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 cytoplamic 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 G?:G?:G? heterotrimers mediate the signal transfer;and the affected target proteins include adenylyl cyclase, phospholipase C?, 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. This project emphasizes continued efforts on three GPCR systems;mammalian neurotransmitter receptors for serotonin (5-hydroxytryptamine (5HT) notably receptor 5HT2c;the human glycoprotein hormone receptors, FSHR and LHR;and human chemokine receptors that are co-opted by HIV, CCR5 and CXCR4. Continued studies are also planned for other GPCRs. GPCRs are used widely in eukaryotic ells, and processes controlled by these receptors are relevant in human disease;notably, serotonin receptors are involved in disorders of mood such as depression, FSHR and LHR are crucial for human reproduction, and CCR5 and CXCR4 are implicated in HIV/AIDs. GPCRs are actual and prospective pharmaceutical targets.
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