mice. Moreover, we observe that moxonidine lowers blood pressure without sedation in wild-type mice, consistent with the above hypothesis regarding partial agonists. Our findings suggest that weak partial agonists can evoke response-selective pathways and might be exploited successfully to achieve alpha(2A)-AR pharmacotherapy where concomitant sedation is undesirable, i.e., in treatment of depression or attention deficit hyperactivity disorder, in suppression of epileptogenesis, or enhancement of cognition. Furthermore, rigorous physiological and behavioral assessment of mice heterozygous for particular receptors provides a general strategy for elucidation of pathways that might be selectively activated by partial agonists, thus achieving response-specific therapy.

    View details for DOI 10.1073/pnas.122368499

    View details for Web of Science ID 000178187000086

    View details for PubMedID 12205290

  • The PDZ binding motif of the beta(1) adrenergic receptor modulates receptor trafficking and signaling in cardiac myocytes JOURNAL OF BIOLOGICAL CHEMISTRY Xiang, Y., Devic, E., Kobilka, B. 2002; 277 (37): 33783-33790

    Abstract

    Beta(1) and beta(2) adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse cardiac myocytes through distinct signaling pathways. It has been shown that stimulation of beta(1)ARs leads to a protein kinase A-dependent increase in contraction rate. In contrast, stimulation of beta(2)ARs has a biphasic effect on contraction rate, with an initial protein kinase A-independent increase followed by a sustained decrease that is blocked by pertussis toxin. The beta(2)AR undergoes agonist-induced endocytosis in cardiac myocytes while the beta(1)AR remains on the cell surface. It has been shown that a PDZ domain binding motif at the carboxyl terminus of beta(1)AR interacts with the postsynaptic density protein PSD-95 when both are expressed in HEK293 cells. We found that mutation of this PDZ binding motif in the beta(1)AR (beta(1)AR-PDZ) enabled agonist-induced internalization in cardiac myocytes. Moreover, stimulation of beta(1)AR-PDZ had a biphasic effect on the myocyte contraction rate similar to that observed following stimulation of the beta(2)AR. The secondary decrease in the contraction rate was mediated by G(i) and could be blocked by pertussis toxin. Furthermore, a non-selective endocytosis inhibitor, concanavalin A, inhibited the internalization of wild type beta(2)AR and the mutated beta(1)AR-PDZ, and blocked the coupling of both receptors to G(i). Finally, treating myocytes with a membrane-permeable peptide representing beta(1)AR PDZ motif caused the endogenous beta(1)AR to behave like beta(1)AR-PDZ. These studies suggest that association of the beta(1)AR with PSD-95 or a related protein dictates signaling specificity by retaining the receptor at the cell surface and preventing interaction with G(i).

    View details for DOI 10.1074/jbc.M204136200

    View details for Web of Science ID 000177959100035

    View details for PubMedID 12097326

  • Caveolar localization dictates physiologic signaling of beta(2)-adrenoceptors in neonatal cardiac myocytes JOURNAL OF BIOLOGICAL CHEMISTRY Xiang, Y., Rybin, V. O., Steinberg, S. F., Kobilka, B. 2002; 277 (37): 34280-34286

    Abstract

    There is a growing body of evidence that G protein-coupled receptors function in the context of plasma membrane signaling compartments. These compartments may facilitate interaction between receptors and specific downstream signaling components while restricting access to other signaling molecules. We recently reported that beta(1)- and beta(2)-adrenergic receptors (AR) regulate the intrinsic contraction rate in neonatal mouse myocytes through distinct signaling pathways. By studying neonatal myocytes isolated from beta(1)AR and beta(2)AR knockout mice, we found that stimulation of the beta(1)AR leads to a protein kinase A-dependent increase in the contraction rate. In contrast, stimulation of the beta(2)AR has a biphasic effect on the contraction rate. The biphasic effect includes an initial protein kinase A-independent increase in the contraction rate followed by a sustained decrease in the contraction rate that can be blocked by pertussis toxin. Here we present evidence that caveolar localization is required for physiologic signaling by the beta(2)AR but not the beta(1)AR in neonatal cardiac myocytes. Evidence for beta(2)AR localization to caveolae includes co-localization by confocal imaging, co-immunoprecipitation of the beta(2)AR and caveolin 3, and co-migration of the beta(2)AR with a caveolin-3-enriched membrane fraction. The beta(2)AR-stimulated increase in the myocyte contraction rate is increased by approximately 2-fold and markedly prolonged by filipin, an agent that disrupts lipid rafts such as caveolae and significantly reduces co-immunoprecipitation of beta(2)AR and caveolin 3 and co-migration of beta(2)AR and caveolin-3 enriched membranes. In contrast, filipin has no effect on beta(1)AR signaling. These observations suggest that beta(2)ARs are normally restricted to caveolae in myocyte membranes and that this localization is essential for physiologic signaling of this receptor subtype.

    View details for DOI 10.1074/jbc.M201644200

    View details for Web of Science ID 000177959100098

    View details for PubMedID 12097322

  • Analysis of bimolecular interactions using a miniaturized surface plasmon resonance sensor ANALYTICAL CHEMISTRY Whelan, R. J., Wohland, T., Neumann, L., Huang, B., Kobilka, B. K., Zare, R. N. 2002; 74 (17): 4570-4576

    Abstract

    A commercially available miniaturized surface plasmon resonance sensor has been investigated for its applicability to biological interaction analysis. The sensor was found to exhibit excellent repeatability and linearity for high-refractive index solutions and good reproducibility for the binding of proteins. Its detection limit for the monoclonal antibody M1 was found to be 2.1 fmol, which corresponds to a surface concentration of 21 pg/mm2. Simple surface immobilization procedures relying on biotin/avidin or glycoprotein/lectin chemistry have been explored. Equilibrium dissociation constants for the binding of the FLAG peptide to its monoclonal antibody (M1) and for the binding of concanavalin A to a glycoprotein have been determined. The close agreement of these measurements with values obtained by surface fluorescence microscopy and fluorescence correlation spectroscopy helps to validate the use of this device. Thus, this sensor shows promise as an inexpensive, portable, and accurate tool for bioanalytical applications in laboratory and clinical settings.

    View details for DOI 10.1021/ac025669y

    View details for Web of Science ID 000177862800039

    View details for PubMedID 12236371

  • beta AR signaling required for diet-induced thermogenesis and obesity resistance SCIENCE Bachman, E. S., Dhillon, H., Zhang, C. Y., Cinti, S., Bianco, A. C., Kobilka, B. K., Lowell, B. B. 2002; 297 (5582): 843-845

    Abstract

    Excessive caloric intake is thought to be sensed by the brain, which then activates thermogenesis as a means of preventing obesity. The sympathetic nervous system, through beta-adrenergic receptor (betaAR) action on target tissues, is likely the efferent arm of this homeostatic mechanism. To test this hypothesis, we created mice that lack the three known betaARs (beta-less mice). beta-less mice on a Chow diet had a reduced metabolic rate and were slightly obese. On a high-fat diet, beta-less mice, in contrast to wild-type mice, developed massive obesity that was due entirely to a failure of diet-induced thermogenesis. These findings establish that betaARs are necessary for diet-induced thermogenesis and that this efferent pathway plays a critical role in the body's defense against diet-induced obesity.

    View details for Web of Science ID 000177192800053

    View details for PubMedID 12161655

  • The ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region - Studies using chimeric human and fly receptors JOURNAL OF BIOLOGICAL CHEMISTRY NISHI, S., Nakabayashi, K., Kobilka, B., Hsueh, A. J. 2002; 277 (6): 3958-3964

    Abstract

    Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.

    View details for DOI 10.1074/jbc.M109617200

    View details for Web of Science ID 000173813900023

    View details for PubMedID 11723133

  • Isoflurane and nociception - Spinal alpha(2A) adrenoceptors mediate antinociception while supraspinal alpha(1) adrenoceptors mediate pronociception ANESTHESIOLOGY Kingery, W. S., Agashe, G. S., Guo, T. Z., Sawamura, S., Davies, M. F., Clark, J. D., Kobilka, B. K., Maze, M. 2002; 96 (2): 367-374

    Abstract

    The authors recently established that the analgesic actions of the inhalation anesthetic nitrous oxide were mediated by noradrenergic bulbospinal neurons and spinal alpha2B adrenoceptors. They now determined whether noradrenergic brainstem nuclei and descending spinal pathways are responsible for the antinociceptive actions of the inhalation anesthetic isoflurane, and which alpha adrenoceptors mediate this effect.After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin coupled to the antibody directed against dopamine beta hydroxylase (DbetaH-saporin), the antinociceptive action of isoflurane was determined. Antagonists for the alpha1 and alpha2 adrenoceptors were injected at spinal and supraspinal sites in intact and spinally transected rats to identify the noradrenergic pathways mediating isoflurane antinociception. Null mice for each of the three alpha2-adrenoceptor subtypes (alpha2A, alpha2B, and alpha2C) and their wild-type cohorts were tested for their antinociceptive response to isoflurane.Both DbetaH-saporin treatment and chronic spinal transection enhanced the antinociceptive effects of isoflurane. The alpha1-adrenoceptor antagonist prazosin also enhanced isoflurane antinociception at a supraspinal site of action. The alpha2-adrenoceptor antagonist yohimbine inhibited isoflurane antinociception, and this effect was mediated by spinal alpha2 adrenoceptors. Null mice for the alpha2A-adrenoceptor subtype showed a reduced antinociceptive response to isoflurane.The authors suggest that, at clinically effective concentrations, isoflurane can modulate nociception via three different mechanisms: (1) a pronociceptive effect requiring descending spinal pathways, brainstem noradrenergic nuclei, and supraspinal alpha1 adrenoceptors; (2) an antinociceptive effect requiring descending noradrenergic neurons and spinal alpha2A adrenoceptors; and (3) an antinociceptive effect mediated within the spinal cord for which no role for adrenergic mechanism has been found.

    View details for Web of Science ID 000173606400019

    View details for PubMedID 11818770

  • Use of fluorescence spectroscopy to study conformational changes in the beta(2)-adrenoceptor G PROTEIN PATHWAYS, PT A, RECEPTORS Kobilka, B. K., Gether, U. 2002; 343: 170-182

    View details for Web of Science ID 000171866900011

    View details for PubMedID 11665566

  • Allosteric modulation of beta(2)-adrenergic receptor by Zn2+ MOLECULAR PHARMACOLOGY Swaminath, G., Steenhuis, J., Kobilka, B., LEE, T. W. 2002; 61 (1): 65-72

    Abstract

    Zn(2+) is abundant in the brain, where it plays a role in the function of a number of enzymes, structural proteins, and transcription factors. Zn(2+) is also found in synaptic vesicles and is released into synapses achieving concentrations in the range of 100 to 300 microM [Proc Natl Acad Sci USA 1997;94:13386-13387; Mol Pharmacol 1997;51:1015-1023]. Therefore, Zn(2+) may play a physiological role in regulating the function of postsynaptic channels and receptors. We characterized the effect of Zn(2+) on the functional properties of the beta2-adrenergic receptor (beta2AR). We found that physiological concentrations of Zn(2+) increased agonist affinity and enhanced cAMP accumulation stimulated by submaximal concentrations of the betaAR agonist isoproterenol. These results provide evidence that Zn(2+) released at nerve terminals may modulate signals generated by the beta2AR in vivo.

    View details for Web of Science ID 000173135000009

    View details for PubMedID 11752207

  • Differential distribution of beta-adrenergic receptor subtypes in blood vessels of knockout mice lacking beta(1)- or beta(2)-adrenergic receptors MOLECULAR PHARMACOLOGY Chruscinski, A., Brede, M. E., Meinel, L., Lohse, M. J., Kobilka, B. K., Hein, L. 2001; 60 (5): 955-962

    Abstract

    beta-Adrenergic receptors (beta-AR) are essential regulators of cardiovascular homeostasis. In addition to their prominent function in the heart, beta-AR are located on vascular smooth muscle cells, where they mediate vasodilating effects of endogenous catecholamines. In this study, we have investigated in an isometric myograph different types of blood vessels from mice lacking beta(1)- and/or beta(2)-adrenergic receptor subtypes (beta(1)-KO, beta(2)-KO, beta(1)beta(2)-KO). In wild-type mice, isoproterenol induced relaxation of segments from thoracic aorta, carotid, femoral and pulmonary arteries, and portal vein. The relaxant effect of beta-receptor stimulation was absent in femoral and pulmonary arteries from beta(1)-KO mice. In aortic and carotid arteries and in portal veins, the vasodilating effect of isoproterenol was reduced in mice lacking beta(1)- or beta(2)-receptors. However, in these vessels the vasodilating effect was only abolished in double KO mice lacking both beta(1)- and beta(2)-receptors. Vessel relaxation induced by forskolin did not differ between wild-type and KO mice. Similar contributions of beta(1)- and beta(2)-receptors to isoproterenol-induced vasorelaxation were found when vessels from KO mice were compared with wild-type arteries in the presence of subtype-selective beta-receptor antagonists. These studies demonstrate that beta(1)-adrenergic receptors play a dominant role in the murine vascular system to mediate vasodilation. Surprisingly, beta(2)-receptors contribute to adrenergic vasodilation only in a few major blood vessels, suggesting that differential distribution of beta-adrenergic receptor subtypes may play an important role in redirection of tissue perfusion.

