Modulation of ligand responses by coupling of alpha(2A)-adrenoceptors to diverse G(alpha)-proteins

The hypothesis that different signalling may be mediated via a single alpha (2A)-adrenoceptor (alpha (2A) AR) subtype was investigated by challenging alpha (2) AR ligands in combination with diverse recombinant wt, mutant, an d chimeric G(alpha)-proteins. Possible coupling of alpha (2A) AR to endogen ous G(alphai/o)-proteins in CHO-K1 cells was excluded by measuring pertussi s toxin (PTX)-resistant [S-35]GTP gammaS-binding responses as a common func tional response to alpha (2A) AR activation. (-)-Adrenaline (10 muM) displa yed the highest magnitude of [S-35]GTP gammaS-binding response in the co-pr esence of a PTX-resistant G(alphao)Cys(351)Ile protein, whereas a decreased response was obtained with the mutant G(alphai 1/2)-proteins. Replacement of the last six amino acids at the C-terminal portion of the G(alphao)-prot ein by the corresponding amino acid region Of either the G(alphaz)-, G(alph as)-, G(alphaq), or G(alpha 15)-protein and co-expression with the alpha (2 A) AR resulted in similar maximal (-)-adrenaline-mediated [S-35]GTP gammaS- binding responses with these chimeric G(alphao)-proteins. The ligands D-med etomidine, BHT 920 (6-allyl-5,6,7,8-tetrahydro-4H-thiazalo[4,5-d]azepin-2-y lsumine) and (+)-RX 811059 (2-(2-ethoxy-2,3-dihydro-benzo[ 1,4]dioxin-2-yl) -4,5-dihydro-1 H-imidazole) were weakly active or virtually inactive at the chimeric G(alphao/s)-, G alphao/q-, and G(alphao/15)-proteins in contrast to the G(alphao/z)-protein. Furthermore, combining the constitutively activ e mutant Thr(373)Lys alpha (2A) AR with these chimeric G(alphao)-proteins e nhanced the apparent intrinsic activity of d-medetomidine and BHT 920. A si milar observation was made using the corresponding fusion proteins, where t he stoichiometry of the mutant alpha (2A) AR to the chimeric G(alphao)-prot ein was fixed at 1.0. These data indicate that a single ligand may display different magnitudes of activation at the alpha (2A) AR subtype coupled to chimeric G(alphao) proteins under controlled conditions of alpha (2A) AR: G (alphao)-protein expression. (C) 2001 Elsevier Science Inc. Ah rights reser ved.