Modulation of ligand responses by coupling of α2A-adrenoceptors to diverse Gα-proteins
ABSTRACT The hypothesis that different signalling may be mediated via a single α2A-adrenoceptor (α2A AR) subtype was investigated by challenging α2 AR ligands in combination with diverse recombinant wt, mutant, and chimeric Gα-proteins. Possible coupling of α2A AR to endogenous Gαi/o-proteins in CHO-K1 cells was excluded by measuring pertussis toxin (PTX)-resistant [35S]GTPγS-binding responses as a common functional response to α2A AR activation. (−)-Adrenaline (10 μM) displayed the highest magnitude of [35S]GTPγS-binding response in the co-presence of a PTX-resistant GαoCys351Ile protein, whereas a decreased response was obtained with the mutant Gαi1/2-proteins. Replacement of the last six amino acids at the C-terminal portion of the Gαo-protein by the corresponding amino acid region of either the Gαz-, Gαs-, Gαq-, or Gα15-protein and co-expression with the α2A AR resulted in similar maximal (−)-adrenaline-mediated [35S]GTPγS-binding responses with these chimeric Gαo-proteins. The ligands d-medetomidine, BHT 920 (6-allyl-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin-2-ylamine) and (+)-RX 811059 (2-(2-ethoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole) were weakly active or virtually inactive at the chimeric Gαo/s-, Gαo/q-, and Gαo/15-proteins in contrast to the Gαo/z-protein. Furthermore, combining the constitutively active mutant Thr373Lys α2A AR with these chimeric Gαo-proteins enhanced the apparent intrinsic activity of d-medetomidine and BHT 920. A similar observation was made using the corresponding fusion proteins, where the stoichiometry of the mutant α2A AR to the chimeric Gαo-protein was fixed at 1.0. These data indicate that a single ligand may display different magnitudes of activation at the α2A AR subtype coupled to chimeric Gαo proteins under controlled conditions of α2A AR: Gαo-protein expression.