    View details for Web of Science ID 000171985300010

    View details for PubMedID 11641423

  • beta-adrenergic receptor subtype-specific signaling in cardiac myocytes from beta(1) and beta(2) adrenoceptor knockout mice MOLECULAR PHARMACOLOGY Devic, E., Xiang, Y., Gould, D., Kobilka, B. 2001; 60 (3): 577-583

    Abstract

    The sympathetic nervous system modulates cardiac contractility and rate by activating beta-adrenergic receptors (beta AR) expressed on cardiac myocytes and specialized cells in the sinoatrial node and the conduction system. Recent clinical studies have suggested that beta-adrenergic receptors also play a role in cardiac remodeling that occurs in the pathogenesis of cardiomyopathy. Both beta(1) and beta(2) adrenergic receptors are expressed in human and murine hearts. We have examined the effect of beta AR activation on the spontaneous contraction rate of neonatal myocyte cultures from wild-type and beta receptor knockout (KO) mice (beta(1)AR-KO, beta(2)AR-KO and beta(1)beta(2)AR-KO mice). Stimulation of the beta(1)AR in beta(2)AR-KO myocytes produces the greatest increase in contraction rate through a signaling pathway that requires protein kinase A (PKA) activation. In contrast, stimulation of the beta(2)AR in beta(1)AR-KO myocytes results in a biphasic effect on contraction rate with an initial increase in rate that does not require PKA, followed by a decrease in rate that involves coupling to a pertussis toxin sensitive G protein. A small isoproterenol-induced decrease in contraction rate observed in beta(1)beta(2)AR-KO myocytes can be attributed to the beta(3)AR. These studies show that all three beta AR subtypes are expressed in neonatal cardiac myocytes, and the beta(1)AR and beta(2)AR couple to distinct signaling pathways.

    View details for Web of Science ID 000170593800022

    View details for PubMedID 11502890

  • A genetically engineered cell-based biosensor for functional classification of agents BIOSENSORS & BIOELECTRONICS Aravanis, A. M., DeBusschere, B. D., Chruscinski, A. J., Gilchrist, K. H., Kobilka, B. K., Kovacs, G. T. 2001; 16 (7-8): 571-577

    Abstract

    Cell-based biosensors (CBBs) utilize whole cells to detect biologically active agents. Although CBBs have shown success in detecting the presence of biological agents, efforts to classify the type of agent based on functional activity have proven difficult because multiple biochemical pathways can lead to the same cellular response. However, a new approach using a genetically-engineered cell-based biosensor (GECBB) described in this paper translates this cross-talk noise into common-mode noise that can be rejected. The GECBB operates by assaying for an agent's ability to differentially activate two populations of cells, wild-type (WT) cells and cells genetically engineered to lack a specific receptor, knockout (KO) cells. Any biological agent that targets the knocked out receptor will evoke a response in the WT but not in the KO. Thus, the GECBB is exquisitely sensitive to agents that effect the engineered pathway. This approach provides the benefits of an assay for specific functional activity while simplifying signal analysis. The GECBB implemented was designed to be sensitive to agents that activate the beta 1-adrenergic receptor (beta 1-AR). This was achieved by using mouse cardiomyocytes in which the beta 1-AR had been knocked out. The cellular signal used in the GECBB was the spontaneous beat rate of the two cardiomyocyte syncitia as measured with microelectrode arrays. The GECBB was able to detect the beta-AR agonist isoproterenol (ISO) at a concentration of 10 microM (P<0.005).

    View details for Web of Science ID 000171257900017

    View details for PubMedID 11544051

  • Single-molecule spectroscopy of the beta(2) adrenergic receptor: Observation of conformational substates in a membrane protein PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Peleg, G., Ghanouni, P., Kobilka, B. K., Zare, R. N. 2001; 98 (15): 8469-8474

    Abstract

    Single-molecule studies of the conformations of the intact beta(2) adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the beta(2) adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

    View details for Web of Science ID 000169967000049

    View details for PubMedID 11438704

  • Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta(2) adrenergic receptor JOURNAL OF BIOLOGICAL CHEMISTRY Ghanouni, P., Gryczynski, Z., Steenhuis, J. J., LEE, T. W., Farrens, D. L., Lakowicz, J. R., Kobilka, B. K. 2001; 276 (27): 24433-24436

    Abstract

    G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.

    View details for Web of Science ID 000169800700001

    View details for PubMedID 11320077

  • Functional differences between full and partial agonists: Evidence for ligand-specific receptor conformations JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Seifert, R., Wenzel-Seifert, K., Gether, U., Kobilka, B. K. 2001; 297 (3): 1218-1226

    Abstract

    The interaction of an agonist-bound G-protein-coupled receptor (GPCR) with its cognate G-protein initiates a sequence of experimentally quantifiable changes in both the GPCR and G-protein. These include the release of GDP from G(alpha), the formation of a ternary complex between the nucleotide-free G-protein and the GPCR, which has a high affinity for agonist, followed by the binding of GTP to G(alpha), the dissociation of the GPCR/G-protein complex, and the hydrolysis of GTP. The efficacy of an agonist is a measure of its ability to activate this cascade. It has been proposed that efficacy reflects the ability of the agonist to stabilize the active state of the GPCR. We examined a series of beta(2)-adrenoceptor (beta(2)AR) agonists (weak partial agonists to full agonists) for their efficacy at promoting two different steps of the G-protein activation/deactivation cycle: stabilizing the ternary complex (high-affinity, GTP-sensitive agonist binding), and steady-state GTPase activity. We obtained results for the wild-type beta(2)AR and a constitutively active mutant of the beta(2)AR (beta(2)AR(CAM)) using fusion proteins between the GPCRs and G(salpha) to facilitate GPCR/G-protein interactions. There was no correlation between efficacy of ligands in activating GTPase and their ability to stabilize the ternary complex at beta(2)AR(CAM). Our results suggest that the GPCR state that optimally promotes the GDP release and GTP binding is different from the GPCR state that stabilizes the ternary complex. By strongly stabilizing the ternary complex, certain partial agonists may reduce the rate of G-protein turnover relative to a full agonist.

    View details for Web of Science ID 000169005200048

    View details for PubMedID 11356949

  • Agonist-induced conformational changes in the G-protein-coupling domain of the beta(2) adrenergic receptor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ghanouni, P., Steenhuis, J. J., Farrens, D. L., Kobilka, B. K. 2001; 98 (11): 5997-6002

    Abstract

    The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the beta(2) adrenergic receptor (beta(2)AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-beta(2)AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the beta(2)AR, which may reflect the different energetics of activation by diffusible ligands.

    View details for Web of Science ID 000168883700014

    View details for PubMedID 11353823

  • Antinociceptive action of nitrous oxide is mediated by stimulation of noradrenergic neurons in the brainstem and activation of alpha(2B) adrenoceptors JOURNAL OF NEUROSCIENCE Sawamura, S., Kingery, W. S., Davies, M. F., Agashe, G. S., Clark, J. D., Kobilka, B. K., Hashimoto, T., Maze, M. 2000; 20 (24): 9242-9251

    Abstract

    Although nitrous oxide (N(2)O) has been used to facilitate surgery for >150 years, its molecular mechanism of action is not yet defined. Having established that N(2)O-induced release of norepinephrine mediates the analgesic action at alpha(2) adrenoceptors in the spinal cord, we now investigated whether activation of noradrenergic nuclei in the brainstem is responsible for this analgesic action and which alpha(2) adrenoceptor subtype mediates this property. In rats, Fos immunoreactivity was examined in brainstem noradrenergic nuclei after exposure to nitrous oxide. After selective lesioning of noradrenergic nuclei by intracerebroventricular application of the mitochondrial toxin saporin, coupled to the antibody directed against dopamine beta hydroxylase (DbetaH-saporin), the analgesic and sedative actions of N(2)O were determined. Null mice for each of the three alpha(2) adrenoceptor subtypes (alpha(2A), alpha(2B), and alpha(2C)), and their wild-type cohorts, were tested for their antinociceptive and sedative response to N(2)O. Exposure to N(2)O increased expression of Fos immunoreactivity in each of the pontine noradrenergic nuclei (A5, locus coeruleus, and A7). DbetaH-saporin treatment eliminated nearly all of the catecholamine-containing neurons in the pons and blocked the analgesic but not the sedative effects of N(2)O. Null mice for the alpha(2B) adrenoceptor subtype exhibited a reduced or absent analgesic response to N(2)O, but their sedative response to N(2)O was intact. Our results support a pivotal role for noradrenergic pontine nuclei and alpha(2B) adrenoceptors in the analgesic, but not the sedative effects of N(2)O. Previously we demonstrated that the analgesic actions of alpha(2) adrenoceptor agonists are mediated by the alpha(2A) subtype; taken together with these data we propose that exogenous and endogenous alpha(2) adrenoceptor ligands activate different alpha(2) adrenoceptor subtypes to produce their analgesic action.

    View details for Web of Science ID 000165976500035

    View details for PubMedID 11125002

  • Cell-type specific targeting of the alpha(2c)-andrenoceptor - Evidence for the organization of receptor microdomains during neuronal differentiation of PC12 cells JOURNAL OF BIOLOGICAL CHEMISTRY Hurt, C. M., Feng, F. Y., Kobilka, B. 2000; 275 (45): 35424-35431

    Abstract

    We have previously shown differences in the intracellular targeting of alpha2a (alpha(2A))- and alpha2c (alpha(2C))-adrenoreceptors expressed in the same cell line (von Zastrow, M., Link, R., Daunt, D. , Barsh, G., and Kobilka, B. (1993) J. Biol. Chem. 268, 763-766; Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720). alpha(2A)-Adrenoreceptors reside primarily in the plasma membrane in HEK 293 cells, while co-expressed alpha(2C)-adrenoreceptors are found mainly in an intracellular compartment. Since alpha(2c)-adrenoreceptors are expressed primarily in the brain, we compared the intracellular targeting of alpha(2C)-adrenoreceptors in two neuroendocrine cell lines with the targeting in three epithelial cell lines and one fibroblast cell line. In transiently transfected COS7 cells, and in stably transfected normal rat kidney cells, Madin-Darby canine kidney cells, and Rat1 fibroblasts, a significant proportion of alpha(2C)-adrenoreceptor detected by immunocytochemistry co-localized with markers for both the endoplasmic reticulum and the cis/medial Golgi compartments. In contrast, both PC12 cells and AtT20 cells efficiently targeted alpha(2C)-adrenoreceptors to the plasma membrane. Ligand binding and Western blot analyses indicate that intracellular receptor in normal rat kidney cells is functional and undergoes normal post-translational processing. In PC12 cells the expressed alpha(2C)-adrenoreceptors become concentrated in neurite outgrowths in discrete regions of the plasma membrane having a high density of F-actin following treatment with nerve growth factor. These findings provide evidence for cell-type specific factors that facilitate the targeting of the G protein-coupled receptors to the plasma membrane.

    View details for Web of Science ID 000165422800079

    View details for PubMedID 10906149

  • Activation of the luteinizing hormone receptor following substitution of Ser-277 with selective hydrophobic residues in the ectodomain hinge region JOURNAL OF BIOLOGICAL CHEMISTRY Nakabayashi, K., Kudo, M., Kobilka, B., Hsueh, A. W. 2000; 275 (39): 30264-30271

    Abstract

    Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in G(s) activation.

    View details for Web of Science ID 000089577900049

    View details for PubMedID 10889210

  • Allosteric activation of the CaR by L-amino acids PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kobilka, B. 2000; 97 (9): 4419-4420

    View details for Web of Science ID 000086703000004

    View details for PubMedID 10781033

  • The effect of pH on beta(2) adrenoceptor function - Evidence for protonation-dependent activation JOURNAL OF BIOLOGICAL CHEMISTRY Ghanouni, P., Schambye, H., Seifert, R., LEE, T. W., Rasmussen, S. G., Gether, U., Kobilka, B. K. 2000; 275 (5): 3121-3127

    Abstract

    The transition of rhodopsin from the inactive to the active state is associated with proton uptake at Glu(134) (1), and recent mutagenesis studies suggest that protonation of the homologous amino acid in the alpha(1B) adrenergic receptor (Asp(142)) may be involved in its mechanism of activation (2). To further explore the role of protonation in G protein-coupled receptor activation, we examined the effects of pH on the rate of ligand-induced conformational change and on receptor-mediated G protein activation for the beta(2) adrenergic receptor (beta(2)AR). The rate of agonist-induced change in the fluorescence of NBD-labeled, purified beta(2)AR was 2-fold greater at pH 6.5 than at pH 8, even though agonist affinity was lower at pH 6.5. This biophysical analysis was corroborated by functional studies; basal (agonist-independent) activation of Galpha(s) by the beta(2)AR was greater at pH 6.5 compared with pH 8.0. Taken together, these results provide evidence that protonation increases basal activity by destabilizing the inactive state of the receptor. In addition, we found that the pH sensitivity of beta(2)AR activation is not abrogated by mutation of Asp(130), which is homologous to the highly conserved acidic amino acids that link protonation to activation of rhodopsin (Glu(134)) and the alpha(1B) adrenergic receptor (Asp(142)).

    View details for Web of Science ID 000085146500017

    View details for PubMedID 10652295

  • The Effect of pH on b2 Adrenoceptor Function. Evidence for protonation-dependent activation J Biol Chem Kobilka, B.K., Ghanouni, P., Schambye, H., Seifert, R., Lee, T.W., Rasmussen, S.G., Gether, U. 2000
  • Two functionally distinct alpha(2)-adrenergic receptors regulate sympathetic neurotransmission NATURE Hein, L., Altman, J. D., Kobilka, B. K. 1999; 402 (6758): 181-184

    Abstract

    The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels and kidney. Alpha2-adrenergic receptors are known to have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system; however, the individual roles of the three highly homologous alpha2-adrenergic-receptor subtypes (alpha2A, alpha2B, alpha2C) in this process are not known. We have now studied neurotransmitter release in mice in which the genes encoding the three alpha2-adrenergic-receptor subtypes were disrupted. Here we show that both the alpha2A- and alpha2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. Alpha2A-adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the alpha2C-subtype modulates neurotransmission at lower levels of nerve activity. Both low- and high-frequency regulation seem to be physiologically important, as mice lacking both alpha2A- and alpha2C-receptor subtypes have elevated plasma noradrenaline concentrations and develop cardiac hypertrophy with decreased left ventricular contractility by four months of age.

