alpha(2A)-adrenoceptor: G(alphai1) protein-mediated pertussis toxin-resistant attenuation of G(s) coupling to the cyclic AMP pathway.
Fusion proteins were constructed between a recombinant human alpha(2A)-adrenoceptor and either a rat wild-type G(alphai1) or putative pertussis toxin-resistant form of the G(alphai1) protein (G(alphai1)Cys(351)Gly). [(3)H]2-[2-(2-Methoxy-1, 4-benzodioxanyl)]imidazoline hydrochloride (RX 821002) saturation binding experiments demonstrated that both fusion proteins were expressed at a similar level as the alpha(2A)-adrenoceptor co-expressed with either a wild-type G(alphai1) or mutant G(alphai1)Cys(351)Gly protein in COS-7 cells, and displayed a ligand binding profile similar to that for the alpha(2A)-adrenoceptor protein. In alpha(2A)-adrenoceptor-transfected COS-7 cells, 5-bromo-6-(2-imidazolin-2-yl-amino) quinoxaline tartrate (brimonidine, 10 microM) induced stimulation (151 +/- 28%) of adenosine 3',5'-cyclic monophosphate (cAMP) formation which was prevented by cholera toxin treatment, demonstrating a direct coupling of the alpha(2A)-adrenoceptor to an endogenous G(alphas) protein in COS-7 cells. Expression of either the wild-type G(alphai1) or mutant G(alphai1)Cys(351)Gly protein in co-expression or fusion with the alpha(2A)-adrenoceptor in COS-7 cells suppressed the brimonidine-induced stimulation of cAMP formation, both in the presence and absence of pertussis toxin pretreatment. Hence, the G(alphai1) protein apparently blocks the G(s)-coupled alpha(2A)-adrenoceptor-mediated pathway in a pertussis toxin-non-sensitive way.
Rauly
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I
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 81106, Castres, France. isabelle.rauly@pierre-fabre.com
Ailhaud
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M
Wurch
T
T
Pauwels
P J
PJ
D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D000242:Cyclic AMP / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D037342:Pertussis Toxin; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
C547768:ADRA2A protein, human; C547770:Adra2a protein, rat; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs