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

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. 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.