Abstract. Adenylyl cyclase in Dictyostelium, as in higher eukaryotes, is activated 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. Neither cAMP treatment of intact cells nor GTP~/S treatment of lysates stimulates adenylyl cyclase activity in dagA mutants. A cytosolic protein that activates adenylyl cyclase, CRAC, has been previously identified. We trace the signaling defect in dagA-cells to the absence of CRAC, and we demonstrate that dagA is the structural gene for CRAC. The 3.2-kb dagA mRNA encodes a predicted 78.5-kD product containing a pleckstrin homology domain, in agreement with the postulated interaction of CRAC with activated G proteins. Although dagA expression is tightly developmentally regulated, the eDNA restores normal development when constitutively expressed in transformed mutant cells. In addition, the megabase region surrounding the dagA locus was mapped.We hypothesize that CRAC acts to connect free G protein ~3' subunits to adenylyl cyclase activation. If so, it may be the first member of an important class of coupling proteins.ESPONSES to light, odorants, chemoattractants, and many hormones and neurotransmitters are mediated by G protein-coupled receptors. When excited, these receptors activate heterotrimeric G proteins, catalyzing the exchange of GTP for GDP on the ot-subunit and the dissociation of the ot from the Bq~-subunit complex. Both of these components can stimulate or inhibit effectors including adenylyl cyclases, phosphodiesterases, phospholipases, and ion channels (Gilman, 1987).Increasing evidence has highlighted the role of B3,-subunits in directly regulating effectors, rather than in merely modulating ot-subunit activity (Birnbaumer, 1992). Several particular adenylyl cyclase subtypes, ion channels, and phospholipases are activated by free B7 complexes (Tang and Gilman, 1991;Logothetis et al., 1987). The phospholipases share a region, the pleckstrin homology (PH) 1 domain, which is also found in a variety of other signal transduction Address all correspondence to Peter Devreotes,

Recent studies have demonstrated that G-protein-linked signal transduction pathways play a significant role in the developmental program of the simple eukaryotic organism Dictyostelium. We have reported previously the isolation of a G-protein 13-subunit and present here a more complete analysis of this gene. Low-stringency Southern blots and RFLP mapping studies suggest that the 13-subunit is a unique gene found on linkage group If. Its deduced amino acid sequence of 347 residues is -60% identical to those of the human, Drosophila, and Caenorhabditis elegans 13-subunits. The carboxy-terminal 300 residues are about 70% identical; the amino-terminal 50 residues are quite divergent, containing only 10 identities. At all stages of growth and development, a single 1.9-kb 13-subunit mRNA is present at a high level, and a specific antibody detects a single 37-kD protein. We propose that G-protein heterotrimers are formed when this 13-subunit couples with each of the eight distinct G-protein a-subunits that are transiently expressed during development. Targeted disruption of the 13-subunit gene had no effect on the viability of haploid cells, but resulted in the inability of cells to aggregate.

Abstract. We have previously reported that activation of adenylyl cyclase by chemoattractant receptors in Dictyostelium requires, in addition to a heterotrimeric G-protein, a cytosolic protein, designated CRAC (Lilly, P., and P. N.

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