Differential activation of the stimulatory and inhibitory guanine nucleotide-binding proteins by fluoroaluminate in cells and in membranes.

Fluoroaluminate had no effect on cAMP levels in cells but inhibited agonist-stimulated cAMP accumulation. In membranes, fluoroaluminate stimulated adenylate cyclase activity between 1 and 10 mM but not at higher concentrations, and it inhibited agonist-stimulated adenylate cyclase activity at concentrations greater than 1 mM. Fluoroaluminate is known to activate Gs and Gi, the guanine nucleotide-binding (G) proteins that stimulate and inhibit adenylate cyclase. G proteins are heterotrimeric, with unique alpha and common beta gamma subunits, and activation involves dissociation of alpha from beta gamma. Pertussis toxin catalyzes ADP-ribosylation of alpha i of heterotrimeric Gi but not free alpha i. Fluoroaluminate prevented pertussis toxin-catalyzed ADP-ribosylation of Gi in cells and membranes, suggesting that Gi is activated by fluoroaluminate in both. Cholera toxin catalyzes ADP-ribosylation of the alpha s subunit of Gs. In cells, agonist often increased cholera toxin-catalyzed ADP-ribosylation of Gs, but fluoroaluminate decreased ADP-ribosylation even in the presence of agonist, suggesting that Gs cannot be activated in the presence of fluoroaluminate. In membranes, both agonist and fluoroaluminate increased cholera toxin-catalyzed ADP-ribosylation, suggesting that Gs is activated by these agents. We conclude that fluoroaluminate activates Gi but not Gs in cells and activates both G proteins in membranes. The value of bacterial toxins in assessing the state of G protein in cells and membranes is demonstrated.