The overall objective of this research proposal is to investigate two essential aspects of the function and structure of the thyrotropin- releasing hormone receptor (TRHR) in pituitary cells. This receptor is a prototype for the class of 7 transmembrane receptors which couple to G(q/11) and regulate cytosolic free Ca2+ concentration and protein kinase C in target cells. For this class of receptors, the mechanism by which signalling is terminated and identification of the molecular interaction sites between agonist ligands and the receptor are much less well understood than for certain receptors that couple to Gs. The experiments proposed are made possible by the availability of several new reagents, including epitope tagged TRHRs, antibodies to the receptor, homologous cells which lack the receptor as recipients for transfection, specific G protein knockout strains of these cells, cells which express specific G protein receptor kinase (GRK) dominant negative mutations and overexpress specific GRK isoforms. In addition, this laboratory has extensive experience in the cell biology, physiology, biochemistry and pharmacology of the TRH-TRHR signalling system. The two specific aims are directed at answering questions that are essential in understanding the molecular mechanisms by which agonist binding regulates receptor internalization, desensitization, and initiation of signal transduction. Results of the first specific aim will define the pathway of TRHR internalization, and determine the roles of signalling and heterotrimeric G proteins in internalization and intracellular trafficking of the TRHR. Experiments to define signal termination (desensitization) will investigate the role of internalization using new methods to induce internalization independent of signalling, and will determine whether and how covalent modification of the TRHR occurs during desensitization. The second specific aim derives from our recent identification of Asn289 in extracellular loop 3 of the receptor as a direct contact site for the pGlu N-H of TRH, and the development of a new model for the agonist binding domain of the TRHR. The proposed experiments will test this model by investigating the interactions of Ser29O with pGlu, of the His residue in TRH with aromatic residues in extracellular loop 2, of the -ProNH2 with Tyr181, and finally to test a new proposal which can explain the importance of Tyr106. Results of the proposed experiments will fill major gaps in current knowledge and will have significance because they will yield new insights into the mechanisms of signal termination and receptor activation for a physiologically important neuropeptide receptor, and probably for other Gq-coupled receptors as well.
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