CRAC, A CYTOSOLIC PROTEIN CONTAINING A PLECKSTRIN HOMOLOGY DOMAIN, ISREQUIRED FOR RECEPTOR AND G-PROTEIN-MEDIATED ACTIVATION OF ADENYLYL-CYCLASE IN DICTYOSTELIUM

Adenylyl cyclase in Dictyostelium, as in higher eukaryotes, is activat ed through G protein-coupled receptors. Insertional mutagenesis into a gene designated dagA resulted in cells that cannot activate adenylyl cyclase, but have otherwise normal responses to exogenous cAMP. Neithe r cAMP treatment of intact cells nor GTP gamma S treatment of lysates stimulates adenylyl cyclase activity in dagA mutants. A cytosolic prot ein that activates adenylyl cyclase, CRAC, has been previously identif ied. We trace the signaling defect in dagA(-) cells to the absence of CRAC, and we demonstrate that dagA is the structural gene for CRAC. Th e 3.2-kb dagA mRNA encodes a predicted 78.5-kD product containing a pl eckstrin homology domain, in agreement with the postulated interaction of CRAC with activated G proteins. Although dagA expression is tightl y developmentally regulated, the cDNA restores normal development when constitutively expressed in transformed mutant cells. In addition, th e megabase region surrounding the dagA locus was mapped. We hypothesiz e that CRAC acts to connect free G protein beta gamma subunits to aden ylyl cyclase activation. If so, it may be the first member of an impor tant class of coupling proteins.