Is MR-guided High-intensity Focused Ultrasound a Feasible Treatment Modality for Desmoid Tumors?
CLINICAL ORTHOPAEDICS AND RELATED RESEARCH
2016; 474 (3): 697-704
Improving thermal dose accuracy in magnetic resonance-guided focused ultrasound surgery: Long-term thermometry using a prior baseline as a reference.
Journal of magnetic resonance imaging
2016; 43 (1): 181-189
MR-guided high-intensity focused ultrasound is a noninvasive treatment modality that uses focused ultrasound waves to thermally ablate tumors within the human body while minimizing side effects to surrounding healthy tissues. This technology is FDA-approved for certain tumors and has potential to be a noninvasive treatment option for extremity soft tissue tumors. Development of treatment modalities that achieve tumor control, decrease morbidity, or both might be of great benefit for patients. We wanted to assess the potential use of this technology in the treatment of extremity desmoid tumors.(1) Can we use MR-guided high-intensity focused ultrasound to accurately ablate a predetermined target volume within a human cadaver extremity? (2) Does MR-guided high-intensity focused ultrasound treatment stop progression and/or cause regression of extremity desmoid tumors?Simulated tumor volumes in four human cadavers, created by using plastic markers, were ablated using a commercially available focused ultrasound system. Accuracy was determined in accordance with the International Organization of Standards location error by measuring the farthest distance between the ablated tissue and the plane corresponding to the target. Between 2012 and 2014, we treated nine patients with desmoid tumors using focused ultrasound ablation. Indications for this were tumor-related symptoms or failure of conventional treatment. Of those, five of them were available for MRI followup at 12 months or longer (mean, 18.2 months; range, 12-23 months). The radiographic and clinical outcomes of five patients who had desmoid tumors treated with focused ultrasound were prospectively recorded. Patients were assessed preoperatively with MRI and followed at routine intervals after treatment with MRI scans and clinical examination.The ablation accuracy for the four cadaver extremities was 5 mm, 3 mm, 8 mm, and 8 mm. Four patients' tumors became smaller after treatment and one patient has slight progression at the time of last followup. The mean decrease in tumor size determined by MRI measurements was 36% (95% confidence interval, 7%-66%). No patient has received additional adjuvant systemic or local treatment. Treatment-related adverse events included first- and second-degree skin burns occurring in four patients, which were managed successfully without further surgery.This preliminary investigation provides some evidence that MR-guided high-intensity focused ultrasound may be a feasible treatment for desmoid tumors. It may also be of use for other soft tissue neoplasms in situations in which there are limited traditional treatment options such as recurrent sarcomas. Further investigation is necessary to better define the indications, efficacy, role, and long-term oncologic outcomes of focused ultrasound treatment.Level IV, therapeutic study.
View details for DOI 10.1007/s11999-015-4364-0
View details for Web of Science ID 000370150000018
View details for PubMedID 26040967
Transcranial MRI-Guided Focused Ultrasound: A Review of the Technologic and Neurologic Applications
AMERICAN JOURNAL OF ROENTGENOLOGY
2015; 205 (1): 150-159
To investigate thermal dose volume (TDV) and non-perfused volume (NPV) of magnetic resonance-guided focused ultrasound (MRgFUS) treatments in patients with soft tissue tumors, and describe a method for MR thermal dosimetry using a baseline reference.Agreement between TDV and immediate post treatment NPV was evaluated from MRgFUS treatments of five patients with biopsy-proven desmoid tumors. Thermometry data (gradient echo, 3T) were analyzed over the entire course of the treatments to discern temperature errors in the standard approach. The technique searches previously acquired baseline images for a match using 2D normalized cross-correlation and a weighted mean of phase difference images. Thermal dose maps and TDVs were recalculated using the matched baseline and compared to NPV.TDV and NPV showed between 47%-91% disagreement, using the standard immediate baseline method for calculating TDV. Long-term thermometry showed a nonlinear local temperature accrual, where peak additional temperature varied between 4-13°C (mean?=?7.8°C) across patients. The prior baseline method could be implemented by finding a previously acquired matching baseline 61%?±?8% (mean?±?SD) of the time. We found 7%-42% of the disagreement between TDV and NPV was due to errors in thermometry caused by heat accrual. For all patients, the prior baseline method increased the estimated treatment volume and reduced the discrepancies between TDV and NPV (P?=?0.023).This study presents a mismatch between in-treatment and post treatment efficacy measures. The prior baseline approach accounts for local heating and improves the accuracy of thermal dose-predicted volume. J. MAGN. RESON. IMAGING 2016;43:181-189.
View details for DOI 10.1002/jmri.24978
View details for PubMedID 26119129
TU-B-210-01: MRg HIFU - Bone and Soft Tissue Tumor Ablation.
2015; 42 (6): 3598-?
This article reviews the physical principles of MRI-guided focused ultra-sound and discusses current and potential applications of this exciting technology.MRI-guided focused ultrasound is a new minimally invasive method of targeted tissue thermal ablation that may be of use to treat central neuropathic pain, essential tremor, Parkinson tremor, and brain tumors. The system has also been used to temporarily disrupt the blood-brain barrier to allow targeted drug delivery to brain tumors.
View details for DOI 10.2214/AJR.14.13632
View details for Web of Science ID 000356781000041
Optimization of White-Matter-Nulled Magnetization Prepared Rapid Gradient Echo (MP-RAGE) Imaging
MAGNETIC RESONANCE IN MEDICINE
2015; 73 (5): 1786-1794
MR guided focused ultrasound (MRgFUS), or alternatively high-intensity focused ultrasound (MRgHIFU), is approved for thermal ablative treatment of uterine fibroids and pain palliation in bone metastases. Ablation of malignant tumors is under active investigation in sites such as breast, prostate, brain, liver, kidney, pancreas, and soft tissue. Hyperthermia therapy with MRgFUS is also feasible, and may be used in conjunction with radiotherapy and for local targeted drug delivery. MRI allows in situ target definition and provides continuous temperature monitoring and subsequent thermal dose mapping during HIFU. Although MRgHIFU can be very precise, treatment of mobile organs is challenging and advanced techniques are required because of artifacts in MR temperature mapping, the need for intercostal firing, and need for gated HIFU or tracking of the lesion in real time. The first invited talk, "MR guided Focused Ultrasound Treatment of Tumors in Bone and Soft Tissue", will summarize the treatment protocol and review results from treatment of bone tumors. In addition, efforts to extend this technology to treat both benign and malignant soft tissue tumors of the extremities will be presented. The second invited talk, "MRI guided High Intensity Focused Ultrasound - Advanced Approaches for Ablation and Hyperthermia", will provide an overview of techniques that are in or near clinical trials for thermal ablation and hyperthermia, with an emphasis of applications in abdominal organs and breast, including methods for MRTI and tracking targets in moving organs.1.Learn background on devices and techniques for MR guided HIFU for cancer therapy2.Understand issues and current status of clinical MRg HIFU3.Understand strategies for compensating for organ movement during MRgHIFU4.Understand strategies for strategies for delivering hyperthermia with MRgHIFUCM - research collaboration with Philips.
View details for DOI 10.1118/1.4925563
View details for PubMedID 26128866
International consensus on use of focused ultrasound for painful bone metastases: Current status and future directions
INTERNATIONAL JOURNAL OF HYPERTHERMIA
2015; 31 (3): 251-259
To optimize the white-matter-nulled (WMn) Magnetization Prepared Rapid Gradient Echo (MP-RAGE) sequence at 7 Tesla (T), with comparisons to 3T.Optimal parameters for maximizing signal-to-noise ratio (SNR) efficiency were derived. The effect of flip angle and repetition time (TR) on image blurring was modeled using simulations and validated in vivo. A novel two-dimensional (2D) -centric radial fan beam (RFB) k-space segmentation scheme was used to shorten scan times and improve parallel imaging. Healthy subjects as well as patients with multiple sclerosis and tremor were scanned using the optimized protocols.Inversion repetition times (TS) of 4.5 s and 6 s were found to yield the highest SNR efficiency for WMn MP-RAGE at 3T and 7T, respectively. Blurring was more sensitive to flip in WMn than in CSFn MP-RAGE and relatively insensitive to TR for both regimes. The 2D RFB scheme had 19% and 47% higher thalamic SNR and SNR efficiency than the 1D centric scheme for WMn MP-RAGE. Compared with 3T, SNR and SNR efficiency were higher for the 7T WMn regime by 56% and 41%, respectively. MS lesions in the cortex and thalamus as well as thalamic subnuclei in tremor patients were clearly delineated using WMn MP-RAGE.Optimization and new view ordering enabled MP-RAGE imaging with 0.8-1 mm(3) isotropic spatial resolution in scan times of 5 min with whole brain coverage.
View details for DOI 10.1002/mrm.25298
View details for Web of Science ID 000353240600010
T2-based temperature monitoring in abdominal fat during MR-guided focused ultrasound treatment of patients with uterine fibroids.
Journal of therapeutic ultrasound
2015; 3: 15-?
Focused ultrasound surgery (FUS), in particular magnetic resonance guided FUS (MRgFUS), is an emerging non-invasive thermal treatment modality in oncology that has recently proven to be effective for the palliation of metastatic bone pain. A consensus panel of internationally recognised experts in focused ultrasound critically reviewed all available data and developed consensus statements to increase awareness, accelerate the development, acceptance and adoption of FUS as a treatment for painful bone metastases and provide guidance towards broader application in oncology. In this review, evidence-based consensus statements are provided for (1) current treatment goals, (2) current indications, (3) technical considerations, (4) future directions including research priorities, and (5) economic and logistical considerations.
View details for DOI 10.3109/02656736.2014.995237
View details for Web of Science ID 000355926300005
View details for PubMedID 25677840
Magnetic Resonance-Guided Focused Ultrasound for Patients With Painful Bone Metastases: Phase II Trial Results
JNCI-JOURNAL OF THE NATIONAL CANCER INSTITUTE
2014; 106 (5)
Magnetic resonance-guided focused ultrasound for patients with painful bone metastases: phase III trial results.
Journal of the National Cancer Institute
2014; 106 (5)
Near-field heating is a potential problem in focused ultrasound treatments, as it can result in thermal injury to skin, subcutaneous fat, and other tissues. Our goals were to determine if T2-based temperature mapping could be used reliably to measure near-field heating in adipose tissue and whether it is practical to perform such mapping during focused ultrasound treatments.We investigated the dependence of T2 on temperature in ex vivo adipose tissue at 3T using a double-echo fast spin echo (FSE) sequence. We implemented and evaluated the T2-based temperature mapping technique in the adipose tissue of two healthy volunteers. Finally, we applied the technique during magnetic resonance-guided focused ultrasound (MRgFUS) treatments to measure near-field heating in eight patients with uterine fibroids.Calibration experiments in porcine adipose tissue determined a temperature coefficient of 6.16 ms/°C during heating and 5.37 ms/°C during cooling. The volunteer experiments demonstrated a strong correlation between the skin temperature and T2-based temperature measurements in the fat layer. During the treatments of patients with uterine fibroids, we observed a measurable change in the T2 of fat tissue within the path of the ultrasound beam and a temperature increase of up to 15 °C with sustained heating of more than 10 °C.Our results demonstrate the feasibility and importance of monitoring near-field heating in fatty tissues. The implementation of near-field monitoring between sonications can shorten treatments by reducing the cooling time. It can help improve safety by avoiding excessive heating in the near field.
View details for DOI 10.1186/s40349-015-0036-5
View details for PubMedID 26366288
Respiration based steering for high intensity focused ultrasound liver ablation.
Magnetic resonance in medicine
2014; 71 (2): 797-806
Pain due to bone metastases is a common cause of cancer-related morbidity, with few options available for patients refractory to medical therapies and who do not respond to radiation therapy. This study assessed the safety and efficacy of magnetic resonance-guided focused ultrasound surgery (MRgFUS), a noninvasive method of thermal tissue ablation for palliation of pain due to bone metastases.Patients with painful bone metastases were randomly assigned 3:1 to receive MRgFUS sonication or placebo. The primary endpoint was improvement in self-reported pain score without increase of pain medication 3 months after treatment and was analyzed by Fisher's exact test. Components of the response composite, Numerical Rating Scale for pain (NRS) and morphine equivalent daily dose intake, were analyzed by t test and Wilcoxon rank-sum test, respectively. Brief Pain Inventory (BPI-QoL), a measure of functional interference of pain on quality of life, was compared between MRgFUS and placebo by t test. Statistical tests were two-sided.One hundred forty-seven subjects were enrolled, with 112 and 35 randomly assigned to MRgFUS and placebo treatments, respectively. Response rate for the primary endpoint was 64.3% in the MRgFUS arm and 20.0% in the placebo arm (P < .001). MRgFUS was also superior to placebo at 3 months on the secondary endpoints assessing worst score NRS (P < .001) and the BPI-QoL (P < .001). The most common treatment-related adverse event (AE) was sonication pain, which occurred in 32.1% of MRgFUS patients. Two patients had pathological fractures, one patient had third-degree skin burn, and one patient suffered from neuropathy. Overall 60.3% of all AEs resolved on the treatment day.This multicenter phase III trial demonstrated that MRgFUS is a safe and effective, noninvasive treatment for alleviating pain resulting from bone metastases in patients that have failed standard treatments.
