Cell surface receptor-ligand recognition and activation in systems relevant to human health and disease
Cell surface receptors represent the gateway through which the cell senses and responds to its environment. Most physiologically important processes are initiated by the interaction of cell-surface receptors with extracellular mediators. This recognition event is communicated across the membrane, resulting in activation of intracellular signal transduction cascades. Molecular insight into recognition and activation of receptors implicated in human disease could reveal new strategies, or better inform current strategies, for therapeutic intervention using protein engineering. In this way, our structural and mechanistic studies inform translational efforts to exploit this information into the therapeutic realm. We view structural information on receptor-ligand complexes as molecular blueprints to then allow us to penetrate the biology of these systems for both basic and practical advances.
Thematically, we are interested in shared receptors that appear to differentially respond to multiple ligands, and protein-protein interaction systems that play central roles in Immunology, Neurobiology, and Development. In addition to understanding basic aspects of receptor signaling and structural biology, we are also interested in the creation of novel receptor ligands using protein engineering, and deorphanization of new ligand-receptor systems using proteomics. A major forward thrust of our laboratory is to synthetically leverage natural biological signaling mechanisms in such a way as to alter and manipulate cell fate and function.
We approach our studies using a range of methodologies including protein biochemistry, protein engineering, combinatorial biology, X-ray crystallography, proteomics, cell biology, electron microscopy and a wide range of in vitro and in vivo translational approaches.
We have a long-standing interest in molecules that mediate, regulate and intersect with the antigen-specific and cytokine-driven phases of cellular and humoral immunity. We are interested in how receptors in these systems contribute or mediate diseases such as autoimmunity and cancer,
In the antigen-specific phase of Immunity we are interested in how both alpha-beta and gamma-delta T cell receptors recognize and signal in response to their self and foreign ligands, comprising both MHC and non-MHC antigens. A question that is currently of high interest in the lab is the relationship between the structural mode of TCR/MHC interaction to signaling and cross-reactivity. We are approaching this question using novel methods of peptide ligand discovery based on yeast surface display of peptide-MHC, which allows us to quantify TCR cross-reactivity. This system is also being applied to the identification of endogenous peptide ligands for “orphan” TCRs that are important in immune disease, such as autoimmune TCRs, TCRs resident on tumor infiltrating lymphocytes, and protective TCRs expanded during viral infection.
T cell and B cell homeostasis is largely determined by the actions of cytokines. We are interested in how shared cytokine receptors, such as gp130, common gamma chain, Interferons, and Interleukin-17 receptors are activated by a range of structurally diverse Interleukins and cytokines, and subsequently communicate intracellular signals to membrane-proximal second messengers in a context-dependent fashion. We have an emerging effort to engineer cytokines with novel signaling activities using combinatorial biology in order to better understand the relationship between extracellular receptor-ligand complex architecture, and intracellular signaling potency.
Stem cell biology and regenerative medicine share an intimate relationship with factors that control Immune homeostasis. We have several programs underway to investigate molecular aspects of receptor-ligand systems that control lineage decisions of stem cells, and influence stem cell engraftment.
A relatively new area involves a collaboration with the group of Professor Irv Weissman to investigate molecular aspects of macrophage phagocytosis mediated by the “don’t eat me” signal. The CD47/SIRP receptor-ligand axis serves as a toggle switch that, on one hand, enables macrophages to destroy cancer cells. However, this same mechanism results in the destruction of newly engrafted stems cells, which is a major problem for bone marrow transplantation. We are attempting to use structure and engineering to toggle the “don’t eat me” signal on or off, depending on the desired therapeutic endpoint.
In keeping with the theme of receptor-ligand pleiotropy and cross-reactivity, we are attempting to make inroads into understanding the molecular basis of signaling in the Wnt/Frizzled and Notch systems, which are sometimes referred to as the Yin/Yang of developmental biology. These powerful receptor-ligand systems have so far eluded therapeutic control, and we aim to exploit structural information on Wnt and Notch complexes to engineer new therapeutic molecules as well as dissect basic developmental biology.
Wnts have diverse roles in governing cell fate, proliferation, migration, polarity, and death. In adults, Wnts function in homeostasis, cell fate determination and stem/progenitor self-renewal, and inappropriate activation of the Wnt pathway is implicated in a variety of cancers. Mammals encode 20 Wnts and 10 Frizzled receptors, yet the nature of Wnt/Fz specificity and its importance in function remains an elusive question. Furthermore, Wnts can signal through a variety of receptors to generate canonical (beta-catenin) or non-canonical downstream signals. While the intracellular signaling modules and their interactions have been well characterized, the nature of the extracellular structures and their interactions has remained almost a complete mystery. We have determined a structural basis for how Wnt engages a Frizzled receptor, and we continue to seek a higher level of understanding of this process in order to design Fz sub-type specific ligands. We hope that we can then utilize this information to carry out functional studies aimed at gleaning the role of specific Wnt/Fz pairs in different diseases, and to perhaps harness this system for therapeutic applications in regenerative medicine.
Similarly, Notch receptors see a diverse milieu of ligands (e.g. Jagged, Delta-like, etc) that control tissue growth and differentiation. Based on structural information for how Notch sees its ligands determined in this laboratory, we are now attempting to engineer Notch ligands to controllaby activate Notch signaling in a sub-type specific manner.
One of the most compelling questions in receptor biology is whether, and how, the structural and chemical features of a receptor-ligand complex can qualitatively and/or quantitatively influence downstream signaling.That is, does extracellular structure matter ? or is intracellular signaling agnostic to the structural and geometric details of the extracellular engagement event ? Most transmembrane receptors we study, such as cytokine and growth factor receptors, are activated through some form of ligand-induced dimerization. This begs the question, will any dimer do ?
We are using protein engineering approaches, namely combinatorial biology methods such as yeast surface display to interrogate fundamental mechanistic questions about receptor signaling and molecular recognition. This involves a synergy with our structural targets to guide development of molecules with interesting signaling, functional and therapeutic properties. We are primarily integrating this approach into our studies of T cell receptor and cytokine receptor signaling.
A large fraction of cell surface receptors and secreted ligands encoded in the human genome do not have known binding partners. A pilot study in our laboratory established a robust methodology for screening pairwise receptor-ligand interactions, and we discovered many new receptor-ligand pairs in Drosophila melanogaster (Ozkan et al., Cell 2013; 2014). We are now embarking on a new program to map all cell surface interactions encoded in the human genome -- interactions between cell surface proteins expressed on opposing cells, as well as, interactions between cell surface proteins and secreted molecules. To this end, we are employing a variety of parallel screening platforms, using assays we developed, to perform high-throughput receptor-ligand screens. While the primary focus of our near-term efforts is to discover new receptor-ligand interactions, our long term vision is to pursue translational efforts aimed at exploiting these cell surface interactions which have the potential to hold broad therapeutic value across multiple disease states. The generation of a database of all receptor-ligand interactions encoded in the human genome will not only have immediate practical value for drug discovery but will also be an invaluable resource for the biomedical and basic science research communities.