    View details for Web of Science ID 000083716400050

    View details for PubMedID 10647009

  • ERK plays a regulatory role in induction of LTP by theta frequency stimulation and its modulation by beta-adrenergic receptors NEURON Winder, D. G., Martin, K. C., Muzzio, I. A., Rohrer, D., Chruscinski, A., Kobilka, B., Kandel, E. R. 1999; 24 (3): 715-726

    Abstract

    MAP kinase (ERK) translates cell surface signals into alterations in transcription. We have found that ERK also regulates hippocampal neuronal excitability during 5 Hz stimulation and thereby regulates forms of long-term potentiation (LTP) that do not require macromolecular synthesis. Moreover, ERK-mediated changes in excitability are selectively required for some forms of LTP but not others. ERK is required for the early phase of LTP elicited by brief 5 Hz stimulation, as well as for LTP elicited by more prolonged 5 Hz stimulation when paired with beta1-adrenergic receptor activation. By contrast, ERK plays no role in LTP elicited by a single 1 s 100 Hz train. Consistent with these results, we find that ERK is activated by beta-adrenergic receptors in CA1 pyramidal cell somas and dendrites.

    View details for Web of Science ID 000083913000025

    View details for PubMedID 10595521

  • Restricting the mobility of G(s)alpha: Impact on receptor and effector coupling BIOCHEMISTRY LEE, T. W., Seifert, R., Guan, X. M., Kobilka, B. K. 1999; 38 (42): 13801-13809

    Abstract

    The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.

    View details for Web of Science ID 000083288400005

    View details for PubMedID 10529225

  • GPCR-G alpha fusion proteins: molecular analysis of receptor-G-protein coupling TRENDS IN PHARMACOLOGICAL SCIENCES Seifert, R., Wenzel-Seifert, K., Kobilka, B. K. 1999; 20 (9): 383-389

    Abstract

    The efficiency of interactions between G-protein-coupled receptors (GPCRs) and heterotrimeric guanine nucleotide-binding proteins (G proteins) is greatly influenced by the absolute and relative densities of these proteins in the plasma membrane. The study of these interactions has been facilitated by the use of GPCR-Galpha fusion proteins, which are formed by the fusion of GPCR to Galpha. These fusion proteins ensure a defined 1:1 stoichiometry of GPCR to Galpha and force the physical proximity of the signalling partners. Thus, fusion of GPCR to Galpha enhances coupling efficiency can be used to study aspects of receptor-G-protein coupling that could not otherwise be examined by co-expressing GPCRs and G proteins as separate proteins. The results of studies that have made use of GPCR-Galpha fusion proteins will be discussed in this article, along with the strengths and limitations of this approach.

    View details for Web of Science ID 000082561500009

    View details for PubMedID 10462762

  • Effects of guanine, inosine, and xanthine nucleotides on beta(2)-adrenergic receptor/G(s) interactions: Evidence for multiple receptor conformations MOLECULAR PHARMACOLOGY Seifert, R., Gether, U., Wenzel-Seifert, K., Kobilka, B. K. 1999; 56 (2): 348-358

    Abstract

    The aim of our study was to examine the effects of different purine nucleotides [GTP, ITP, and xanthosine 5'-triphosphate (XTP)] on receptor/G protein coupling. As a model system, we used a fusion protein of the beta(2)-adrenergic receptor and the alpha subunit of the G protein G(s). GTP was more potent and efficient than ITP and XTP at inhibiting ternary complex formation and supporting adenylyl cyclase (AC) activation. We also studied the effects of several beta(2)-adrenergic receptor ligands on nucleotide hydrolysis and on AC activity in the presence of GTP, ITP, and XTP. The efficacy of agonists at promoting GTP hydrolysis correlated well with the efficacy of agonists for stimulating AC in the presence of GTP. This was, however, not the case for ITP hydrolysis and AC activity in the presence of ITP. The efficacy of ligands at stimulating AC in the presence of XTP differed considerably from the efficacies of ligands in the presence of GTP and ITP, and there was no evidence for receptor-regulated XTP hydrolysis. Our findings support the concept of multiple ligand-specific receptor conformations and demonstrate the usefulness of purine nucleotides as tools to study conformational states of receptors.

    View details for Web of Science ID 000081646500013

    View details for PubMedID 10419554

  • Abnormal regulation of the sympathetic nervous system in alpha(2A)-adrenergic receptor knockout mice MOLECULAR PHARMACOLOGY Altman, J. D., Trendelenburg, A. U., MacMillan, L., Bernstein, D., Limbird, L., Starke, K., Kobilka, B. K., Hein, L. 1999; 56 (1): 154-161

    Abstract

    alpha2-Adrenergic receptors (ARs) play a key role in regulating neurotransmitter release in the central and peripheral sympathetic nervous systems. To date, three subtypes of alpha2-ARs have been cloned (alpha2A, alpha2B, and alpha2C). Here we describe the physiological consequences of disrupting the gene for the alpha2A-AR. Mice lacking functional alpha2A subtypes were compared with wild-type (WT) mice, with animals lacking the alpha2B or alpha2C subtypes, and with mice carrying a point mutation in the alpha2A-AR gene (alpha2AD79N). Deletion of the alpha2A subtype led to an increase in sympathetic activity with resting tachycardia (knockout, 581 +/- 21 min-1; WT, 395 +/- 21 min-1), depletion of cardiac tissue norepinephrine concentration (knockout, 676 +/- 31 pg/mg protein; WT, 1178 +/- 98 pg/mg protein), and down-regulation of cardiac beta-ARs (Bmax: knockout, 23 +/- 1 fmol/mg protein; WT, 31 +/- 2 fmol/mg protein). The hypotensive effect of alpha2 agonists was completely absent in alpha2A-deficient mice. Presynaptic alpha2-AR function was tested in two isolated vas deferens preparations. The nonsubtype-selective alpha2 agonist dexmedetomidine completely blocked the contractile response to electrical stimulation in vas deferens from alpha2B-AR knockout, alpha2C-AR knockout, alpha2AD79N mutant, and WT mice. The maximal inhibition of vas deferens contraction by the alpha2 agonist in alpha2A-AR knockout mice was only 42 +/- 9%. [3H]Norepinephrine release studies performed in vas deferens confirmed these findings. The results indicate that the alpha2A-AR is a major presynaptic receptor subtype regulating norepinephrine release from sympathetic nerves; however, the residual alpha2-mediated effect in the alpha2A-AR knockout mice suggests that a second alpha2 subtype (alpha2B or alpha2C) also functions as a presynaptic autoreceptor to inhibit transmitter release.

    View details for Web of Science ID 000081240600019

    View details for PubMedID 10385696

  • Cardiovascular and metabolic alterations in mice lacking both beta 1-and beta 2-adrenergic receptors JOURNAL OF BIOLOGICAL CHEMISTRY Rohrer, D. K., Chruscinski, A., Schauble, E. H., Bernstein, D., Kobilka, B. K. 1999; 274 (24): 16701-16708

    Abstract

    The activation state of beta-adrenergic receptors (beta-ARs) in vivo is an important determinant of hemodynamic status, cardiac performance, and metabolic rate. In order to achieve homeostasis in vivo, the cellular signals generated by beta-AR activation are integrated with signals from a number of other distinct receptors and signaling pathways. We have utilized genetic knockout models to test directly the role of beta1- and/or beta2-AR expression on these homeostatic control mechanisms. Despite total absence of beta1- and beta2-ARs, the predominant cardiovascular beta-adrenergic subtypes, basal heart rate, blood pressure, and metabolic rate do not differ from wild type controls. However, stimulation of beta-AR function by beta-AR agonists or exercise reveals significant impairments in chronotropic range, vascular reactivity, and metabolic rate. Surprisingly, the blunted chronotropic and metabolic response to exercise seen in beta1/beta2-AR double knockouts fails to impact maximal exercise capacity. Integrating the results from single beta1- and beta2-AR knockouts as well as the beta1-/beta2-AR double knock-out suggest that in the mouse, beta-AR stimulation of cardiac inotropy and chronotropy is mediated almost exclusively by the beta1-AR, whereas vascular relaxation and metabolic rate are controlled by all three beta-ARs (beta1-, beta2-, and beta3-AR). Compensatory alterations in cardiac muscarinic receptor density and vascular beta3-AR responsiveness are also observed in beta1-/beta2-AR double knockouts. In addition to its ability to define beta-AR subtype-specific functions, this genetic approach is also useful in identifying adaptive alterations that serve to maintain critical physiological setpoints such as heart rate, blood pressure, and metabolic rate when cellular signaling mechanisms are perturbed.

    View details for Web of Science ID 000080780400008

    View details for PubMedID 10358009

  • Targeted disruption of the beta 2 adrenergic receptor gene JOURNAL OF BIOLOGICAL CHEMISTRY Chruscinski, A. J., Rohrer, D. K., Schauble, E., Desai, K. H., Bernstein, D., Kobilka, B. K. 1999; 274 (24): 16694-16700

    Abstract

    beta-Adrenergic receptors (beta-ARs) are members of the superfamily of G-protein-coupled receptors that mediate the effects of catecholamines in the sympathetic nervous system. Three distinct beta-AR subtypes have been identified (beta1-AR, beta2-AR, and beta3-AR). In order to define further the role of the different beta-AR subtypes, we have used gene targeting to inactivate selectively the beta2-AR gene in mice. Based on intercrosses of heterozygous knockout (beta2-AR +/-) mice, there is no prenatal lethality associated with this mutation. Adult knockout mice (beta2-AR -/-) appear grossly normal and are fertile. Their resting heart rate and blood pressure are normal, and they have a normal chronotropic response to the beta-AR agonist isoproterenol. The hypotensive response to isoproterenol, however, is significantly blunted compared with wild type mice. Despite this defect in vasodilation, beta2-AR -/- mice can still exercise normally and actually have a greater total exercise capacity than wild type mice. At comparable workloads, beta2-AR -/- mice had a lower respiratory exchange ratio than wild type mice suggesting a difference in energy metabolism. beta2-AR -/- mice become hypertensive during exercise and exhibit a greater hypertensive response to epinephrine compared with wild type mice. In summary, the primary physiologic consequences of the beta2-AR gene disruption are observed only during the stress of exercise and are the result of alterations in both vascular tone and energy metabolism.

    View details for Web of Science ID 000080780400007

    View details for PubMedID 10358008

  • Examining the efficiency of receptor/G-protein coupling with a cleavable beta(2)-adrenoceptor-G(s alpha) fusion protein EUROPEAN JOURNAL OF BIOCHEMISTRY Seifert, R., Wenzel-Seifert, K., Gether, U., Lam, V. T., Kobilka, B. K. 1999; 260 (3): 661-666

    Abstract

    Reconstitution of high-affinity agonist binding at the beta2-adrenoceptor (beta2AR) expressed in Sf9 insect cells requires a large excess of the stimulatory G-protein of adenylyl cyclase, Gsalpha, relative to receptor [R. Seifert, T. W. Lee, V. T. Lam & B. K. Kobilka, (1998) Eur. J. Biochem. 255, 369-382]. In a fusion protein of the beta2AR and Gsalpha (beta2AR-Gsalpha), which has only a 1 : 1 stoichiometry of receptor and G-protein, high-affinity agonist binding and agonist-stimulated GTP hydrolysis, guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding and adenylyl cyclase (AC) activation are more efficient than in the nonfused coexpression system. In order to analyze the stability of the receptor/G-protein interaction, we constructed a fusion protein with a thrombin-cleavage site between beta2AR and Gsalpha (beta2AR-TS-Gsalpha). beta2AR-TS-Gsalpha efficiently reconstituted high-affinity agonist binding, agonist-stimulated GTP hydrolysis, GTP[S] binding and AC activation. Thrombin cleaves approximately 70% of beta2AR-TS-Gsalpha molecules in Sf9 membranes. Thrombin cleavage did not impair high-affinity agonist binding and GTP[S] binding but strongly reduced ligand-regulated GTPase activity and AC activity. We conclude that fusion of the beta2AR to Gsalpha promotes tight physical association of the two partners and that this association remains stable for a single activation/deactivation cycle even after cleavage of the link between the receptor and G-protein. Dilution of Gsalpha in the membrane and release of activated Gsalpha into the cytosol can both prevent cleaved beta2AR-TS-Gsalpha from undergoing multiple activation/deactivation cycles.

    View details for Web of Science ID 000079371200009

    View details for PubMedID 10102993

  • Cardiovascular and metabolic alterations in mice lacking both b1- and b2-adrenergic receptors J Biol Chem Kobilka, B.K., Rohrer, D.K., Chruscinski, A., Schauble, E.H., Bernstein, D. 1999; 274 (24): 16701
  • Restricting mobility of G(s)alpha relative to the beta(2)-adrenoceptor enhances adenylate cyclase activity by reducing G(s)alpha GTPase activity BIOCHEMICAL JOURNAL Wenzel-Seifert, K., LEE, T. W., Seifert, R., Kobilka, B. K. 1998; 334: 519-524

    Abstract

    The beta2-adrenoceptor (beta2AR) activates the G-protein Gsalpha to stimulate adenylate cyclase (AC). Fusion of the beta2AR C-terminus to the N-terminus of Gsalpha (producing beta2ARGsalpha) markedly increases the efficiency of receptor/G-protein coupling compared with the non-fused state. This increase in coupling efficiency can be attributed to the physical proximity of receptor and G-protein. To determine the optimal length for the tether between receptor and G-protein we constructed fusion proteins from which 26 [beta2AR(Delta26)Gsalpha] or 70 [beta2AR(Delta70)Gsalpha] residues of the beta2AR C-terminus had been deleted and compared the properties of these fusion proteins with the previously described beta2ARGsalpha. Compared with beta2ARGsalpha, basal and agonist-stimulated GTP hydrolysis was markedly decreased in beta2AR(Delta70)Gsalpha, whereas the effect of the deletion on binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was relatively small. Surprisingly, deletions did not alter the efficiency of coupling of the beta2AR to Gsalpha as assessed by GTP[S]-sensitive high-affinity agonist binding. Moreover, basal and ligand-regulated AC activities in membranes expressing beta2AR(Delta70)Gsalpha and beta2AR(Delta26)Gsalpha were higher than in membranes expressing beta2ARGsalpha. These findings suggest that restricting the mobility of Gsalpha relative to the beta2AR results in a decrease in G-protein inactivation by GTP hydrolysis and thereby enhanced activation of AC.

    View details for Web of Science ID 000076087600004

    View details for PubMedID 9729456

  • G protein-coupled receptors - II. Mechanism of agonist activation JOURNAL OF BIOLOGICAL CHEMISTRY Gether, U., Kobilka, B. K. 1998; 273 (29): 17979-17982

    View details for Web of Science ID 000074828500001

    View details for PubMedID 9660746

  • Reconstitution of beta(2)-adrenoceptor-GTP-binding-protein interaction in Sf9 cells - High coupling efficiency in a beta(2)-adrenoceptor-G(s alpha) fusion protein EUROPEAN JOURNAL OF BIOCHEMISTRY Seifert, R., LEE, T. W., Lam, V. T., Kobilka, B. K. 1998; 255 (2): 369-382

    Abstract

    In most studies, coupling of the beta2-adrenoceptor (beta2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which beta2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the beta2AR alone, in combination with the alpha-subunit of Gs (G(s alpha)), and as fusion protein with G(s alpha) (beta2AR-G(s alpha)) in Sf9 insect cells. The beta2AR expressed alone couples poorly to the endogenous G(s alpha)-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding were small. Beta2AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta2AR co-expressed with a large excess of G(s alpha). In membranes expressing beta2AR-G(s alpha), highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing beta2AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta2AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta2AR-G(s alpha) fusion protein provides a very sensitive model system to study the regulation of Gs function by beta2AR agonists and inverse agonists directly at the level of the G-protein.

    View details for Web of Science ID 000074954500006

    View details for PubMedID 9716378

  • Neuropeptide Y receptor 1 (NPY-Y1) expression in human heart failure and heart transplantation JOURNAL OF THE AUTONOMIC NERVOUS SYSTEM Gullestad, L., Aass, H., Ross, H., Ueland, T., Geiran, O., KJEKSHUS, J., Simonsen, S., Fowler, M., Kobilka, B. 1998; 70 (1-2): 84-91

    Abstract

    Neuropeptide Y (NPY) is a neurotransmitter released from cardiac sympathetic nerve terminals along with catecholamines. It influences vascular tone and cardiac function, probably through the receptor subtype Y1. The present study examined the expression of Y1 in patients with end-stage heart failure and in heart transplant recipients. Y1 mRNA was analyzed in right ventricular endomyocardial biopsies taken from 12 donor hearts prior to implantation (controls), 15 patients with end stage heart failure at time of transplantation, and 16 patients more than 1 year after transplantation. RT-PCR (reverse transcription polymerase chain reaction) was used to detect mRNA for the Y1 receptor, the beta1-adrenergic-receptor, and beta-actin. Y1 mRNA was present in biopsies of all donor hearts, but was observed significantly less frequently in the two patient groups; only 5 out of 15 (P < 0.01) heart failure and 9 out of 16 (P < 0.05) transplant recipients demonstrated visible PCR product. In contrast, mRNA for the beta1-adrenergic receptor and beta-actin were detected by RT-PCR in all samples. Our results provide the first evidence for altered regulation of the neuropeptide Y1 receptor in heart failure and transplant patients, and suggests that loss of signal transduction by this receptor may be adaptive in both groups.

    View details for Web of Science ID 000074669200012

    View details for PubMedID 9686908

  • Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor. journal of biological chemistry Seifert, R., Wenzel-Seifert, K., LEE, T. W., Gether, U., Sanders-Bush, E., Kobilka, B. K. 1998; 273 (18): 5109-5116

    Abstract

    The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gs alpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gs alpha (Gs alphaL) binds GDP with lower affinity than the short splice variant (Gs alphaS), but the impact of this difference on the interaction of Gs alpha with the beta 2AR is not known. We studied the beta2 AR/Gs alpha interaction using receptor/G-protein fusion proteins (beta2 AR Gs alphaS and beta2 AR Gs alphaL) expressed in Sf9 cells. Fusion of the beta2 AR to Gs alpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2 AR or Gs alpha. The beta2 AR in beta2 AR Gs alphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2 AR in beta2 AR Gs alphaS. The apparent constitutive activity of the beta2 AR in beta2 AR Gs alphaL may be due to the lower GDP affinity of Gs alphaL compared with Gs alphaS, i.e. Gs alphaL is more often nucleotide-free than Gs alphaS and, therefore, more frequently available to stabilize the beta2 AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

    View details for PubMedID 9556548

  • Alterations in dynamic heart rate control in the beta(1)-adrenergic receptor knockout mouse AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Rohrer, D. K., Schauble, E. H., Desai, K. H., Kobilka, B. K., Bernstein, D. 1998; 274 (4): H1184-H1193

    Abstract

    beta 1-Adrenergic receptors (beta 1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a beta 1-AR gene knockout model to test the hypothesis that beta 1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in beta 1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in beta 1-AR knockouts is markedly limited, with peak heart rates approximately 200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in beta 1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the beta 1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.

    View details for Web of Science ID 000072839800015

    View details for PubMedID 9575921

  • Different effects of G(s)alpha splice variants on beta(2)-adrenoreceptor-mediated signaling - The beta(2)-adrenoreceptor coupled to the long splice variant of G(s)alpha has properties of a constitutively active receptor JOURNAL OF BIOLOGICAL CHEMISTRY Seifert, R., Wenzel-Seifert, K., LEE, T. W., Gether, U., Sanders-Bush, E., Kobilka, B. K. 1998; 273 (9): 5109-5116

    Abstract

    The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsalpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gsalpha (GsalphaL) binds GDP with lower affinity than the short splice variant (GsalphaS), but the impact of this difference on the interaction of Gsalpha with the beta2AR is not known. We studied the beta2AR/Gsalpha interaction using receptor/G-protein fusion proteins (beta2ARGsalphaS and beta2ARGsalphaL) expressed in Sf9 cells. Fusion of the beta2AR to Gsalpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2AR or Gsalpha. The beta2AR in beta2ARGsalphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2AR in beta2ARGsalphaS. The apparent constitutive activity of the beta2AR in beta2ARGsalphaL may be due to the lower GDP affinity of GsalphaL compared with GsalphaS, i.e. GsalphaL is more often nucleotide-free than GsalphaS and, therefore, more frequently available to stabilize the beta2AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

    View details for Web of Science ID 000072310400047

    View details for PubMedID 9478963

  • The developmental and physiological consequences of disrupting genes encoding beta 1 and beta 2 adrenoceptors. Advances in pharmacology (San Diego, Calif.) Rohrer, D. K., Bernstein, D., Chruscinski, A., Desai, K. H., Schauble, E., Kobilka, B. K. 1998; 42: 499-501

    View details for PubMedID 9327949

  • G protein-coupled receptors: Functional and mechanistic insights through altered gene expression PHYSIOLOGICAL REVIEWS Rohrer, D. K., Kobilka, B. K. 1998; 78 (1): 35-52

    Abstract

    G protein-coupled receptors (GPCRs) comprise a large and diverse family of molecules that play essential roles in signal transduction. In addition to a constantly expanding pharmacological repertoire, recent advances in the ability to manipulate GPCR expression in vivo have provided another valuable approach in the study of GPCR function and mechanism of action. Current technologies now allow investigators to manipulate GPCR expression in a variety of ways. Graded reductions in GPCR expression can be achieved through antisense strategies or total gene ablation or replacement can be achieved through gene targeting strategies, and exogenous expression of wild-type or mutant GPCR isoforms can be accomplished with transgenic technologies. Both the techniques used to achieve these specific alterations and the consequences of altered expression patterns are reviewed here and discussed in the context of GPCR function and mechanism of action.

    View details for Web of Science ID 000071629200002

    View details for PubMedID 9457168

  • Insights from in vivo modification of adrenergic receptor gene expression ANNUAL REVIEW OF PHARMACOLOGY AND TOXICOLOGY Rohrer, D. K., Kobilka, B. K. 1998; 38: 351-373

    Abstract

    Adrenergic receptors are key targets within the autonomic nervous system, regulating a wide variety of physiological processes. The ability to modify adrenergic receptor expression patterns in vivo has added a powerful new tool to the functional analysis of these receptors. Modification of adrenergic receptor gene expression by overexpression, genetic ablation, or site-specific mutation has added new insight to models of receptor coupling behavior, pharmacology, and subtype-specific physiological function. This review highlights some of the recent advances resulting from such genetic approaches to the study of adrenergic receptors.

    View details for Web of Science ID 000073483100014

    View details for PubMedID 9597159

  • Site-specific fluorescence labeling of the beta(2) adrenergic receptor amino terminus ANALYTICAL BIOCHEMISTRY PAROLA, A. L., Lin, S. S., Kobilka, B. K. 1997; 254 (1): 88-95

    Abstract

    A modified human beta2 receptor, designated 0K-beta2, was developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes. 0K-beta2 has the following modifications: (1) all 16 lysines in the wild-type beta2 receptor were mutated to arginines, (2) a FLAG epitope preceded by a cleaved hemagglutinin signal sequence was fused to the amino terminus, and (3) a hexahistidine tail was added to the carboxyl terminus. The FLAG epitope and hexahistidine tail were added to facilitate purification while lysine to arginine mutations eliminate potential labeling sites for amine-reactive fluorescent probes. The remaining primary amines in the 0K-beta2 receptor, the amino terminal amine and the epsilon-amine of Lys3, both reside in the amino-terminal FLAG epitope. The 0K-beta2 receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type beta2 receptor. The modified receptor was labeled with fluorescamine, an amine-reactive fluorescent probe. Proteolysis with factor Xa showed that labeling was confined to the amino terminus of the 0K-beta2 receptor. Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the 0K-beta2 receptor, a lysine-depleted beta2 receptor that retains functional characteristics of the wild-type receptor.

    View details for Web of Science ID A1997YK71500013

    View details for PubMedID 9398350

  • Agonists induce conformational changes in transmembrane domains III and VI of the beta(2) adrenoceptor EMBO JOURNAL Gether, U., Lin, S., Ghanouni, P., Ballesteros, J. A., Weinstein, H., Kobilka, B. K. 1997; 16 (22): 6737-6747

    Abstract

    Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.

    View details for Web of Science ID A1997YJ20700013

    View details for PubMedID 9362488

  • Co-expression of defective luteinizing hormone receptor fragments partially reconstitutes ligand-induced signal generation JOURNAL OF BIOLOGICAL CHEMISTRY Osuga, Y., Hayashi, M., Kudo, M., Conti, M., Kobilka, B., Hsueh, A. J. 1997; 272 (40): 25006-25012

    Abstract

    Gonadotropin receptors are unique members of the seven-transmembrane (TM), G protein-coupled receptor family with a large extracellular (EC) sequence forming the high-affinity ligand binding domain. In a patient with Leydig cell hypoplasia, we identified a mutant LH receptor that is truncated at TM5. This protein retains limited ligand binding ability but cannot mediate cAMP responses. To study interactions between receptor fragments defective in either ligand binding or signal transduction, we co-expressed this truncated receptor together with a chimeric receptor containing the EC region of the FSH receptor and the TM region of the LH receptor. Although the chimeric receptor could not respond to human chorionic gonadotropin in producing cAMP, co-expression with the truncated LH receptor allowed partial restoration of ligand signaling through intermolecular interactions. In addition, co-expression of the same truncated LH receptor with an N-terminally truncated LH receptor that lacked the EC ligand binding domain also partially restored ligand signaling. Further shortening of the TM region in the mutant receptor found in the patient indicated that the EC domain and TM1 were sufficient for interactions with the N terminally truncated receptor. In contrast, co-expression of the N terminally truncated receptor together with cell-associated or soluble EC region of the LH receptor did not allow ligand signaling. Unlike thrombin receptors, co-expression of the anchored EC region of the LH receptor together with the N-terminally truncated receptor did not allow ligand signaling despite moderate levels of human chorionic gonadotropin binding in transfected cells. These studies demonstrate that the co-expression of binding (+)/signaling (-) and binding (-)/signaling (+) receptor fragments partially restores ligand-induced signal generation and indicate the importance of TM1 of the LH receptor in the proper orientation of the EC ligand binding domain.

    View details for Web of Science ID A1997XY97000046

    View details for PubMedID 9312107

  • Derivation of functional antagonists using N-terminal extracellular domain of gonadotropin and thyrotropin receptors MOLECULAR ENDOCRINOLOGY Osuga, Y., Kudo, M., Kaipia, A., Kobilka, B., Hsueh, A. J. 1997; 11 (11): 1659-1668

    Abstract

    Receptors for the glycoprotein hormones, LH/CG, FSH, and TSH, are a unique subclass of the seven-transmembrane, G protein-coupled proteins with a large N-terminal extracellular (ecto-) domain. Although ecto-domains of gonadotropin receptors confer ligand binding, expression of soluble binding proteins has been difficult. We fused the ecto-domains of LH or FSH receptors to the single-transmembrane domain of CD8 and found that hybrid proteins anchored on the cell surface retained high-affinity ligand binding. Inclusion of a junctional thrombin cleavage site in the hybrids allowed generation of soluble receptor fragments that interfered with gonadotropin binding to their receptors and blocked cAMP production stimulated by gonadotropins. Cross-linking analyses confirmed the formation of high molecular weight complexes between receptor ecto-domains and their specific ligands. A similar approach also generated a soluble TSH receptor fragment capable of blocking TSH-induced signal transduction. When administered to rats, the soluble FSH receptor fragment retarded testis growth and induced testis cell apoptosis. These findings demonstrate the feasibility of generating ligand-binding regions of glycoprotein hormone receptors to selectively neutralize actions of gonadotropins and TSH, thus allowing future design of novel contraceptives and management of different gonadal and thyroid dysfunction. The present study represents the first successful derivation of soluble, ligand-binding domains from glycoprotein hormone receptors as functional antagonists. Similar approaches could allow generation of ecto-domains of related receptors to neutralize actions of ligands or receptor antibodies and to facilitate structural-functional analysis.

