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.
Inoue
Y
Y
Membrane Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Fishman
P H
PH
Rebois
R V
RV
D037361:Adenylate Cyclase Toxin; D000535:Aluminum / Q000494:pharmacology*; D000077410:Aluminum Chloride; D017607:Aluminum Compounds; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000494:pharmacology; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D003676:Deferoxamine / Q000494:pharmacology; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology; D005461:Fluorine / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D037342:Pertussis Toxin; D010712:Phosphatidic Acids / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013806:Theophylline / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D017607:Aluminum Compounds; D002712:Chlorides; D006063:Chorionic Gonadotropin; D010712:Phosphatidic Acids; D010566:Virulence Factors, Bordetella; C053629:fluoroaluminum; D005461:Fluorine; D003995:Dibutyryl Cyclic GMP; D000077410:Aluminum Chloride; D012969:Sodium Fluoride; D013806:Theophylline; D000535:Aluminum; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D003676:Deferoxamine; D002118:Calcium