Cells utilize relatively few second messenger molecules including cyclic nucleotides, calcium, diacylglycerol, phospho-inositides, and arachidonic acid metabolites to orchestrate responses to extracellular signals and integrate cellular mechanisms. Therefore, it is not surprising that one of these second messengers, cAMP, plays a role in virtually every complex cellular response and often interacts with other second messenger pathways. The goal of our research program is to develop and utilize molecular genetic approaches to answer questions about the mechanisms by which cAMP acts in mammalian cells. Cyclic AMP-dependent protein kinases (cA-PKs) are the major transducers of cAMP action and clones for 2 type I (RI) regulatory subunits, 2 type II (RII) regulatory subunits and 3 catalytic (C) subunits have been identified and studied extensively. Further work is proposed to (1) determine the sites of interaction between C and R subunits, (2) characterize potential intracellular localization signals in C, (3) inhibit formation of type II kinase in cultured cells and disrupt RII interactions with intracellular binding sites, (4) isolate novel R cDNA clones, (5) express wild type or mutant R and C subunits in specific tissues using transgenic mice, and (6) disrupt genes for RI-a, RI-beta, and C-beta by homologous recombination to test for physiological roles in vivo. The regulation of gene expression, peptide secretion, and steroid biosynthesis have all been shown to directly involve the cA-PKs and these specific cellular responses are emphasized in this proposal. In addition the neural specific isoforms of R and C subunits are of special interest and their role in neuronal regulation is explored in transgenic and gene-disrupted mice.
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