View details for DOI 10.1093/jnci/dju082
View details for PubMedID 24760791
Respiration Based Steering for High Intensity Focused Ultrasound Liver Ablation
MAGNETIC RESONANCE IN MEDICINE
2014; 71 (2): 797-806
In vivo USPIO magnetic resonance imaging shows that minocycline mitigates macrophage recruitment to a peripheral nerve injury
PURPOSE: Respiratory motion makes hepatic ablation using high intensity focused ultrasound (HIFO) challenging. Previous HIFU liver treatment had required apnea induced during general anesthesia. We describe and test a system that allows treatment of the liver in the presence of breathing motion. METHODS: Mapping a signal from an external respiratory bellow to treatment locations within the liver allows the ultrasound transducer to be steered in real time to the target location. Using a moving phantom, three metrics were used to compare static, steered, and unsteered sonications: the area of sonications once a temperature rise of 15°C was achieved, the energy deposition required to reach that temperature, and the average rate of temperature rise during the first 10 s of sonication. Steered HIFU in vivo ablations of the porcine liver were also performed and compared to breath-hold ablations. RESULTS: For the last phantom metric, all groups were found to be statistically significantly different (P ? 0.003). However, in the other two metrics, the static and unsteered sonications were not statistically different (P > 0.9999). Steered in vivo HIFU ablations were not statistically significantly different from ablations during breath-holding. CONCLUSIONS: A system for performing HIFU steering during ablation of the liver with breathing motion is presented and shown to achieve results equivalent to ablation performed with breath-holding. Magn Reson Med 000:000-000, 2012. © 2012 Wiley Periodicals, Inc.
View details for DOI 10.1002/mrm.24695
View details for PubMedID 23460510
Rapid MR venography in children using a blood pool contrast agent and multi-station fat-water-separated volumetric imaging
2012; 42 (2): 242-248
Minocycline has proven anti-nociceptive effects, but the mechanism by which minocycline delays the development of allodynia and hyperalgesia after peripheral nerve injury remains unclear. Inflammatory cells, in particular macrophages, are critical components of the response to nerve injury. Using ultrasmall superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI) to monitor macrophage trafficking, the purpose of this project is to determine whether minocycline modulates macrophage trafficking to the site of nerve injury in vivo and, in turn, results in altered pain thresholds.Animal experiments were approved by Stanford IACUC. A model of neuropathic pain was created using the Spared Nerve Injury (SNI) model that involves ligation of the left sciatic nerve in the left thigh of adult Sprague-Dawley rats. Animals with SNI and uninjured animals were then injected with/without USPIOs (300??mol/kg i.v.) and with/without minocycline (50?mg/kg i.p.). Bilateral sciatic nerves were scanned with a volume coil in a 7?T magnet 7?days after USPIO administration. Fluid-sensitive MR images were obtained, and ROIs were placed on bilateral sciatic nerves to quantify signal intensity. Pain behavior modulation by minocycline was measured using the Von Frey filament test. Sciatic nerves were ultimately harvested at day 7, fixed in 10% buffered formalin and stained for the presence of iron oxide-laden macrophages. Behavioral measurements confirmed the presence of allodynia in the neuropathic pain model while the uninjured and minocycline-treated injured group had significantly higher paw withdrawal thresholds (p?0.011). Decreased MR signal is observed in the SNI group that received USPIOs (3.3+/-0.5%) compared to the minocycline-treated SNI group that received USPIOs (15.2+/-4.5%) and minocycline-treated group that did not receive USPIOs (41.2+/-2.3%) (p?0.04). Histology of harvested sciatic nerve specimens confirmed the presence USPIOs at the nerve injury site in the SNI group without minocycline treatment.Animals with neuropathic pain in the left hindpaw show increased trafficking of USPIO-laden macrophages to the site of sciatic nerve injury. Minocycline to retards the migration of macrophages to the nerve injury site, which may partly explain its anti-nociceptive effects. USPIO-MRI is an effective in vivo imaging tool to study the role of macrophages in the development of neuropathic pain.
