alpha(2A)-adrenoceptor: G(alpha i1) 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)-adre noceptor and either a rat wild-type G(alpha il) or putative pertussis toxin -resistant form of the G(alpha il) protein (G(alpha il)Cys(351)Gly). [H-3]2 -[2-(2-Methoxy-1,4-benzodioxanyl)]imidazoline hydrochloride (RX 821002) sat uration binding experiments demonstrated that both fusion proteins were exp ressed at a similar level as the alpha(2A)-adrenoceptor co-expressed with e ither a wild-type G(alpha il) or mutant G(alpha il)Cys(351)Gly protein in C OS-7 cells, and displayed a ligand binding profile similar to chat for the alpha(2A)-adrenoccptor protein. In alpha(2A)-adrenoceptor-transfected COS-7 cells, 5-bromo-6-(2-imidazolin-2-yl-amino) quinoxaline tartrate (brimonidi ne, 10 mu M) induced stimulation (151 +/- 28%) of adenosine 3',5'-cyclic mo nophosphate: (cAMP) formation which was prevented by cholera toxin treatmen t, demonstrating a direct coupling of the alpha(2A)-adrenoceptor to an endo genous G(alpha s) protein in COS-7 cells. Expression of either the wild-typ e G(alpha il) or mutant G(alpha il)Cys(351)Gly protein in co-expression or fusion with the alpha(2A)-adrenoceptor in COS-7 cells suppressed the brimon idine-induced stimulation of cAMP formation, both in che presence and absen ce of pertussis toxin pretreatment. Hence, the G(alpha il) protein apparent ly blocks the G(s)-coupled alpha(2A)-adrenoceptor-mediated pathway in a per tussis toxin-non-sensitive way. (C) 2000 Elsevier Science Inc.