The intent of this proposal is to test our hypothesis that cAmp- dependent protein kinase (PKA) mediates the rapid physiological heterologous desensitization of hormonal stimulation of adenylate cyclase in the S49 wild type (WT) lymphoma cell line. Previous work had demonstrated only a homologous desensitization in response to relatively high hormone concentrations, which was independent of Gs' cAMP, and PKA. We have recently found that cAMP activation of PKA plays a crucial role in the heterologous desensitization of the beta-adrenergic receptor (beta-AR) and the prostaglandin E1 (PGE1) receptor in WT cells in response to physiological concentr- ations of epinephrine or PGE1 (0-20 nM). Use of Mg2+ levels in our adenylate cyclase assays which mimic intracellular concentrations (0.1-0.5 mM) revealed: 1) heterologous desensitization of the WT adenylate cyclase, but not that of cyc or kin cells, and 2) that addition of dibutyryl cAMP along with epinephrine to the cyc cells caused heterologous desensitization. We will pursue a genetic-biochemical approach using the S49 WT and mutant cells to carry out the following specific aims: 1) characterization of the heterologous desensitization of the WT cells and derived clones deficient in either adenylate cyclase or PKA in response to the treatment of these cells with 0-20 nM PGE1 or epinephrine, with cAMP analogues and forskolin, and with the hormones in combination with these agents; 2) elucidation of conditions for cell-free heterologous desensitization of adenylate cyclase, and use of this system to examine the effects of purified PKA and phosphatases on the phosphorylation-dephosphorylation of the beta-AR and the correlation with heterologous desensitization- resensitization; 3) characterization of the phosphorylation of the beta-AR in response to treatment of intact cells with low concentrations of hormones and forskolin; and 4) determination of the generality of the role of PKA in the heterologous desensitization of receptors which stimulate adenylate cyclase in mammalian cell lines.
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