View details for DOI 10.1186/1744-8069-8-49
View details for Web of Science ID 000309839300001
View details for PubMedID 22742763
MR Imaging-guided Cryoablation for the Treatment of Benign Prostatic Hyperplasia
JOURNAL OF VASCULAR AND INTERVENTIONAL RADIOLOGY
2011; 22 (10): 1427-1430
A rapid, reliable radiation-free method of pediatric body venography might complement US by evaluating veins in the abdomen and pelvis and by providing a global depiction of venous anatomy. We describe a MR venography technique utilizing gadofosveset, a blood pool contrast agent, in children. The technique allows high-spatial-resolution imaging of the veins from the diaphragm to the knees in less than 15 min of total exam time.
View details for DOI 10.1007/s00247-011-2254-5
View details for Web of Science ID 000301664100015
View details for PubMedID 21989981
In vivo MR acoustic radiation force imaging in the porcine liver
2011; 38 (9): 5081-5089
A patient with benign prostatic hyperplasia presented with chronic lower urinary tract symptoms despite prior surgery and continued medical therapy. Using a magnetic resonance imaging-guided transperineal approach, two cryoprobes were placed into the transition zone of the prostate gland, and two cryoablation freeze-thaw cycles were performed. At 10 weeks after treatment, the frequency of nocturia had decreased from once every 1.5 hours to once per night, urinary peak flow rates had increased from 5.1 mL/s to 10.3 mL/s, and postvoid residual urinary bladder volume had decreased from 187 mL to 58 mL. Improved flow rates and symptoms remained stable 16 weeks after treatment.
View details for DOI 10.1016/j.jvir.2011.08.010
View details for Web of Science ID 000295708400013
View details for PubMedID 21961982
Computed Tomographic Diagnosis of Appendicitis Within a Spigelian Hernia
JOURNAL OF COMPUTER ASSISTED TOMOGRAPHY
2010; 34 (2): 199-200
Early Sonographic Diagnosis of Intrauterine Device Migration to the Adnexa
JOURNAL OF CLINICAL ULTRASOUND
2009; 37 (7): 414-419
High intensity focused ultrasound (HIFU) in the abdomen can be sensitive to acoustic aberrations that can exist in the beam path of a single sonication. Having an accurate method to quickly visualize the transducer focus without damaging tissue could assist with executing the treatment plan accurately and predicting these changes and obstacles. By identifying these obstacles, MR acoustic radiation force imaging (MR-ARFI) provides a reliable method for visualizing the transducer focus quickly without damaging tissue and allows accurate execution of the treatment plan.MR-ARFI was used to view the HIFU focus, using a gated spin echo flyback readout-segmented echo-planar imaging sequence. HIFU spots in a phantom and in the livers of five live pigs under general anesthesia were created with a 550 kHz extracorporeal phased array transducer initially localized with a phase-dithered MR-tracking sequence to locate microcoils embedded in the transducer. MR-ARFI spots were visualized, observing the change of focal displacement and ease of steering. Finally, MR-ARFI was implemented as the principle liver HIFU calibration system, and MR-ARFI measurements of the focal location relative to the thermal ablation location in breath-hold and breathing experiments were performed.Measuring focal displacement with MR-ARFI was achieved in the phantom and in vivo liver. In one in vivo experiment, where MR-ARFI images were acquired repeatedly at the same location with different powers, the displacement had a linear relationship with power [y?=?0.04x?+?0.83 ?m (R(2)?=?0.96)]. In another experiment, the displacement images depicted the electronic steering of the focus inside the liver. With the new calibration system, the target focal location before thermal ablation was successfully verified. The entire calibration protocol delivered 20.2 J of energy to the animal (compared to greater than 800 J for a test thermal ablation). ARFI displacement maps were compared with thermal ablations during seven breath-hold ablations. The error was 0.83?±?0.38 mm in the S/I direction and 0.99?±?0.45 mm in the L/R direction. For six spots in breathing ablations, the mean error in the nonrespiration direction was 1.02?±?0.89 mm.MR-ARFI has the potential to improve free-breathing plan execution accuracy compared to current calibration and acoustic beam adjustment practices. Gating the acquisition allows for visualization of the focal spot over the course of respiratory motion, while also being insensitive to motion effects that can complicate a thermal test spot. That MR-ARFI measures a mechanical property at the focus also makes it insensitive to high perfusion, of particular importance to highly perfused organs such as the liver.
View details for DOI 10.1118/1.3622610
View details for Web of Science ID 000294482900019
View details for PubMedID 21978053
Ductal pattern enhancement on magnetic resonance imaging of the breast due to ductal lavage
2007; 13 (3): 281-286
Uterine perforation is an uncommon complication of intrauterine devices (IUDs). Perforating IUDs can migrate to various locations but paradoxically are rarely found in ovaries or broad ligament. We describe an unusual case of a 23-year-old woman 1-month postpartum with an IUD translocation to the right adnexa. The IUD was inserted only 1 week prior to presentation, and she experienced pain on insertion. After visualization by ultrasound, the IUD was laparoscopically removed. We suggest early use of ultrasound in cases of potential IUD migration, particularly in high-risk patients and when IUD insertion causes pain.
View details for DOI 10.1002/jcu.20591
View details for Web of Science ID 000269365100011
View details for PubMedID 19484740
General purpose, field-portable cell-based biosensor platform
BIOSENSORS & BIOELECTRONICS
2001; 16 (7-8): 557-564
Our purpose is to describe the appearance of breast ductal enhancement found on magnetic resonance imaging (MRI) after breast ductal lavage (DL). We describe a novel etiology of enhancement in a ductal pattern on postcontrast MRI of the breast. Knowledge of the potential for breast MRI enhancement subsequent to DL, which can mimic the appearance of a pathologic lesion, is critical to the care of patients who undergo breast MRI and DL or other intraductal cannulation procedures.
View details for Web of Science ID 000245992200010
View details for PubMedID 17461903
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
2001; 98 (15): 8469-8474
There are several groups of researchers developing cell-based biosensors for chemical and biological warfare agents based on electrophysiologic monitoring of cells. In order to transition such sensors from the laboratory to the field, a general-purpose hardware and software platform is required. This paper describes the design, implementation, and field-testing of such a system, consisting of cell-transport and data acquisition instruments. The cell-transport module is a self-contained, battery-powered instrument that allows various types of cell-based modules to be maintained at a preset temperature and ambient CO(2) level while in transit or in the field. The data acquisition module provides 32 channels of action potential amplification, filtering, and real-time data streaming to a laptop computer. At present, detailed analysis of the data acquired is carried out off-line, but sufficient computing power is available in the data acquisition module to enable the most useful algorithms to eventually be run real-time in the field. Both modules have sufficient internal power to permit realistic field-testing, such as the example presented in this paper.
View details for Web of Science ID 000171257900015
View details for PubMedID 11544049
Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta(2) adrenergic receptor
JOURNAL OF BIOLOGICAL CHEMISTRY
2001; 276 (27): 24433-24436
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
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
2001; 98 (11): 5997-6002
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
The effect of pH on beta(2) adrenoceptor function - Evidence for protonation-dependent activation
JOURNAL OF BIOLOGICAL CHEMISTRY
2000; 275 (5): 3121-3127
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
Examination of ligand-induced conformational changes in the beta(2) adrenergic receptor
1998; 62 (17-18): 1509-1512
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
Agonists induce conformational changes in transmembrane domains III and VI of the beta(2) adrenoceptor
1997; 16 (22): 6737-6747
The environmentally sensitive and cysteine reactive fluorescent probe, IANBD, was used to monitor ligand-induced structural changes in the beta2 adrenergic receptor (beta2AR) by fluorescent spectroscopy. We found that agonists caused a dose-dependent and reversible decrease in fluorescence from the purified IANBD-labeled beta2AR. This suggested that agonists promote a conformational change in the receptor that leads to an increase in the polarity of the environment around one or more IANBD labeled cysteines. The wildtype receptor contains eight free cysteines and mutagenesis and peptide mapping experiments have indicated that several of these sites are accessible for chemical derivatization. Thus, to identify the cysteine(s) involved in the agonist-induced change in fluorescence and thereby map agonist-induced conformational changes in the beta2AR, we generated a series of mutant receptors having limited numbers of cysteines available for fluorescent labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants showed that IANBD labeled 125Cys and 285Cys are responsible for the observed changes in fluorescence consistent with movements of TM III and VI in response to agonist binding.
View details for Web of Science ID 000072850800013
View details for PubMedID 9585127
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