Glucagon receptor-mediated activation of Gs is accompanied by subunit dissociation.
The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.
Iyengar
R
R
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York 10029.
Rich
K A
KA
Herberg
J T
JT
Premont
R T
RT
Codina
J
J
D000262:Adenylyl Cyclases / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018027:Receptors, Glucagon; D013873:Thionucleotides / Q000378:metabolism
D046911:Macromolecular Substances; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D013873:Thionucleotides; D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D008274:Magnesium; D005033:Ethylmaleimide