Agonist potency differentiates G protein activation and Ca2+ signalling by the orexin receptor type 1

The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with G(q)/G(11) proteins as well as with G(s) and G(i) proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate G(q)/G(11) proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+](i). No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the G alpha(s) protein backbone with various G alpha donor sequences (G alpha(s/x)) at the N and C termini, and measured cAMP as functional output. The G alpha(s/x) chimeric proteins with G alpha(11)(G alpha(q)) and G alpha(16) structure in the C terminus were stimulated by the OX1R. Concentration-response curves with G alpha(s/16) revealed an agonist potency correlation between G protein activation and the elevation of [Ca2+](i) via discharge of intracellular Ca2+ stores, a feature also recognized for the muscarinic M-3 receptor. However, in contrast to the M-3 receptor, the OX1R elevated [Ca2+](i) via influx from extracellular space at about 30-fold lower agonist concentration. The results suggest that the OX1R is linked to influx of Ca2+ through a signal pathway independent of G(q)/G(11) protein activation. (c) 2005 Elsevier Inc. All rights reserved.