    View details for Web of Science ID A1997XY29500009

    View details for PubMedID 9328348

  • Intracellular trafficking of angiotensin II and its AT(1) and AT(2) receptors: Evidence for selective sorting of receptor and ligand MOLECULAR ENDOCRINOLOGY Hein, L., Meinel, L., Pratt, R. E., Dzau, V. J., Kobilka, B. K. 1997; 11 (9): 1266-1277

    Abstract

    Angiotensin II (Ang II) binds to two different receptor subtypes, AT1 and AT2 receptors. In many cases, receptor stimulation by Ang II is followed by a rapid desensitization of the intracellular signal transduction and a decrease in cell surface receptor number. The present study was designed to examine by immunofluorescence microscopy the cellular trafficking pathways of Ang II and its AT1a and AT2 receptors in human embryonal kidney 293 cells stably expressing these receptor subtypes. Fluorescently labeled Ang II and AT1a receptors were rapidly internalized into endosomes. AT2 receptors were localized in the plasma membrane and did not undergo endocytosis upon agonist stimulation. After removal of agonist, AT1a receptors recycled to the plasma membrane, whereas fluorescently labeled Ang II was targeted to the lysosomal pathway. Even though no further loss of surface receptor was measurable by ligand binding at steady state, fluorescein-Ang II was continuously internalized, and cycling of receptor between endosomal vesicles and the plasma membrane was detected by antibody feeding. These experiments provide evidence for subtype-specific receptor sorting and internalization of Ang II and its AT1a receptor as a receptor-ligand complex, and they suggest that the sequestration of receptors into endosomes is in dynamic equilibrium with receptor cycling to the plasma membrane. Finally, internalization of AT1a receptors and Ang II persists after desensitization mechanisms have attenuated Ca2+ and inositol 1,4,5-trisphosphate signaling.

    View details for Web of Science ID A1997XN62300008

    View details for PubMedID 9259318

  • A novel interaction between adrenergic receptors and the alpha-subunit of eukaryotic initiation factor 2B JOURNAL OF BIOLOGICAL CHEMISTRY Klein, U., Ramirez, M. T., Kobilka, B. K., VONZASTROW, M. 1997; 272 (31): 19099-19102

    Abstract

    The alpha-subunit of eukaryotic initiation factor 2B (eIF-2B), a guanine nucleotide exchange protein that functions in regulation of translation, was observed to associate with the carboxyl-terminal cytoplasmic domains of the alpha2A- and alpha2B-adrenergic receptors in a yeast two-hybrid screen of a cDNA library prepared from 293 cells. This protein association was confirmed in vitro by affinity chromatography and was shown to be specific for a subset of G protein-coupled receptors, including the alpha2A-, alpha2B-, alpha2C-, and beta2-adrenergic receptors, but not the vasopressin (V2) receptor. Association of these proteins in vivo was confirmed by specific co-immunoprecipitation of eIF-2Balpha with full-length beta2-adrenergic receptors expressed in transfected 293 cells and by fluorescence microscopy showing co-localization of these proteins in intact cells. Remarkably, eIF-2Balpha co-localized with receptors exclusively in regions of the plasma membrane that are in contact with the extracellular medium, but failed to associate with membranes making cell-cell contacts. Overexpression of eIF-2Balpha in 293 cells caused a small (approximately 15%) but significant enhancement of beta2-adrenergic receptor-mediated activation of adenylyl cyclase, without affecting forskolin or V2 receptor-mediated activation. These observations suggest a new role for a previously identified guanine nucleotide exchange protein in membrane biology and cell signaling.

    View details for Web of Science ID A1997XP06300002

    View details for PubMedID 9235896

  • Adrenergic receptors - From molecular structure to in vivo function TRENDS IN CARDIOVASCULAR MEDICINE Hein, L., Kobilka, B. K. 1997; 7 (5): 137-145

    Abstract

    Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system as well as many endocrine and parenchymal tissues. Although several hundred G-protein-coupled receptors have been identified, adrenergic receptors, along with the visual pigment rhodopsin, have been among the most extensively studied members of this family of receptors. This review focuses on recent advances in understanding the molecular structure, function, and regulation of adrenergic receptors using in vitro systems and integrates recent transgenic animal models that were generated to study the adrenergic system in vivo. (Trends Cardiovasc Med 1997;7:137-145). © 1997, Elsevier Science Inc.

    View details for Web of Science ID A1997XM99100001

    View details for PubMedID 21235877

  • Overexpression of angiotensin AT(1) receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hein, L., Stevens, M. E., Barsh, G. S., Pratt, R. E., Kobilka, B. K., Dzau, V. J. 1997; 94 (12): 6391-6396

    Abstract

    Previous studies have suggested that angiotensin II (Ang II) modulates cardiac contractility, rhythm, metabolism, and structure. However, it is unclear whether the cardiac effects are due to direct actions of Ang II on the myocardium or if they are due to secondary effects mediated through the hemodynamic actions of Ang II. In this study, we used the alpha-myosin heavy chain (alphaMHC) promoter to generate transgenic mice overexpressing angiotensin II type 1 (AT1a) receptor selectively in cardiac myocytes. The specificity of transgene expression in the transgenic offspring was confirmed by radioligand binding studies and reverse transcription-PCR. The offspring displayed massive atrial enlargement with myocyte hyperplasia at birth, developed significant bradycardia with heart block, and died within the first weeks after birth. Thus, direct activation of AT1 receptor signaling in cardiac myocytes in vivo is sufficient to induce cardiac myocyte growth and alter electrical conduction.

    View details for Web of Science ID A1997XD84400077

    View details for PubMedID 9177228

  • Subtype-specific intracellular trafficking of alpha(2)-adrenergic receptors MOLECULAR PHARMACOLOGY Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., Kobilka, B. K. 1997; 51 (5): 711-720

    Abstract

    The three alpha2-adrenergic receptor subtypes (alpha2a, alpha2b, and alpha2c) are highly homologous G protein-coupled receptors. These receptors all couple to pertussis toxin-sensitive G proteins and have relatively similar pharmacological properties. To further explore functional differences between these receptors, we used immunocytochemical techniques to compare the ability of the three alpha2-receptor subtypes to undergo agonist-mediated internalization. The alpha2a-receptor does not internalize after agonist treatment. In contrast, we observed that the alpha2b-receptor is able to undergo agonist-induced internalization and seems to follow the same endosomal pathway used by the beta2-adrenergic receptor. Attempts to examine internalization of the alpha2c-receptor were complicated by the fact that the majority of the alpha2c receptor resides in the endoplasmic reticulum and cis/media Golgi and there is relatively little cell surface localization. Nevertheless, we were able to detect some internalization of the alpha2c-receptor after prolonged agonist treatment. However, we observed no significant movement of alpha2c-receptor from the intracellular pool to the plasma membrane during a 4-hr treatment of cells with cycloheximide, suggesting that these cells are unable to process alpha2c-receptors in the same way they process the alpha2a or alpha2b subtypes.

    View details for Web of Science ID A1997WY28000003

    View details for PubMedID 9145909

  • Cardiovascular indexes in the mouse at rest and with exercise: New tools to study models of cardiac disease AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Desai, K. H., Sato, R., Schauble, E., Barsh, G. S., Kobilka, B. K., Bernstein, D. 1997; 272 (2): H1053-H1061

    Abstract

    Manipulations of the murine genome that alter cardiovascular function have created the need for methods to study cardiovascular physiology in genetically altered animals in vivo. We adapted chronic physiological measurement techniques to the nonanesthetized, nonrestrained murine model, established strain-specific cardiovascular and metabolic norms, and evaluated responses to anesthesia, exercise, and adrenergic stimulation. Anesthesia resulted in alterations in heart rate (HR), blood pressure (BP), and O2 consumption (V(O2)) and CO2 production (V(CO2)) for up to 6 h postoperatively. There were significant interstrain differences in resting values of HR and BP Graded treadmill exercise resulted in linear increases in HR, V(O2), V(CO2), and respiratory exchange ratio (RER) similar to those seen in larger species. Response to beta-adrenergic stimulation showed a classic sigmoidal dose-response curve; however, there was very little tachycardiac response to vagal blockade, indicating low resting vagal tone. This study demonstrates the feasibility of performing chronic cardiovascular measurements in nonanesthetized mice and stresses the importance of allowing for anesthetic recovery and strain variability. Murine cardiovascular responses to exercise can be reliably measured and are qualitatively similar to those in humans.

    View details for Web of Science ID A1997WJ80900057

    View details for PubMedID 9124413

  • Structural instability of a constitutively active G protein-coupled receptor - Agonist-independent activation due to conformational flexibility JOURNAL OF BIOLOGICAL CHEMISTRY Gether, U., Ballesteros, J. A., Seifert, R., SANDERSBUSH, E., Weinstein, H., Kobilka, B. K. 1997; 272 (5): 2587-2590

    Abstract

    Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.

    View details for Web of Science ID A1997WE66700002

    View details for PubMedID 9006889

  • Ligand stabilization of the beta(2) adrenergic receptor: Effect of DTT on receptor conformation monitored by circular dichroism and fluorescence spectroscopy BIOCHEMISTRY Lin, S. S., Gether, U., Kobilka, B. K. 1996; 35 (46): 14445-14451

    Abstract

    Treatment of the beta 2 adrenergic receptor with the reducing agent dithiothreitol (DTT) is known to abolish ligand binding to the receptor. Interestingly, the loss of binding can be prevented by preoccupation of the receptor with ligand. It is unclear, however, whether the ligand blocks access of DTT to the receptor, or the ligand stabilizes the receptor structure. In the present study, we have utilized circular dichroism (CD) and intrinsic tryptophan fluorescence to directly probe structural changes in the beta 2 adrenergic receptor in response to DTT treatment. Analysis of CD spectra of purified beta 2 receptor in the detergent micelle indicated that the receptor has an alpha-helix content of 60%, which is substantially more than what would be attributed to the seven transmembrane domains. The alpha-helix content was unchanged in the presence of DTT, suggesting that DTT treatment does not alter the secondary structure of the receptor. In contrast, the tryptophan fluorescence spectra demonstrated that DTT induces a reversible conformational change of the beta 2 receptor. Thus, DTT caused a red-shift in the maximum emission wavelength of the intrinsic tryptophan fluorescence. The change in emission spectrum correlated with a loss in the ability of the receptor to bind antagonist. Both changes in receptor binding and fluorescence emission were reversible, as removal of DTT allowed the receptor to restore 70% of ligand binding and return to the initial emission spectrum. Furthermore, we found adrenergic antagonists were able to slow the rate of the conformational change induced by DTT but not the rate of disulfide reduction, suggesting that the antagonists stabilize the structure of the reduced receptor.

    View details for Web of Science ID A1996VU22400001

    View details for PubMedID 8931540

  • Transmembrane regions V and VI of the human luteinizing hormone receptor are required for constitutive activation by a mutation in the third intracellular loop JOURNAL OF BIOLOGICAL CHEMISTRY Kudo, M., Osuga, Y., Kobilka, B. K., Hsueh, A. J. 1996; 271 (37): 22470-22478

    Abstract

    Gonadotropin receptors are members of the seven-transmembrane (TM) receptor family. Several point mutations in TM V and VI and the intracellular loop 3 (i3) have been identified in the luteinizing hormone (LH) receptor gene, leading to constitutive activation of the receptor. Because gonadotropin receptors are highly conserved, we mutated the follicle-stimulating hormone (FSH) receptor at the corresponding amino acids. However, the FSH receptor mutants showed minimal increases in basal cAMP production. Taking advantage of this difference between the two receptors, we designed chimeric receptors with or without a point mutation in the i3 to identify the region in the LH receptor important for its constitutive activation. Introduction of the point mutation into chimeric receptors containing only TM V to VI from the LH receptor led to major increases in ligand-independent cAMP production. Furthermore, a chimeric receptor with only TM V and VI derived from the LH receptor can be rendered constitutively active by the mutation in the i3 from the FSH receptor. These results suggest that interactions between TM V and VI of the FSH receptor are essential for maintaining the receptor in the more constrained state, whereas interactions between these domains of the LH receptor are permissive for constitutively activating mutations in the i3.

    View details for Web of Science ID A1996VG67200031

    View details for PubMedID 8798412

  • Cardiovascular regulation in mice lacking alpha(2)-adrenergic receptor subtypes b and c SCIENCE Link, R. E., Desai, K., Hein, L., Stevens, M. E., Chruscinski, A., Bernstein, D., Barsh, G. S., Kobilka, B. K. 1996; 273 (5276): 803-805

    Abstract

    alpha2-Adrenergic receptors (alpha2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific alpha2AR subtypes (alpha2a, alpha2b, and alpha2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either alpha2b or alpha2c receptors. Stimulation of alpha2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating alpha2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the alpha2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective alpha2AR drugs.

