I propose to study the mechanism of regulation of G-protein-linked receptors (GPLR); the structural determinants of the C-terminus of GPLR enabling counterregulation by tyrosine kinases; and the organization of GPLR complexes involved in signaling. The proposal focuses upon the C-terminal, cytoplasmic """"""""tail"""""""" of the mammalian beta2-adrenergic receptor (beta2AR), approaching the analysis of its structure and function as a discrete domain. (1) We will define the structural determinants by which tyrosine kinases (e.g., insulin receptors) phosphorylate the beta2AR and provoke protein-protein interactions with adaptor molecules and enzymes relevant to counterregulation of the beta2AR by insulin. The ability of counterregulatory growth factors to stimulate sequestration of the beta2AR will be tested using mutagenesis in parallel with phosphorylation as well as confocal microscopy with the GFP-beta2AR. (2) We will analyze the macromolecular nature of beta2AR in complexation with protein kinases, phosphatases, enzymes, adaptor and scaffold proteins (as shown for AKAP 250). Dynamic association will be tested under conditions of activation, desensitization, and sequestration exploiting protein-protein interactions amenable to co-immuneprecipitation and/or isolation to immobilized affinity matrices. Functional roles for proteins in complex with beta2AR will be tested using several complementary approaches including elimination of target proteins by antisense technology, expression of fragments of the domain or protein of interest as possible inhibitors/activators, and via mutagenesis of specific domains of the target protein. (3) We will express the C-terminal tail in E.coli in sufficient quantities to allow its detailed analysis as a substrate for protein phosphorylation by several kinases and in mammalian cells to test the ability of this region (or specific fragments) of the beta2AR to alter signaling. In addition, we will collaborate on large-scale isolation, crystallization, and X-ray analysis of the C-terminal domain. Complementary studies performed with epifluorescence confocal microscopy and antibodies specific to complex elements and/or GFP-tagged fusion proteins of interest will complement and test further the data derivative of biochemical and cell biology studies on the role of the C-terminus in the regulation of beta2AR by counterregulatory growth factors.
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