The goal is to develop a detailed understanding of the molecular mechanisms involved in desensitization of cell surface signal transduction systems that use G-proteins. For the coming term two major issues in desensitization will be addressed: The role of post-receptor sites as the loci for the desensitizing effects, and the multiplicity of distinct mechanisms that are capable of producing a similar response. The first issue will be experimentally addressed by studying the catalyst of adenylyl cyclase as target. These studies will be conducted on the liver and S49 murine lymphoma cell adenylyl cyclases. We hope to establish that phosphorylation of the catalyst by the cAMP-dependent protein kinase results in decreased sensitivity to Gs. This done both in vivo and in cell free studies using purified components. We will also study the role of C- kinase in sensitizing the catalyst and whether such sensitizing results in a blockade of the PKA effects. The post-receptor effects will also be studied at the level of G-proteins. For these studies we have chosen three tissues/cells that show very differing time courses for the onset of desensitization: chick hepatocytes, the MDCK cell and the luteinized rat ovary. All three systems show decreased Gs mediated stimulation of adenylyl cyclase, but the molecular basis may be different in the various systems. This will be analyzed by activity measurements, quantitative immunoblotting of the protein levels of various G alpha and G beta gamma subunits, as well as mRNA level determination by solution hybridization and transcriptional run-on assays. We will also use the muscarinic receptor evoked C1- current in Xenopus oocytes to address the relative contribution of the various components of system to produce the desensitizing response. In the oocyte we will analyze changes at the cell surface signal machinery as well as intracellular sites (the IP3 regulated Ca2+ store) in the development of the desensitized state by intracellular injection of activated alpha-subunit, IP3 or Ca2+ to identify the locus of change. This analysis will be done as a function of time of agonist exposure to test, if for differing times of exposures, differing mechanisms may be involved. From these studies we hope to ascertain the relative importance of changes at G-protein and effector levels in the modulation of cellular responsiveness and provide useful information for the development of drugs that would bypass the cell surface receptors to have their effect on cellular metabolism.
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