    View details for Web of Science ID A1996VB42900045

    View details for PubMedID 8670422

  • Targeted disruption of the mouse beta 1-adrenergic receptor gene: Developmental and cardiovascular effects PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rohrer, D. K., Desai, K. H., Jasper, J. R., Stevens, M. E., Regula, D. P., Barsh, G. S., Bernstein, D., Kobilka, B. K. 1996; 93 (14): 7375-7380

    Abstract

    At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.

    View details for Web of Science ID A1996UW79200098

    View details for PubMedID 8693001

  • Arrangement of transmembrane domains in adrenergic receptors - Similarity to bacteriorhodopsin JOURNAL OF BIOLOGICAL CHEMISTRY Mizobe, T., Maze, M., Lam, V., Suryanarayana, S., Kobilka, B. K. 1996; 271 (5): 2387-2389

    Abstract

    G protein-coupled receptors (GPCRs) have seven hydrophobic domains, which are thought to span the lipid bilayer as alpha helical transmembrane domains (TMDs). The tertiary structure of GPCRs has not been determined; however, molecular models of GPCRs have generally been based on bacteriorhodopsin, which is functionally unrelated to GPCRs but has a similar secondary structure. We sought to examine the validity of using bacteriorhodopsin as a scaffold for GPCR model building by experimentally determining the orientation of the TMDs of adrenergic receptors in the plasma membrane. In separate experiments, three sequential amino acid residues (Leu-310, Leu-311, Asn-312) in TMD VII of the beta 2 adrenoreceptors were mutated to the amino acids found in the homologous domain of the alpha 2 adrenoceptor (Phe, Phe, Phe). Exchange of Asn-312 and Leu-311 in the beta 2 adrenoceptor resulted in nonfunctional proteins, most likely due to incompatibility of the introduced bulky phenylalanine side chain with adjacent structural domains in the beta 2 adrenoreceptor. This structural incompatibility was "repaired" by replacing the specific beta 2 TMD sequence with an alpha 2 receptor sequence. TMD I and TMD II complemented the Asn-312-->Phe mutation, and TMD III and TMD VI complemented the Leu-311-->Phe mutation. These results indicate that TMDs I, II, III, and VI surround TMD VII in a counter-clockwise orientation analogous to the orientation of TMDs in bacteriorhodopsin.

    View details for Web of Science ID A1996TT48800010

    View details for PubMedID 8576196

  • FLUORESCENT LABELING OF PURIFIED BETA(2) ADRENERGIC-RECEPTOR - EVIDENCE FOR LIGAND-SPECIFIC CONFORMATIONAL-CHANGES JOURNAL OF BIOLOGICAL CHEMISTRY Gether, U., Lin, S. S., Kobilka, B. K. 1995; 270 (47): 28268-28275

    Abstract

    The purpose of the present study was to develop an approach to directly monitor structural changes in a G protein-coupled receptor in response to drug binding. Purified human beta 2 adrenergic receptor was covalently labeled with the cysteine-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)ethylenediamine (IANBD). IANBD is characterized by a fluorescence which is highly sensitive to the polarity of its environment. We found that the full agonist, isoproterenol, elicited a stereoselective and dose-dependent decrease in fluorescence from IANBD-labeled beta 2 receptor. The change in fluorescence could be plotted against the concentration of isoproterenol as a simple hyperbolic binding isotherm demonstrating interaction with a single binding site in the receptor. The ability of several adrenergic antagonists to reverse the response confirmed that this binding site is identical to the well described binding site in the beta 2 receptor. Comparison of the response to isoproterenol with a series of adrenergic agonists, having different biological efficacies, revealed a linear correlation between biological efficacy and the change in fluorescence. This suggests that the agonist-mediated decrease in fluorescence from IANBD-labeled beta 2 receptor is due to the same conformational change as involved in receptor activation and G protein coupling. In contrast to agonists, negative antagonists induced a small but significant increase in base-line fluorescence. Despite the small amplitude of this response, it supports the notion that antagonists by themselves may alter receptor structure. In conclusion, our data provide the first direct evidence for ligand-specific conformational changes occurring in a G protein-coupled receptor. Furthermore, the data demonstrate the potential of fluorescence spectroscopy as a tool for further delineating the molecular mechanisms of drug action at G protein-coupled receptors.

    View details for Web of Science ID A1995TG21000049

    View details for PubMedID 7499324

  • BEHAVIORAL AND CARDIOVASCULAR EFFECTS OF DISRUPTING THE ANGIOTENSIN-II TYPE-2 RECEPTOR GENE IN MICE NATURE Hein, L., Barsh, G. S., Pratt, R. E., Dzau, V. J., Kobilka, B. K. 1995; 377 (6551): 744-747

    Abstract

    Angiotensin II, a potent regulator of blood pressure and of water and electrolyte balance, binds to two different G-protein-coupled receptors. The type-1 receptor (AT1) mediates the vasopressive and aldosterone-secreting effects of angiotensin II, but the function of the type-2 receptor (AT2) is unknown, although it is expressed in both adult and embryonic life. To address this question, we have generated mice lacking the gene encoding the AT2 receptor. Mutant mice develop normally, but have an impaired drinking response to water deprivation as well as a reduction in spontaneous movements. Their baseline blood pressure is normal, but they show an increased vasopressor response to injection of angiotensin II. Thus, although the AT2 receptor is not required for embryonic development, it plays a role in the central nervous system and cardiovascular functions that are mediated by the renin-angiotensin system.

    View details for Web of Science ID A1995TB46900060

    View details for PubMedID 7477266

  • AMINO AND CARBOXYL-TERMINAL MODIFICATIONS TO FACILITATE THE PRODUCTION AND PURIFICATION OF A G-PROTEIN-COUPLED RECEPTOR ANALYTICAL BIOCHEMISTRY Kobilka, B. K. 1995; 231 (1): 269-271

    View details for Web of Science ID A1995RZ60200039

    View details for PubMedID 8678314

  • TARGETED INACTIVATION OF THE GENE ENCODING THE MOUSE ALPHA(2C)-ADRENOCEPTOR HOMOLOG MOLECULAR PHARMACOLOGY Link, R. E., Stevens, M. S., KULATUNGA, M., Scheinin, M., Barsh, G. S., Kobilka, B. K. 1995; 48 (1): 48-55

    Abstract

    alpha 2-Adrenergic receptors (alpha 2-ARs) regulate a wide range of physiological functions and are targets for clinically important antihypertensive and anesthetic agents. Three genes encoding alpha 2-AR subtypes have been cloned in humans and mice, but the physiological significance of each subtype has not been completely characterized. The available agonist and antagonist compounds are not sufficiently subtype selective to allow the unambiguous dissection of these receptors in vivo. As an alternative approach, we have used gene targeting in embryonic stem cells to disrupt the Adra2c gene, which encodes the alpha 2c-AR subtype in mice. Adra2c-/Adra2c- animals do not express a functional alpha 2c-AR transcript, as detected by Northern blotting or reverse transcription-polymerase chain reaction analysis. In addition, these mice have markedly reduced [3H]rauwolscine binding in their caudate putamen and in other brain regions normally expressing Adra2c binding sites. Adra2c-/Adra2c- mice, however, are viable and fertile and appear grossly normal. Expression levels of Adra2a and Adra2b mRNA in brain and kidney are not altered by the Adra2c knockout. These data suggest that up-regulation of Adra2a or Adra2b does not compensate for the Adra2c deficiency and that the receptor encoded by Adra2c is not required for normal mouse development or for survival in a laboratory environment.

    View details for Web of Science ID A1995RK03200007

    View details for PubMedID 7623774

  • Adrenergic receptor signal transduction and regulation. Neuropharmacology Hein, L., Kobilka, B. K. 1995; 34 (4): 357-366

    View details for PubMedID 7566466

  • GENETIC MODELS OF HUMAN VASCULAR-DISEASE CIRCULATION Dzau, V. J., Gibbons, G. H., Kobilka, B. K., Lawn, R. M., Pratt, R. E. 1995; 91 (2): 521-531

    Abstract

    The use of genetic models has greatly assisted investigations of the natural history, mechanisms, and potential therapy for human vascular disease. In the past, genetic models of vascular disease were obtained through serendipity and/or selective breeding to obtain inbred lines that express the phenotype of interest. This approach has yielded several valuable models of atherosclerosis and hypertension. In the past several years, the advent of molecular techniques has enabled investigators to produce additional novel genetic models of disease that have further enhanced the study of vascular biology and medicine. Transgenic techniques and the techniques of homologous recombination have allowed researchers to alter the genotype of an animal in a precise manner and to study the resultant change in phenotype. More recently, techniques of in vivo gene transfer have also accelerated and enhanced the development of novel models. The application of these methodologies has resulted in important breakthroughs in our understanding of the pathogenesis and treatment of vascular diseases. In this review, we compare and contrast these technologies along with examples of their use in the studies of vascular biology and medicine.

    View details for Web of Science ID A1995QB42600036

    View details for PubMedID 7805258

  • INTRACELLULAR TARGETING AND TRAFFICKING OF THROMBIN RECEPTORS - A NOVEL MECHANISM FOR RESENSITIZATION OF A G-PROTEIN-COUPLED RECEPTOR JOURNAL OF BIOLOGICAL CHEMISTRY Hein, L., Ishii, K., Coughlin, S. R., Kobilka, B. K. 1994; 269 (44): 27719-27726

    Abstract

    The receptor for the protease thrombin is a member of the G protein-coupled receptor family, but is activated by a unique proteolytic mechanism. The irreversibility of this proteolytic mechanism and the fact that the ligand is tethered to its receptor raise special questions about inactivation of cleaved receptors and recovery of thrombin responsiveness. We compared the intracellular trafficking of the thrombin receptor to that of the beta 2-adrenergic receptor in transfected Rat1 fibroblasts. In unstimulated cells almost all beta 2 receptors were located on the plasma membrane; by contrast, part of a cell's thrombin receptors were found in an intracellular membrane compartment which co-localized with Golgi markers. Stimulation by agonist caused internalization and subsequent recycling of the beta 2-adrenergic receptor, but most activated thrombin receptors were internalized and targeted to lysosomes. The intracellular pool of thrombin receptors found in unstimulated cells was protected from activation by thrombin, but was translocated to the plasma membrane upon activation of cell surface thrombin receptors. Replenishment of plasma membrane thrombin receptors correlated with recovery of thrombin responsiveness. These observations reveal a novel trafficking mechanism for resensitizing the thrombin receptor as opposed to the internalization/recycling pathway of other G protein-coupled receptors.

    View details for Web of Science ID A1994PV77100087

    View details for PubMedID 7961693

  • ANTAGONIST-DEPENDENT AND ANTAGONIST-INDEPENDENT STEPS IN THE MECHANISM OF ADRENERGIC-RECEPTOR INTERNALIZATION JOURNAL OF BIOLOGICAL CHEMISTRY VONZASTROW, M., Kobilka, B. K. 1994; 269 (28): 18448-18452

    Abstract

    Epitope tagging and immunocytochemical techniques were used to examine the agonist-regulated internalization of human beta 2-adrenergic receptors in 293 cells. In the absence of agonist, receptors tagged with monoclonal antibody remain in the plasma membrane for > 1 h. In the presence of agonist, tagged receptors are endocytosed within 10 min. Endocytosed receptors are located in endosomes and can be recycled to the plasma membrane. In the prolonged presence of agonist, receptor endocytosis continues even after maximum sequestration of surface receptors (measured by radioligand binding to intact cells) has occurred. The process of receptor endocytosis requires cellular ATP and is temperature-dependent. At 4 degrees C, no agonist-induced redistribution of receptors located in the plasma membrane is observed. At 16 degrees C, agonist causes receptors to cluster in and around coated invaginations of the plasma membrane, but receptor endocytosis does not occur. Agonist treatment of cells at 16 degrees C, but not 4 degrees C, predisposes receptors to agonist-independent endocytosis upon warming to 37 degrees C. These studies suggest that: 1) beta 2-adrenergic receptors reside stably in the plasma membrane of untreated cells, while they continuously cycle between the plasma membrane and endosomes in the presence of agonist; 2) agonist regulates an early step in the endocytosis mechanism, which is associated with the redistribution of adrenergic receptors between distinct microdomains of the plasma membrane; and 3) later steps in the endocytosis mechanism do not require agonist and may utilize the same endocytic machinery that mediates the endocytosis of constitutively recycling receptors.

    View details for Web of Science ID A1994NW79800036

    View details for PubMedID 7518433

  • THE PEPTIDE PRODUCT OF A 5' LEADER CISTRON IN THE BETA(2) ADRENERGIC-RECEPTOR MESSENGER-RNA INHIBITS RECEPTOR SYNTHESIS JOURNAL OF BIOLOGICAL CHEMISTRY PAROLA, A. L., Kobilka, B. K. 1994; 269 (6): 4497-4505

    Abstract

    The 5' leader region of mammalian beta 2 adrenergic receptor messenger RNAs (mRNA) have a short open reading frame (sORF) preceding the receptor cistron. Mutational inactivation of the sORF start codon increased beta 2 receptor expression and translation 1.9-fold from beta 2 receptor genes transfected into COS-7 cells. sORF inactivation also increased receptor synthesis 2.4-fold in a cell-free expression system that synthesizes functional beta 2 receptor in vitro. Translational initiation at the sORF was demonstrated both in vitro and in transfected COS-7 cells using an epitope-tagged fusion protein. Using the fusion protein as a reporter for initiation at the sORF shows that 5' leader mutations which increase translation of the sORF decrease receptor translation. Mutation analysis of the 5' leader region and peptide coding sequences suggests the peptide itself inhibits beta 2 receptor expression. Consistent with this hypothesis, a synthetic peptide corresponding to the peptide encoded by the beta 2 receptor sORF potently inhibits translation in vitro. Our results suggest that a nonoverlapping cistron in the beta 2 receptor mRNA 5' leader region is translated and the resulting peptide inhibits receptor translation.

