The objective of the research proposal is to understand the regulation of adenosine receptor signal transduction. These mechanisms could be fundamental in the roles of adenosine as a cardioprotector and a smooth muscle and endothelial cell mitogen. A1 and A2a adenosine receptor subtypes have opposite effects on adenylyl cyclase. Subtypes also appear to have distinct desensitization profiles. These features will be exploited in designing canine A1/A2a receptor chimeric receptors in whiCh the intracellular domains thought to regulate G protein coupling and desensitization have been switched from one subtype to the other. The chimeric receptors will be compared to wildtype recombinant receptors using a number of different experimental approaches to determine G protein coupling selectivity and desensitization patterns. Classic indirect markers for coupling to particular G proteins, such as alterations in high affinity ligand binding in the presence of Gi-selective uncoupling agents, and changes in adenylyl cyclase responses, will be supplemented with newer, more direct methodologies based on purification of receptor-G protein complexes. The G proteins associated with the purified complexes will be identified and quantitated using subtype specific antibodies. Protein phosphorylation is thought to play an important role in agonist- induced receptor desensitization. The kinetics of desensitization for chimeric and wildtype adenosine receptors will be compared in CHO cells and related to the extent and location of phosphorylation of the receptors and their associated G proteins. Changes in native receptor mRNA levels and G protein mRNA levels during desensitization will be assayed using solution hybridization. Immunocytochemistry will be used to identify changes in the localization of G protein subunits following adenosine receptor stimulation. Finally, using ligand binding and cAMP assays, the chimeras will be used to explore the mechanism of action of compounds which are allosteric enhancers of agonist ligand binding to A1 adenosine receptors.
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