The basic mechanisms by which many hormones, neurotransmitters, odorants, and chemokines control physiological events in health and disease ar fundamental responses of all eukaryotic cells: The receptors for these cell-cell signaling molecules activate heterotrimeric G-proteins that regulate effectors, including adenylyl cyclases. Members of the superfamily of adenylyl cyclases, including nine mammalian subtypes as well as the D. discoideum enzyme, ACA, share a twelve transmembrane, dual cytoplasmic domain topology. I is proposed that enzyme activity is controlled by formation of a """"""""heterodimer' of the cytoplasmic domains. The critical role that ACA plays in aggregation and differentiation of D. discoideum cells has been exploited to develop a powerful system for genetic analysis of the regulation of these important signaling pathways. Random mutagenesis and phenotypic screening will be used to isolate uncoupled and constitutively active mutations in ACA and mammalian Type II adenylyl cyclases Allele-specific second-site suppressors will be selected to investigate intramolecular interactions within the adenylyl cyclase """"""""heterodimer.' The mutations will be mapped onto structural models of the dimer to understand how the enzymes are regulated. A related series of studies are designed to reveal how receptors, such as thos for chemokines, that are linked to G-alpha(i) (G-alpha-q) rather than G-alpha-activate adenylyl cyclases. Chemoattractant and G-alpha-beta -mediated activation of ACA requires the rapid translocation of the pleckstrin homology (PH)-domain containing protein, CRAC, from the cytosol to the plasma membrane. The domains of CRAC required for its relocalization and for activation of ACA will be delineated by site directed mutagenesis using HA- and GFP-tagged constructs in living cells. The membrane binding site for CRAC will be determined and its regulation by chemoattractants and G-proteins will be investigated. Taken together, proposed studies will provide insights into the functions of PH-domains in vivo as well as into the mechanisms of activation and regulation of adenylyl cyclases.
