Reconstitution of cyc- S49 membranes by in vitro translated Gs alpha. Membrane anchorage and functional implications.
After ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the alpha-subunit of Gs (Gs alpha) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH greater than or equal to 11.5. In vitro translated Gs alpha could interact with plasma membranes from the cyc- mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTP gamma S did not alter anchorage of Gs alpha. The characteristics of the association of in vitro translated Gs alpha with cyc- membranes after GTP gamma S treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc- membranes by in vitro translated alpha-subunit of Gs.
Journot
L
L
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
Bockaert
J
J
Audigier
Y
Y
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007545:Isoproterenol / Q000494:pharmacology; D008223:Lymphoma; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D009243:NAD / Q000378:metabolism; D009994:Osmolar Concentration; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D013873:Thionucleotides; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol