Adenylyl cyclases are central to one of the most important transmembrane signal transduction pathways in virtually all biological systems and constitute a family of isozymes catalyzing the formation of adenosine 3': 5'-monophosphate (cAMP) from 5'ATP. Mammalian forms of the enzyme are stimulated or inhibited by hormones via G-protein-linked cell-surface receptors and are inhibited by intracellular adenine nucleoside 3'-phosphates via an enzyme conformation that is distinct from that through which catalysis occurs. The proposal is based on observations made with and unique applications for conformation-specific inhibitors of adenylyl cyclases we have synthesized: a) competitive inhibitors targeting the pre-transition, catalytically competent conformation, exemplified by b-L-2',3'-dideoxyadenosine-5'-triphosphate (IC50 about 24nM), and b) non-competitive inhibitors targeting the post-transition product leaving conformation, exemplified by 2',5'-dideoxyadenosine-3'-triphosphate (IC50 about 40nM). The goals of the proposed investigations include: i) to use these ligands and derivatives of them to define structural differences between pre- and post-transition configurations of adenylyl cyclase and differences among selected wild type and mutated adenylyl cyclase isozymes, by biochemical and biophysical techniques; ii) to establish in intact cells and tissues the consequences of adenylyl cyclase inhibition by prodrug derivatives of these nucleotides; iii) to identify and quantify naturally occurring adenine nucleoside 3'-polyphosphates and to identify the pathways of their synthesis and degradation; and iv) to isolate other enzymes through which they may act to alter cell function. The proposed approaches build on the applicant's experience with adenylyl cyclases, with its regulation by adenine nucleotides, and with the synthesis of unique, labeled and unlabeled nucleotide derivatives targeted to specific configurations of this key transmembrane signal transduction enzyme. The proposed studies will provide unique ligands and approaches to address key questions regarding the structure and regulation of adenylyl cyclases, will establish previously unexplored links between cell-initiated regulation of adenylyl cyclases and 3'-nucleotide metabolism, and will lead to the identification of other target proteins with which adenine nucleoside 3'-polyphosphates interact and regulate.
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