CYCLIC NUCLEOTIDE--RECEPTOR INTERACTIONS

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The long range goal of this research is to understand the molecular mechanisms by which cyclic nucleotides regulate cell function through interaction with diverse receptors. This project will establish the cAMP-binding domains of cAMP-dependent protein kinase (cAMP kinase) as model systems for examining cyclic nucleotide-receptor interactions in related proteins such as the cGMP-dependent protein kinase (cGMP kinase) and cyclic nucleotide-gated cation channels. Features of the type Ialpha regulatory subunit (RIalpha) of mammalian cAMP kinase will be identified that make it highly selective for cAMP binding as opposed to cGMP binding. The contribution of the carboxyl terminus of RIalpha to selective high affinity binding of cyclic nucleotides will be determined. An isolated cAMP-binding domain of cAMP kinase will be developed as a simple model for studying cyclic nucleotide receptor structure and function. Finally, the cAMP/cGMP-binding selectivities of the A and B domains of the yeast R subunit will be characterized. Many of the proposed experiments have been designed based on comparisons of molecular models, amino acid sequence alignments, and cyclic nucleotide-binding properties of cAMP kinase and cGMP kinase cyclic nucleotide-binding domains. Point mutations or truncations will be introduced into the RIalpha cAMP-binding domains by oligonucleotide-directed mutagenesis of the RIalpha cDNA. Recombinant R subunits will be produced in Escherichia coli, purified and characterized for their cyclic nucleotide-binding properties. specific residues in cAMP kinase (which binds cAMP with 200- fold greater affinity than cGMP) will be changed to the corresponding residues in the homologous cGMP kinase, which binds cGMP with 270-fold greater affinity than it binds cAMP, with the ultimate objective of changing cAMP kinase into an enzyme that is selectively activated by cGMP. Isolated cAMP-binding domains of RIalpha will be generated either by proteolytic digestion of the intact protein, or de novo expression of a recombinant isolated cAMP-binding domain. Wild-type and mutant yeast R subunits will be produced in bacteria. Since cAMP kinase and cGMP kinase are both involved in regulating cardiovascular function, detailed knowledge of the structural determinants that confer cyclic nucleotide selectivity to each kinase will be invaluable for the rational design of drugs that may be useful in treating cardiovascular disease.