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Updated: Aug 9, 2026

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
Published on: June 9, 2017
Modulation of ligand responses by coupling of alpha(2A)-adrenoceptors to diverse G(alpha)-proteins
P J Pauwels1, S Tardif, F C Colpaert
1Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17 Avenue Jean Moulin, F-81106, Castres Cédex, France. peter.pauwels@pierre-fabre.com
Abstract:
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, and chimeric G(alpha)-proteins. Possible coupling of alpha(2A) AR to endogenous G(alphai/o)-proteins in CHO-K1 cells was excluded by measuring pertussis toxin (PTX)-resistant [(35)S]GTPgammaS-binding responses as a common functional response to alpha(2A) AR activation. (-)-Adrenaline (10 microM) displayed the highest magnitude of [(35)S]GTPgammaS-binding response in the co-presence of a PTX-resistant G(alphao)Cys(351)Ile protein, whereas a decreased response was obtained with the mutant G(alphai1/2)-proteins. Replacement of the last six amino acids at the C-terminal portion of the G(alphao)-protein by the corresponding amino acid region of either the G(alphaz)-, G(alphas)-, G(alphaq)-, or G(alpha15)-protein and co-expression with the alpha(2A) AR resulted in similar maximal (-)-adrenaline-mediated [(35)S]GTPgammaS-binding responses with these chimeric G(alphao)-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 G(alphao/s)-, G(alphao/q)-, and G(alphao/15)-proteins in contrast to the G(alphao/z)-protein. Furthermore, combining the constitutively active mutant Thr(373)Lys alpha(2A) AR with these chimeric G(alphao)-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 alpha(2A) AR to the chimeric G(alphao)-protein 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.
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