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Cyclic AMP (cAMP), a prototypic second messenger, is involved in a plethora of physiological responses (i.e. the control of heart rate and locomotion) and in certain pathophysiology conditions (i.e. drug abuse and addiction). Adenylyl cyclase synthesizes cAMP and its activity is the key step in regulating cAMP level. Hormones and neurotransmitters bind heptahelical receptors to activate G proteins. An activated G protein can release both alpha and beta-gamma subunits; both can regulate adenylyl cyclase activity. Mammalian adenylyl cyclase has at least nine isoforms (AC1-AC9). Each isoform differs significantly in its protein sequence ( about35-70 percent of sequence similarity), tissue distribution, and pattern of regulation. The long-term goal of this project is to elucidate the underlying molecular mechanism in the regulation of mammalian adenylyl cyclases. All nine isoforms share a common structure, including two highly conserved domains (C1a and C2a) connected by the less conserved C1b and transmembrane domains. C1a and C2a form a soluble, Gs-alpha-sensitive enzyme. This soluble enzyme model has been used to identify the sites for catalysis and for binding of Gs-alpha forskolin, and Gi-alpha. This soluble enzyme has also been used to elucidate the molecular structure of activated state of adenylyl cyclase (C1a and C2a with forskolin and Gs-alpha). The model suggests that the regulation of adenylyl cyclase is mediated by domain interaction of C1a/C2a and domain interaction between Gs-alpha and C1a/C2a. To test this hypothesis, the Gs-alpha-activated state needs to be compared with the inactive state and with one that has higher activity than the Gs-alpha activated state. The soluble AC2 and AC7 models that can better represent membrane-bound adenylyl cyclase will be constructed. This model will include the C1b region that is important in regulating adenylyl cyclase activity. G protein beta-gamma subunit potentiates Gs-alpha activated AC2 and AC7 5-20 fold. The C1a/C2a/C1b complex of AC2 and AC7 will represent the basal state. Gs-alpha/C1a/C2a will be the activated state and Gs-alpha/G-beta gamma/C1a/C2a/C1b complex will represent the enzyme state that has higher activity than that activated by Gs-alpha. All three models will be analyzed by biochemical and structural analysis. Success in our analysis will not only provide a framework for understanding how G proteins regulate their effectors but also offer a model in analyzing how domain interaction between proteins and within a protein alters their biological activity. The C-terminus of adenylyl cyclase, C2b, is poorly conserved and it may provide an isoform-specific signature sequence to interact with other signaling molecules. AC9 C2b has the consensus sequence for the binding of PDZ domains. Thus AC9 C2b interacting proteins have been screened by yeast two-hybrid and by hypothesis-driven bioinformatics searches. Two candidates have been identified and they are involved in modulating programmed cell death and activation of MAP kinase pathway. Experiments will be performed to test whether AC9 interacts with these two proteins and whether their interaction leads to regulation of apoptotic and MAP kinase pathways. Success in our search will provide novel ways for crosstalk between adenylyl cyclase and other signaling pathways.

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