Regulation of intracellular cyclic AMP concentrations is principally controlled at the level of its synthesis, through the hormonal regulation of adenylylcyclase, the enzyme responsible for the conversion of ATP into cyclic AMP. The adenylylcyclase system is comprised of three components: heptahelical, G protein-coupled receptors for a variety of hormones, neurotransmitters, and autocoids; heterotrimeric G proteins; and the catalytic entity itself. The G proteins regulate the activity of the enzyme in response to the interaction of ligands with an appropriate receptor. This molecular architecture is common to all G protein regulated effector systems identified to date and include hormone regulated phospholipases and ion channels, as well as the light activated cyclic GMP phosphodiesterases. Central to the regulation of adenylylcyclase, or any of the other effector molecules, is the specificity of the G proteins to couple an appropriate receptor to the correct effector. An additional level of regulation is achieved by the cross-talk of different G protein-coupled effector systems, often mediated by the action of downstream protein kinases. The recent cloning of multiple isoforms of adenylylcyclases has permitted the biochemical demonstration that these cyclases are regulated by G proteins and protein kinases in an isoform specific fashion. The two aims of this project are: 1) to determine the precise molecular signals underlying the specificity of recognition of adenylylcyclase by G proteins, and 2) to characterize the role of phosphorylation in the regulation of adenylylcyclase isoforms. Two genetic system (based on the expression of mammalian adenylylcyclases and G protein subunits in yeast) will be used to select for regulatory mutants of both adenylylcyclases and G protein subunits. Isolation of mutant defective in coupling G protein subunits to adenylylcyclases will allow to probe the molecular basis of G protein recognition and regulation of adenylylcyclase. In addition, in vitro and in vivo approaches will be used to elucidate the regulation of specific adenylylcyclase isoforms by phosphorylation. These studies will have a significant impact on the understanding of the mechanisms underlying the regulation of effector systems by G proteins and the intricate cross-talk among different hormone regulated signaling pathways.
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