    View details for Web of Science ID A1994MW98900091

    View details for PubMedID 8308019

  • LINKAGE MAPPING OF ALPHA-2-ADRENERGIC RECEPTOR GENES TO MOUSE CHROMOSOME-2 AND CHROMOSOME-5 MAMMALIAN GENOME Link, R. E., Kobilka, B. K., Barsh, G. S. 1993; 4 (11): 650-655

    Abstract

    alpha-2 adrenergic receptors can be subdivided into three related subtypes which are conserved in humans, rats, and mice. In the mouse, these receptors are encoded by three genes (Adra-2a, Adra-2b, Adra-2c). To gain insight into the evolution of this multigene family and to investigate whether these genes are candidates for previously identified mouse mutations, we have determined the map positions of the Adra-2b and Adra-2c genes. The Adra-2a gene has been previously mapped to mouse Chromosome (Chr) 19 (Oakey et al. Genomics 10, 338-344, 1991). Using segregation among recombinant inbred strains of a single-stranded conformational polymorphism specific for alleles of Adra-2b and Adra-2c, we present map positions for these genes on mouse Chrs 2 and 5, respectively. In the case of Adra-2b, these results have been confirmed by an analysis of somatic cell hybrids. In addition, we generate AKXD recombinant inbred strain distribution patterns for 11 previously defined SSLP microsatellite markers, further refining the haplotype maps for these chromosomes. Finally, several candidate mouse mutations that map close to Adra-2b and Adra-2c are discussed.

    View details for Web of Science ID A1993MF57400005

    View details for PubMedID 8281014

  • PRIMARY STRUCTURE OF THE MOUSE BETA(1)-ADRENERGIC RECEPTOR GENE BIOCHIMICA ET BIOPHYSICA ACTA Jasper, J. R., Link, R. E., Chruscinski, A. J., Kobilka, B. K., Bernstein, D. 1993; 1178 (3): 307-309

    Abstract

    The mouse beta 1-adrenergic receptor was isolated from a genomic library and cloned into pBluescript SK-. Characterization of the clone revealed an open reading frame which encodes a predicted protein of 466 amino acids. The mouse beta 1 receptor is 92.7% identical to the human sequence, 98.5% identical to the rat sequence, and contains a consensus site for N-linked glycosylation at Asn-15 and a cAMP-dependent protein kinase phosphorylation site at Ser-301.

    View details for Web of Science ID A1993LX80300011

    View details for PubMedID 8395893

  • AMINO-ACID SUBSTITUTIONS AT POSITION 312 IN THE 7TH HYDROPHOBIC SEGMENT OF THE BETA(2)-ADRENERGIC RECEPTOR MODIFY LIGAND-BINDING SPECIFICITY MOLECULAR PHARMACOLOGY Suryanarayana, S., Kobilka, B. K. 1993; 44 (1): 111-114

    Abstract

    We previously reported that Asn312 of the beta 2-adrenergic receptor and Asn385 in the homologous position in the 5-hydroxytryptamine1A receptor are important for binding to a class of beta-adrenergic receptor antagonists including propranolol and alprenolol. We proposed that the asparagine may be forming a hydrogen bond with the phenoxy oxygen common to these ligands. To further test this hypothesis we made alanine, threonine, phenylalanine, and glutamine substitutions at position 312 in the beta 2-adrenergic receptor. We observed that substitution with amino acids that permit formation of hydrogen bonds (threonine and glutamine) supported binding to aryloxyalkylamines, whereas substitution with amino acids that cannot form hydrogen bonds (alanine and phenylalanine) did not permit binding to these compounds. We were surprised to find that two of these substitutions led to an increase in affinity for alpha-adrenergic ligands. Substitution with glutamine and threonine at position 312 led to a 11-15-fold increase in affinity for yohimbine and enabled p-aminoclonidine to act as an agonist. These results further emphasize the role of position 312 in the formation of the ligand binding site for multiple ligands.

    View details for Web of Science ID A1993LN99200015

    View details for PubMedID 8101966

  • SUBTYPE-SPECIFIC DIFFERENCES IN THE INTRACELLULAR SORTING OF G-PROTEIN-COUPLED RECEPTORS JOURNAL OF BIOLOGICAL CHEMISTRY VONZASTROW, M., Link, R., Daunt, D., Barsh, G., Kobilka, B. 1993; 268 (2): 763-766

    Abstract

    We have examined the subcellular distribution of three subtypes of adrenergic receptor by immunocytochemical localization of wild-type and epitope-tagged proteins expressed in Cos-7 and K293 cells. Two subtypes (beta 2 and M alpha 2-10H) are localized in the plasma membrane at steady state in untreated cells, while another subtype (M alpha 2-4H) is found both in the plasma membrane and in a population of intracellular vesicles. Within 15 min following the addition of adrenergic agonists, beta 2 and M alpha 2-10H receptors are differentially sorted; beta 2 receptors are selectively internalized to intracellular vesicles, which are distinct from those containing M alpha 2-4H receptors, while M alpha 2-10H receptors remain in the plasma membrane. Subtype-specific sorting suggests a new class of functional properties that may differentiate the signaling and regulation of homologous G protein-coupled receptors.

    View details for Web of Science ID A1993KG07700003

    View details for PubMedID 7678260

  • ENHANCEMENT OF MEMBRANE INSERTION AND FUNCTION IN A TYPE IIIB MEMBRANE-PROTEIN FOLLOWING INTRODUCTION OF A CLEAVABLE SIGNAL PEPTIDE JOURNAL OF BIOLOGICAL CHEMISTRY Guan, X. M., Kobilka, T. S., Kobilka, B. K. 1992; 267 (31): 21995-21998

    Abstract

    The human beta 2 adrenergic receptor is a type IIIb membrane protein. It has a putative seven-transmembrane topology but lacks an amino-terminal cleavable signal sequence. The mechanism by which the amino terminus of the beta 2 receptor is translocated across the endoplasmic reticulum membrane is unknown. Furthermore, it is not known if translocation as a type IIIb protein is essential for the proper folding. Our studies indicate that conversion of beta 2 receptor from a type IIIb to a type IIIa membrane protein by introducing an NH2-terminal cleavable signal sequence enhances translocation of the receptor into the endoplasmic reticulum membrane, thereby facilitating expression of functional receptor.

    View details for Web of Science ID A1992JW71900003

    View details for PubMedID 1331042

  • IDENTIFICATION OF INTRAMOLECULAR INTERACTIONS IN ADRENERGIC-RECEPTORS JOURNAL OF BIOLOGICAL CHEMISTRY Suryanarayana, S., VONZASTROW, M., Kobilka, B. K. 1992; 267 (31): 21991-21994

    Abstract

    Adrenergic receptors are representative of a large family of plasma membrane receptors that interact with G proteins during the process of transmembrane signal transduction. G protein-coupled receptors have a primary structure that is homologous to bacteriorhodopsin and are proposed to have a similar three-dimensional structure; however, it has not yet been possible to examine this hypothesis experimentally. We have used a novel mutagenesis approach to identify intramolecular interactions. Our results indicate that specific amino acids in the seventh hydrophobic segment of alpha 2 and beta 2 adrenergic receptors lie adjacent to the first hydrophobic segment. These studies provide the first experimental evidence defining spatial relationships that exist in the three-dimensional structure of adrenergic receptors.

    View details for Web of Science ID A1992JW71900002

    View details for PubMedID 1331041

  • CLONING OF 2 MOUSE GENES ENCODING ALPHA-2-ADRENERGIC RECEPTOR SUBTYPES AND IDENTIFICATION OF A SINGLE AMINO-ACID IN THE MOUSE ALPHA-2-C10 HOMOLOG RESPONSIBLE FOR AN INTERSPECIES VARIATION IN ANTAGONIST BINDING MOLECULAR PHARMACOLOGY Link, R., Daunt, D., Barsh, G., Chruscinski, A., Kobilka, B. 1992; 42 (1): 16-27

    Abstract

    Molecular cloning and ligand binding studies have shown the alpha 2 class of adrenergic receptor (alpha 2-AR) to be a family of at least three related subtypes in humans. These studies have not, however, identified distinct subtype-specific functions for these receptors in vivo. It should be possible to extend the analysis of alpha 2-AR subtype function to the animal level through the use of experimental mammalian embryology in mice. To begin this process, we have isolated two mouse genomic clones encoding alpha 2-AR subtypes and expressed these genes in COS-7 cells for binding studies. Sequence homology and ligand binding data allow the assignment of one clone (M alpha 2-4H) as the mouse homolog of the human alpha 2-C4 subtype. The other clone (M alpha 2-10H) closely resembles the human alpha 2-C10 subtype in sequence but binds with significantly lower affinity to yohimbine and rauwolscine, members of a distinct class of bulky alpha 2-selective antagonists commonly used to evaluate alpha 2-AR function in vivo. To define the domain(s) responsible for this unusual binding property, we constructed a series of M alpha 2-10H/human alpha 2-C10 chimeric receptors. Analysis of these receptors identified a conservative Cys201 to Ser201 change in the fifth transmembrane domain of M alpha 2-10H as being responsible for the low affinity of the mouse receptor for yohimbine.

    View details for Web of Science ID A1992JE03200004

    View details for PubMedID 1353249

  • IDENTIFICATION OF A SINGLE AMINO-ACID RESIDUE RESPONSIBLE FOR THE BINDING OF A CLASS OF BETA-ADRENERGIC-RECEPTOR ANTAGONISTS TO 5-HYDROXYTRYPTAMINE1A RECEPTORS MOLECULAR PHARMACOLOGY Guan, X. M., Peroutka, S. J., Kobilka, B. K. 1992; 41 (4): 695-698

    Abstract

    The 5-hydroxytryptamine1A (5-HT1A) receptor can bind certain beta-adrenergic receptor antagonists, such as pindolol, with high affinity. Such pharmacological cross-reactivity suggests a structural similarity in the ligand binding site between the two receptors. To identify this structural entity, we mutated Asn385 in the seventh transmembrane domain of the human 5-HT1A receptor, based on the observation that this residue is conserved in all 5-HT1A and beta-adrenergic receptors of different species but is absent in all other cloned guanine nucleotide-binding protein-coupled receptors. This single point mutation (Asn385 to valine) causes a highly selective decrease in the affinity of pindolol and other aryloxyalkylamines for the mutant receptor (about 100-fold), while producing only minor changes in the binding of other 5-HT agonists and antagonists. The results provide direct evidence that Asn385 is responsible for the high affinity interaction between 5-HT1A receptors and aryloxyalkylamine beta-adrenergic antagonists but is not required for the binding of other chemical classes of ligands.

    View details for Web of Science ID A1992HP40500016

    View details for PubMedID 1349154

  • LIGAND-REGULATED INTERNALIZATION AND RECYCLING OF HUMAN BETA-2-ADRENERGIC RECEPTORS BETWEEN THE PLASMA-MEMBRANE AND ENDOSOMES CONTAINING TRANSFERRIN RECEPTORS JOURNAL OF BIOLOGICAL CHEMISTRY VONZASTROW, M., Kobilka, B. K. 1992; 267 (5): 3530-3538

    Abstract

    Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.

    View details for Web of Science ID A1992HD15400107

    View details for PubMedID 1371121

  • ADRENERGIC-RECEPTORS AS MODELS FOR G PROTEIN-COUPLED RECEPTORS ANNUAL REVIEW OF NEUROSCIENCE Kobilka, B. 1992; 15: 87-114

    View details for Web of Science ID A1992HF92100004

    View details for PubMedID 1575451

  • A POINT MUTATION IN THE 7TH HYDROPHOBIC DOMAIN OF THE ALPHA-2 ADRENERGIC-RECEPTOR INCREASES ITS AFFINITY FOR A FAMILY OF BETA-RECEPTOR-ANTAGONISTS JOURNAL OF BIOLOGICAL CHEMISTRY Suryanarayana, S., Daunt, D. A., VONZASTROW, M., Kobilka, B. K. 1991; 266 (23): 15488-15492

    Abstract

    Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.

    View details for Web of Science ID A1991GB09700098

    View details for PubMedID 1678390

  • MOLECULAR AND CELLULAR BIOLOGY OF ADRENERGIC-RECEPTORS TRENDS IN CARDIOVASCULAR MEDICINE Kobilka, B. 1991; 1 (5): 189-194

    Abstract

    Adrenergic receptors form the interface between the sympathetic nervous system and the cardiovascular system. Genomic or cDNA clones for 8 types of mammalian adrenergic receptors have been obtained. Much has been learned about the structure and functional properties of the ?(2)-adrenergic receptor. Less is known about the functional properties and the physiologic role of the other adrenergic receptors. Further progress in this field may lead to the development of more selective drugs to modify the physiologic processes controlled by these receptors.

    View details for Web of Science ID A1991GD51800003

    View details for PubMedID 21239309

  • THE ROLE OF CYTOSOLIC AND MEMBRANE FACTORS IN PROCESSING OF THE HUMAN BETA-2 ADRENERGIC-RECEPTOR FOLLOWING TRANSLOCATION AND GLYCOSYLATION IN A CELL-FREE SYSTEM JOURNAL OF BIOLOGICAL CHEMISTRY Kobilka, B. K. 1990; 265 (13): 7610-7618

    Abstract

    The beta-2 adrenergic receptor has been proposed to have seven membrane-spanning domains. Expression of functional beta-2 adrenergic receptor was achieved in a heterologous cell-free system composed of rabbit reticulocyte lysate and microsomal membranes from Xenopus laevis oocytes. The functional state of the receptor protein can be determined by ligand-binding assays and by the ability of ligands to alter the susceptibility of the receptor to proteinase K digestion. The process by which functional receptor is made was studied. The receptor protein remains nonfunctional immediately following translocation and glycosylation, and additional processing steps are needed before the receptor is able to interact with ligands. These processing steps require intact microsomal membranes as well as several cytosolic factors including ATP and one or more high molecular mass (greater than 30 kDa) factors but do not require receptor glycosylation and are not inhibited by nonhydrolyzable GTP analogues.

