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Structural Biology of Cell Surface Receptor Recognition and Activation
Summary: K. Christopher Garcia studies the structure and function of cell surface receptor recognition and activation, in the immune and nervous systems. His laboratory uses structural biology, protein engineering, combinatorial biology, and proteomics to interrogate receptor activation mechanisms and discover new ligand-receptor interactions.
Extracellular information is communicated to intracellular mediators by activation of membrane-embedded proteins, called receptors, on the cell surface. Receptors represent the gateway through which the cell senses and responds to its environment. Most physiologically important processes are, at some level, initiated by the engagement of an extracellular molecule with the extracellular regions of cell surface receptors in a highly specific fashion, in order to activate intracellular signal transduction cascades and subsequent genetic programs. Cell surface receptors are also the major therapeutic entry points for drug development by the pharmaceutical industry.
My laboratory is investigating structural and functional aspects of cell surface receptor recognition and activation, in receptor-ligand systems with relevance to human health and disease. Our goal is to paint a detailed mechanistic picture—from the outside to the inside of a cell—of how ligand binding is structurally coupled to receptor activation. How is the static aspect of recognition linked with the dynamic aspects of activation? We hope to combine this structural and mechanistic information with protein engineering to modulate the strength and specificity of downstream intracellular signaling.
The mechanisms of cell surface receptor activation, which are highly diverse, usually involve some combination of ligand-induced oligomerization and conformational change. Yet, we currently have an appreciation for only a small fraction of the potential receptor-ligand interaction paradigms across the human genome. We have endeavored to characterize novel receptor activation mechanisms by determining crystal structures of extracellular receptor-ligand complexes. The structural studies have been complemented by companion biochemical, functional, and engineering experiments to probe the relevance of the structural information to the proteins in their native environments.
We have described new paradigms for recognition and activation of a range of cell surface receptors that mediate adaptive immunity and neural signaling. Examples include cytokine receptors, vasoactive hormone receptors, neurotrophin receptors, semaphorin receptors, and T cell receptors (TCRs). These receptors play critical roles in immune regulation (e.g., IL-2, IL-4), cancer (gp130, LIFR), autoimmunity (TCR/MHC), blood pressure regulation (ANP), neural growth and repair (neurotrophin, GDNF, and Nogo receptors), and axon guidance (semaphorin/plexin). My lab continues to investigate the diversity of receptor activation mechanisms by targeting receptor systems for which structural information does not exist. Our long-term goal, however, is to use several approaches to probe more deeply the systems for which we have gained structural insight. In one approach we are attempting to use mechanism-based ligand-engineering strategies as well as combinatorial ligand libraries to modulate receptor activation. In a second approach, we are carrying our studies into the membrane, to examine entire receptor molecules fully loaded with both extracellular ligand and intracellular adapter molecules as intact membrane proteins. Finally, we are increasingly using proteomic strategies to identify ligands for "orphan" receptors.
Thematically, my laboratory focuses on "shared" receptors—receptors that are activated by a variety of structurally diverse ligands and give rise to both unique and redundant signaling outcomes. We are particularly interested in shared receptors at the interface between immunity, neurobiology, and microbial pathogenesis. As one example, the shared cytokine receptor gp130 is a signaling receptor for more than 10 different cytokine ligands with critical functions in both the immune and nervous systems. What is the structural basis for this cross-reactivity, and how can the engagement of the different cytokines lead to qualitatively different signaling outcomes? So far, our studies have shown that gp130 cross-reacts with different ligands through a uniquely accommodating surface chemistry in its structurally rigid binding site, rather than through conformational change. It also appears that ligand recognition by gp130 is coupled to a bending of the receptor as it enters the membrane, in order to position the intracellular segments optimally for signaling. Our ongoing studies of the gp130 system include incorporating a second shared receptor, LIFR (the leukemia inhibitory factor receptor), into the heterodimeric signaling complex with gp130 to determine the basis for assembly of an asymmetric signaling complex. We are also interested in visualizing, potentially through electron microscopy, the architecture of the entire transmembrane gp130 receptor in both quiescent and activated forms.
An important example of receptor sharing in the nervous system involves semaphorins, which can act as ligands for both neuropilin and plexin receptors in order to mediate repulsive signaling during axonal growth. However, semaphorins are also active in both the immune and nervous systems, where interactions with plexins mediate a common activity of inducing cell homing and pathfinding through the shared receptor plexinC1. In fact, viruses express mimics of semaphorins in order to interfere with immune semaphorins and enhance the virus survival. We carried out a series of mechanistic and structural studies of both human and viral semaphorins complexed with the shared receptor, plexinC1. The mammalian and viral Sema proteins engaged plexinC1 in nearly identical fashions, inducing plexinC1 into an activated dimer for signaling. The basis of the cross-reactivity for plexinC1 appears to be that the virus hijacked the mammalian Sema and evolved the plexin-binding site so as to increase its affinity and "out-compete" the human Sema for receptor engagement.
My lab also has a long-standing interest in structural aspects of T cell recognition of peptide-MHC (major histocompatibility complex). Currently we are focused on the apparent ability of TCRs to react with any MHC molecule. This property raises the question of whether there is a germline-derived recognition code between TCR and MHC molecules that has so far been elusive. To determine whether conserved "footprints" might emerge from several structures of highly related TCR-pMHC complexes, we are studying TCRs derived from transgenic mice that utilize only a restricted subset of TCR variable-region genes. We are also developing a novel methodology to use yeast-displayed peptide-MHC libraries to discover new TCR ligands. In tandem, we are attempting to reconstitute a functional TCR signaling complex on insect cells to assess the contribution of T cell coreceptors to this recognition process.
Finally, although we have made significant progress in structural studies of receptor extracellular domain complexes, our foray into membrane proteins is a new challenge. The vast majority of receptors are multiple transmembrane helix proteins that must be studied in a lipid environment. Therefore, we are beginning to attempt structural studies of multipass membrane proteins.
This work is also supported by grants from the National Institutes of Health.
As of May 30, 2012
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