Evidence that a beta-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)- dependent adenylate cyclase activity.
The guanine nucleotide regulatory protein component (N) of the frog erythrocyte membrane adenylate cyclase system appears to form a stable complex with the beta-adrenergic receptor (R) in the presence of agonist (H). This agonist-promoted ternary complex HRN can be solubilized with Lubrol. The guanine nucleotide regulatory protein associated with the solubilized complex can be adsorbed either to GTP-Sepharose directly or to wheat germ lectin-Sepharose via its interaction with the receptor which is a glycoprotein. Guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) can be used to elute the guanine nucleotide regulatory protein from either Sepharose derivative. The resulting N.GTP gamma S complex conveys nucleotide-dependent adenylate cyclase activity when combined with a Lubrol-solubilized extract of turkey erythrocyte membranes. The ability to observe GTP gamma S-dependent reconstitution of adenylate cyclase activity in the eluate from either resin required the formation of the HRN complex prior to solubilization. The N protein can be identified by its specific [32P]ADP ribosylation catalyzed by cholera toxin in the presence of [32P]NAD+. The existence of a stable HRN intermediate complex is supported by the observation that agonist pretreatment of frog erythrocyte membranes results in a 100% increase in the amount of 32P-labeled N protein eluted from the lectin-Sepharose in the presence of GTP gamma S compared to membranes pretreated with either antagonist or agonist plus GTP. Our results therefore provide evidence that the same guanine nucleotide-binding protein that associates with the beta-adrenergic receptor in the presence of agonist mediates adenylate cyclase activation.
D000246:Adenosine Diphosphate Ribose / Q000097:blood; D000262:Adenylyl Cyclases / Q000097:blood*; D000818:Animals; D002846:Chromatography, Affinity; D004912:Erythrocytes / Q000378:metabolism*; D019204:GTP-Binding Proteins; D006150:Guanine Nucleotides; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D011898:Ranidae; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology*; D014422:Turkeys
D006150:Guanine Nucleotides; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; D011956:Receptors, Cell Surface; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; C023427:guanosine 5'-O-(2-thiodiphosphate); D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases