This is a proposal to continue studies on the structure, function, and regulation of the CCK-A receptor. The general hypothesis is that the confirmation, post-translational modifications, and cellular compartmentation of this molecule are in dynamic states in the living cell, and that these processes are ligand and cell-dependent. The investigator proposes that receptor activation is conformationally determined, and proposes to use affinity labeling and receptor mutagenesis to explore athe receptor domains occupied by agonists and antagonists. A second proposal is that post-translational modifications of the receptor provide a secondary level of change, which occurs over time in a ligand-dependent manner, which contributes to direct cellular handling of the receptor. This work will focus on receptor phosphorylation and fatty acid acylation in pancreatic acinar cells and cells transfected with the CCK-A receptor. Finally, it is proposed that the cell possesses multiple distinct mechanisms for desensitization and resensitization to hormonal stimulation. Cellular compartments of desensitization such as clatharin-coated endocytic compartments, caveceolae, and a newly described compartment of insulation will all be examined and related to state of receptor phosphorylation. Together, these studies will provide insight into the physiology of G- protein coupled receptors with the CCK receptor as a model, and as an extension of previous work exploring desensitization uncoupling, sequestration and downregulation of the CCK receptor that were initiated in the previous grant cycle.
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