Chemoattractant and GTP gamma S-mediated stimulation of adenylyl cyclase in Dictyostelium requires translocation of CRAC to membranes.

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. Devreotes. 1994. J. Biol. Chem. 269:14123-14129; Insall, R. H., A. Kuspa, P. J. Lilly, G. Schaulsky, L. R. Levin, W. F. Loomis, and P. N. Devreotes. 1994. J. Cell Biol. 126:1537-1545). In this report, we show that in intact cells, chemoattractants promote translocation of CRAC from the cytosolic to the membrane fraction. However, CRAC is not required at the time of receptor stimulation; it can be added to lysates of activated cells. Treatment of membranes with guanine nucleotides creates binding sites for CRAC. These binding sites can be generated in mutants lacking each of the components of the pathway except the beta-subunit, suggesting that free or "activated" beta gamma-subunits may be a part of the binding site. This hypothesis is consistent with previous observations that CRAC contains a pleckstrin homology domain and that the beta gamma-subunits likely mediate activation of adenylyl cyclase in this system. Thus, CRAC may serve as an adapter, linking the G-protein beta gamma-subunits to activation of the enzyme. GTP gamma S cannot generate CRAC-binding sites when the adenylyl cyclase pathway has been adapted by prior chemoattractant stimulation, suggesting that this is a point of downstream adaptation.

CRAC, a cytosolic protein containing a pleckstrin homology domain, is required for receptor and G protein-mediated activation of adenylyl cyclase in Dictyostelium.

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 gamma 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 cDNA 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 beta gamma subunits to adenylyl cyclase activation. If so, it may be the first member of an important class of coupling proteins.

Identification of CRAC, a cytosolic regulator required for guanine nucleotide stimulation of adenylyl cyclase in Dictyostelium.

As previously reported, guanine nucleotide regulation of adenylyl cyclase activity in the Dictyostelium mutant synag 7 can be restored in vitro by addition of a high-speed supernatant prepared from wild-type cells (Theibert, A., and Devreotes, P. N. (1986) J. Biol. Chem. 261, 15121-15125). We have designated this activity CRAC, for cytosolic regulator of adenylyl cyclase. Trypsinization of partially purified material demonstrated that this activity contains a protein. We report here a 50,000-fold purification of this protein using Q and S Sepharose fast flow and P11 cellulose chromatography, followed by sucrose gradient centrifugation and separation on SDS-polyacrylamide gel electrophoresis. Purification of wild-type and mutant supernatants in parallel allowed identification of an 88-kDa protein required for reconstituting activity. A polyclonal antibody was raised against this protein; it stains a band in unfractionated wild-type, but not mutant, supernatants. Immunoblots of fractions from each purification step show that activity and the immunostaining band cochromatograph. We have determined a short N-terminal sequence of the 88-kDa CRAC polypeptide, which matches a portion of the deduced N terminus of a gene, dagA, isolated from a mutant similar to synag 7. Expression of the dagA cDNA in synag 7 cells restores both the 88 kDa band and CRAC activity.