Analysis of the relative interactions between the alpha 2C10 adrenoceptor and the guanine-nucleotide-binding proteins G(o)1 alpha and Gi 2 alpha following co-expression of these polypeptides in rat 1 fibroblasts.
Rat 1 fibroblasts which had been transfected to express the human alpha 2C10 adrenoceptor (clone 1C) were further co-transfected with a plasmid containing the hygromycin-B-resistance gene and a plasmid containing a cDNA encoding the alpha-subunit of the rat pertussis-toxin-sensitive G-protein G(o)1. In clone 3 the receptor was expressed at some 2.2 pmol/mg of membrane protein, and G(o)1 alpha at approx. 100 pmol/mg of membrane protein. The interaction of these two polypeptides and that between the receptor and Gi2 alpha (endogenously expressed at some 50 pmol/mg of membrane protein) were studied. Agonist activation of G(o)1 alpha was observed in membranes of the alpha 2C10-adrenoceptor(+)-G(o)1 alpha+ cells (clone 3), but not in alpha 2C10-adrenoceptor(+)-G(o)alpha-cells (clone 1C), whereas similar agonist-dependent activation of Gi2 alpha was observed in both cell types. alpha 2C10-adrenoceptor activation of G(o)1 alpha and Gi2 alpha in clone-3 membranes was produced with similar agonist-dose-effect curves. These observations indicate that the receptor interacts with equivalent affinity with each of these G-proteins. Agonist-dependent cholera-toxin-catalysed [32P]ADP-ribosylation of G(o)1 alpha was terminated when the alpha 2-adrenoceptor antagonist yohimbine was added subsequent to agonist-induced initiation of the reaction and release of GDP, demonstrating the conformational requirement for this reaction to be the ternary complex of agonist-occupied receptor and guanine-nucleotide-denuded G-protein.
Grassie
M A
MA
Molecular Pharmacology Group, University of Glasgow, Scotland, U.K.
Milligan
G
G
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D000068438:Brimonidine Tartrate; D002772:Cholera Toxin / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D009243:NAD / Q000378:metabolism; D037342:Pertussis Toxin; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D015016:Yohimbine / Q000494:pharmacology
D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D009243:NAD; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D015016:Yohimbine; D000068438:Brimonidine Tartrate; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins