The overall objective of this proposal is to develop an atomic-level understanding of transmembrane signal transduction by G-protein coupled receptors (GPCRs). These receptors are imbedded in the plasma membrane where they can sense specific external signals and transmit this information to associated cytoplasmic heterotrimeric G proteins, which can then mediate changes in the activity of target proteins. Signals detected by GPCRs include neurotransmitters, hormones, light, and odorants; a combinatorial variety of Galpha: Gbeta: G7 heterotrimers mediate the signal transfer; and the affected target proteins include adenylyl cyclase, phospholipase C-beta, and ion channels. The ultimate aim of the proposal is to use x-ray crystallography to determine structures for GPCRs in relevant states, including complexes with natural signaling ligands, with pharmacological agonists and antagonists, and with signaling partner proteins, notably heterotrimeric G proteins. Hypotheses inspired by these structures will be tested through the analysis of site-directed mutant variants, complexes with ligand analogs, and cellular assays of function. Since the crystallographic analysis of membrane protein structure is not routine, methods for the expression cloning, purification, biochemical characterization, complexation with stabilizing ligands, and ultimate crystallization of relevant GPCRs will be developed as required. The primary emphasis in this study will be on mammalian neurotransmitter receptors for serotonin, 5-hydroxytryptamine (5HT), but applications to other GPCRs are also envisioned. GPCRs are used widely in eukaryotic cells. Several processes controlled by these receptors are relevant in human disease, e.g. serotonin receptor involvement in disorders of mood such as depression, and GPCRs provide the sites of action for a number of pharmaceutical reagents.
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