Direct evidence for the role of the coupling proteins in forskolin activation of adenylate cyclase.

Forskolin stimulation of adenylate cyclase in wild type (WT) S49 lymphoma membrane preparations exhibited a lag and biphasic kinetics as a function of the forskolin concentration (i.e., both a high and low affinity component were observed). In contrast to WT, forskolin stimulation of cyc- adenylate cyclase in membranes demonstrated no observable lag and only the low affinity component. Both the lag and the high affinity component characteristic of forskolin activation of WT were observed in cyc- reconstituted with cholate extracts of WT which contained the G/F protein. The potency of forskolin stimulation of reconstituted adenylate cyclase was increased still further if the reconstitution was carried out with epinephrine and Gpp(NH)p. The Vmax of the forskolin stimulation of adenylate cyclase was approximately the same in the reconstituted and unreconstituted cyc-. In addition to the experiments with reconstituted cyc-, we have demonstrated that forskolin increased the apparent affinity of epinephrine for activation of adenylate cyclase in WT, and reciprocally, epinephrine increased the apparent affinity of forskolin for activation. We conclude that the lag, biphasic kinetics of forskolin activation and the synergism of hormone and forskolin activation of WT are attributable to functional G/F and are consistent with the forskolin stabilization of the activated catalytic unit of adenylate cyclase.