The long-term objectives of this proposal are to understand the molecular mechanisms in the regulation of acetylcholine muscarinic receptor (mAChR). Specifically, the aims are to elucidate the mechanisms of protein phosphorylation and dephosphorylation of mAChRs by Ca2+-mediated process involving calmodulin (CaM), calcineurin and cyclic nucleotide (cAMP) and the GTP-binding proteins. It is postulated that the decrease in receptor responsiveness is dependent on the state of receptor phosphorylation- dephosphorylation of the receptor resulting in loss of specific binding affinity to radioligand leading to sensitivity decrease or desensitization. The receptor recovers from such loss by a process of dephosphorylation. Receptor phosphorylation may be mediated by Ca2+/CaM, c-AMP-dependent, C-kinase or other protein kinases. Receptor dephosphorylation may be mediated by the Ca2+-dependent phosphatase in the membrane such as calcineurin, leading to a reversal of specific receptor binding loss. Data obtained from preliminary studies suggest a role in Ca2+/CaM mediation of mAChR binding dependent on the state or receptor phosphorylation-dephosphorylation; Ca2+/CaM may regulate receptor function during senescence. Using solubilized and purified mAChR from brain synaptic membrane labeled with (3H)-PrBCM and/or (gamma-32P)-ATP, synaptic membrane, it was further demonstrated that the mAChR was phosphorylated by cAMP-dependent protein kinase catalytic subunit and the (32P)- mAChR was dephosphorylated by calcineurin. Experiments are designed here to evaluate the alterations in mAChR functions using membrane bound, detergent solubilized and affinity chromatography purified receptors from the brains of rats and mice and in cultured neuroblastoma cells. The contents of membrane CaM calcineurin; protein kinase, and phosphatase activities will be measured. The solubilized and/or purified receptors will be reconstituted into liposomes and the interactions of Ca2+/CaM, calcineurin and GTP-binding proteins will be studied. mAChR-mediated responsiveness as determined by phosphoinositide hydrolysis, adenylate cyclase and PDE activities will be measured. The results obtained will provide important information on the regulation of receptor function by the phosphorylation-dephosphorylation mechanisms.
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