    View details for Web of Science ID A1990DB30700079

    View details for PubMedID 1692024

  • MOLECULAR-CLONING AND EXPRESSION OF THE CDNA FOR THE HAMSTER ALPHA-1-ADRENERGIC RECEPTOR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cotecchia, S., Schwinn, D. A., RANDALL, R. R., Lefkowitz, R. J., Caron, M. G., Kobilka, B. K. 1988; 85 (19): 7159-7163

    Abstract

    The cDNA for the Syrian hamster alpha 1-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the receptor protein purified from DDT1MF-2 smooth muscle cells. The deduced amino acid sequence encodes a 515-residue polypeptide that shows the most sequence identity with the other adrenergic receptors and the putative protein product of the related clone G-21. Similarities with the muscarinic cholinergic receptors are also evident. Expression studies in COS-7 cells confirm that we have cloned the alpha 1-adrenergic receptor that couples to inositol phospholipid metabolism.

    View details for Web of Science ID A1988Q358500025

    View details for PubMedID 2845398

  • THE GENOMIC CLONE G-21 WHICH RESEMBLES A BETA-ADRENERGIC-RECEPTOR SEQUENCE ENCODES THE 5-HT1A RECEPTOR NATURE FARGIN, A., Raymond, J. R., Lohse, M. J., Kobilka, B. K., Caron, M. G., Lefkowitz, R. J. 1988; 335 (6188): 358-360

    Abstract

    The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.

    View details for Web of Science ID A1988Q137300061

    View details for PubMedID 3138543

  • CLONING AND EXPRESSION OF A HUMAN-KIDNEY CDNA FOR AN ALPHA-2-ADRENERGIC RECEPTOR SUBTYPE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Regan, J. W., Kobilka, T. S., YANGFENG, T. L., Caron, M. G., Lefkowitz, R. J., Kobilka, B. K. 1988; 85 (17): 6301-6305

    Abstract

    An alpha 2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet alpha 2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet alpha 2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the alpha 2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the alpha 2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet alpha 2-adrenergic receptor (alpha 2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective alpha-adrenergic ligands.

    View details for Web of Science ID A1988Q047600016

    View details for PubMedID 2842764

  • HUMAN BETA-1-ADRENERGIC AND BETA-2-ADRENERGIC RECEPTORS - STRUCTURALLY AND FUNCTIONALLY RELATED RECEPTORS DERIVED FROM DISTINCT GENES TRENDS IN NEUROSCIENCES Frielle, T., Kobilka, B., Lefkowitz, R. J., Caron, M. G. 1988; 11 (7): 321-324

    View details for Web of Science ID A1988N945500010

    View details for PubMedID 2465637

  • CHIMERIC ALPHA-2-ADRENERGIC, BETA-2-ADRENERGIC RECEPTORS - DELINEATION OF DOMAINS INVOLVED IN EFFECTOR COUPLING AND LIGAND-BINDING SPECIFICITY SCIENCE Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G., Lefkowitz, R. J. 1988; 240 (4857): 1310-1316

    Abstract

    The alpha 2 and beta 2 adrenergic receptors, both of which are activated by epinephrine, but which can be differentiated by selective drugs, have opposite effects (inhibitory and stimulatory) on the adenylyl cyclase system. The two receptors are homologous with each other, rhodopsin, and other receptors coupled to guanine nucleotide regulatory proteins and they contain seven hydrophobic domains, which may represent transmembrane spanning segments. The function of specific structural domains of these receptors was determined after construction and expression of a series of chimeric alpha 2-,beta 2-adrenergic receptor genes. The specificity for coupling to the stimulatory guanine nucleotide regulatory protein lies within a region extending from the amino terminus of the fifth hydrophobic domain to the carboxyl terminus of the sixth. Major determinants of alpha 2- and beta 2-adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane spanning domain. Chimeric receptors should prove useful for elucidating the structural basis of receptor function.

    View details for Web of Science ID A1988N633900023

    View details for PubMedID 2836950

  • FUNCTIONAL-ACTIVITY AND REGULATION OF HUMAN BETA-2-ADRENERGIC RECEPTORS EXPRESSED IN XENOPUS OOCYTES JOURNAL OF BIOLOGICAL CHEMISTRY Kobilka, B. K., MacGregor, C., Daniel, K., Kobilka, T. S., Caron, M. G., Lefkowitz, R. J. 1987; 262 (32): 15796-15802

    Abstract

    The recently cloned human beta-adrenergic cDNA and several mutated forms have been expressed in Xenopus laevis oocytes by injection of RNA made from the cDNA under the control of the bacteriophage SP6 promoter. The cDNA and gene of the beta 2-adrenergic receptor possess the unusual feature of having a second upstream ATG (-101 base pairs) and a 19-codon open reading frame 5' to the initiator methionine codon of the receptor (Kobilka, B. K., Dixon, R. A. F., Frielle, T., Dohlman, H. G., Bolanowski, M., Sigal, I. S., Yang-Feng, T. L., Francke, U., Caron, M. G., and Lefkowitz, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 46-50). RNA lacking this upstream AUG and open reading frame was translated approximately 10-fold more efficiently both in an in vitro rabbit reticulocyte system and in oocytes. Injected oocytes but not water injected controls expressed typical beta 2-adrenergic receptors as assessed by ligand binding (450 fmol/mg membrane protein) and catecholamine-stimulated adenylate cyclase (approximately 20 fold). Moreover, these receptors displayed typical agonist-induced homologous desensitization when oocytes were incubated with isoproterenol at room temperature for 3-24 h. Among a series of mutations, truncations of the membrane-anchored core of the receptor eliminated receptor binding and cyclase stimulating activity. In contrast, disruption of one of the cAMP-dependent protein kinase phosphorylation sites or removal of the serine/threonine-rich carboxyl terminus had little or no effect on these functions or on the extent of agonist-induced desensitization relative to that observed with native receptor. These studies validate the beta 2-adrenergic nature of the cloned human beta-adrenergic cDNA, document the utility of the Xenopus oocyte system for studying functional and regulatory properties of receptors coupled to adenylate cyclase, and suggest the possibility that elements in the 5' untranslated region of the beta 2-adrenergic receptor RNA may regulate its translation in vivo.

    View details for Web of Science ID A1987K886800076

    View details for PubMedID 2824467

  • CLONING OF THE CDNA FOR THE HUMAN BETA-1-ADRENERGIC RECEPTOR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Frielle, T., Collins, S., Daniel, K. W., Caron, M. G., Lefkowitz, R. J., Kobilka, B. K. 1987; 84 (22): 7920-7924

    Abstract

    Screening of a human placenta lambda gt11 library has led to the isolation of the cDNA for the human beta 1-adrenergic receptor (beta 1AR). Used as the probe was the human genomic clone termed G-21. This clone, which contains an intronless gene for a putative receptor, was previously isolated by virtue of its cross hybridization with the human beta 2-adrenergic receptor (beta 2AR). The 2.4-kilobase cDNA for the human beta 1AR encodes a protein of 477 amino acid residues that is 69% homologous with the avian beta AR but only 54% homologous with the human beta 2AR. This suggests that the avian gene encoding beta AR and the human gene encoding beta 1AR evolved from a common ancestral gene. RNA blot analysis indicates a message of 2.5 kilobases in rat tissues, with a pattern of tissue distribution consistent with beta 1AR binding. This pattern is quite distinct from the pattern obtained when the beta 2AR cDNA is used as a probe. Expression of receptor protein in Xenopus laevis oocytes conveys adenylate cyclase responsiveness to catecholamines with a typical beta 1AR specificity. This contrasts with the typical beta 2 subtype specificity observed when the human beta 2AR cDNA is expressed in this system. Mammalian beta 1AR and beta 2AR are thus products of distinct genes, both of which are apparently related to the putative G-21 receptor.

    View details for Web of Science ID A1987L051100026

    View details for PubMedID 2825170

  • CLONING, SEQUENCING, AND EXPRESSION OF THE GENE CODING FOR THE HUMAN-PLATELET ALPHA-2-ADRENERGIC RECEPTOR SCIENCE Kobilka, B. K., MATSUI, H., Kobilka, T. S., YANGFENG, T. L., FRANCKE, U., Caron, M. G., Lefkowitz, R. J., Regan, J. W. 1987; 238 (4827): 650-656

    Abstract

    The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes.

    View details for Web of Science ID A1987K595500029

    View details for PubMedID 2823383

  • AN INTRONLESS GENE ENCODING A POTENTIAL MEMBER OF THE FAMILY OF RECEPTORS COUPLED TO GUANINE-NUCLEOTIDE REGULATORY PROTEINS NATURE Kobilka, B. K., Frielle, T., Collins, S., YANGFENG, T., Kobilka, T. S., FRANCKE, U., Lefkowitz, R. J., Caron, M. G. 1987; 329 (6134): 75-79

    Abstract

    Plasma membrane receptors for hormones, drugs, neurotransmitters and sensory stimuli are coupled to guanine nucleotide regulatory proteins. Recent cloning of the genes and/or cDNAs for several of these receptors including the visual pigment rhodopsin, the adenylate-cyclase stimulatory beta-adrenergic receptor and two subtypes of muscarinic cholinergic receptors has suggested that these are homologous proteins with several conserved structural and functional features. Whereas the rhodopsin gene consists of five exons interrupted by four introns, surprisingly the human and hamster beta-adrenergic receptor genes contain no introns in either their coding or untranslated sequences. We have cloned and sequenced a DNA fragment in the human genome which cross-hybridizes with a full-length beta 2-adrenergic receptor probe at reduced stringency. Like the beta 2-adrenergic receptor this gene appears to be intronless, containing an uninterrupted long open reading frame which encodes a putative protein with all the expected structural features of a G-protein-coupled receptor.

    View details for Web of Science ID A1987J846700060

    View details for PubMedID 3041227

  • DELINEATION OF THE INTRONLESS NATURE OF THE GENES FOR THE HUMAN AND HAMSTER BETA-2-ADRENERGIC RECEPTOR AND THEIR PUTATIVE PROMOTER REGIONS JOURNAL OF BIOLOGICAL CHEMISTRY Kobilka, B. K., Frielle, T., Dohlman, H. G., BOLANOWSKI, M. A., Dixon, R. A., Keller, P., Caron, M. G., Lefkowitz, R. J. 1987; 262 (15): 7321-7327

    Abstract

    The beta 2-adrenergic receptor is the first adenylate cyclase-coupled receptor to be cloned. We provide here a detailed characterization of its complete gene in both the human and hamster which reveals several unusual and provocative features. The genes are present in a single copy, are intronless, and are bounded by homologous 18-bp (base pair) direct repeats. These findings suggest that the beta 2-adrenergic receptor may have arisen as a processed gene for another related gene. Genomic Southern blots done at reduced stringency in fact reveal additional weak signals. The human and hamster gene sequences 5' to the principal site of transcription initiation are highly homologous and share many characteristics of promoters for housekeeping genes. Moreover, there is present in the human genome a long (777 bp) open reading frame which is in frame with the beta-adrenergic receptor coding block and which ends only 234 bp 5' to the initiator methionine of the receptor. An unusual cDNA has been found, transcribed from a putative second more 5' promoter which contains the 5' half of the beta-adrenergic receptor as well as 1065-bp 5' to the receptor coding region, including the entire upstream long open reading frame (sufficient to encode a putative protein of Mr approximately 28,000).

    View details for Web of Science ID A1987H414700060

    View details for PubMedID 3034889

  • CDNA FOR THE HUMAN BETA-2-ADRENERGIC RECEPTOR - A PROTEIN WITH MULTIPLE MEMBRANE-SPANNING DOMAINS AND ENCODED BY A GENE WHOSE CHROMOSOMAL LOCATION IS SHARED WITH THAT OF THE RECEPTOR FOR PLATELET-DERIVED GROWTH-FACTOR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kobilka, B. K., Dixon, R. A., Frielle, T., Dohlman, H. G., BOLANOWSKI, M. A., SIGAL, I. S., YANGFENG, T. L., FRANCKE, U., Caron, M. G., Lefkowitz, R. J. 1987; 84 (1): 46-50

    Abstract

    We have isolated and sequenced a cDNA encoding the human beta 2-adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster beta 2-adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. We have localized the gene for the beta 2-adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor.

    View details for Web of Science ID A1987F667900010

    View details for PubMedID 3025863

  • CLONING OF THE GENE AND CDNA FOR MAMMALIAN BETA-ADRENERGIC-RECEPTOR AND HOMOLOGY WITH RHODOPSIN NATURE Dixon, R. A., Kobilka, B. K., STRADER, D. J., Benovic, J. L., Dohlman, H. G., Frielle, T., BOLANOWSKI, M. A., Bennett, C. D., Rands, E., Diehl, R. E., Mumford, R. A., Slater, E. E., SIGAL, I. S., Caron, M. G., Lefkowitz, R. J., Strader, C. D. 1986; 321 (6065): 75-79

    Abstract

    The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.

    View details for Web of Science ID A1986C169000052

    View details for PubMedID 3010132

Conference Proceedings


  • A SINGLE POINT MUTATION IN THE 7TH HYDROPHOBIC DOMAIN OF THE ALPHA-2-ADRENERGIC RECEPTOR CHANGES ANTAGONIST BINDING-SPECIFICITY TO THAT OF A BETA-RECEPTOR Suryanarayana, S., Daunt, D. A., VONZASTROW, M., Kobilka, B. K. ASSOC AMER PHYSICIANS. 1991: 62-68

    View details for Web of Science ID A1991BV21X00009

    View details for PubMedID 1688262

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