Agonist efficacy at the α2A-adrenoceptor:Gα15 fusion protein:: an analysis based on Ca2+ responses

A fusion protein was constructed between the recombinant human alpha(2A)-adrenoceptor and a mouse G(alpha 15) protein to measure the efficacy of agonist-induced Ca2+ responses at a receptor:G(alpha 15) protein stoichiometry of 1. Activation of this fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pE(50): 7.28 +/- 0.04) increase in the intracellular Ca2+ concentration. The magnitude of the Ca2+ response was related to the amount of the fusion protein and the number of surface alpha(2A)-adrenoceptor binding sites as estimated by [H-3]RX 821002 binding. Whereas UK 14304 was as efficacious as (-)-adrenaline, the following ligands displayed partial agonist properties [E-max in percentage vs. (-)-adrenaline: d-medetomidine (76 +/- 3) > BHT 920 (53 +/- 3)> clonidine (39 +/- 4) >> oxymetazoline (10 +/- 1)]. This ligand activation profile was not affected over a 30-fold range of expression of the fusion protein in contrast to the observed enhancement of the partial agonists' maximal responses by co-expression of the alpha(2A)-adrenoceptol with increasing amounts of the G(alpha 15) protein. In conclusion, the fusion protein approach opens perspectives to quantify intrinsic activities of ligands under controlled experimental conditions of a fixed receptor:G(alpha 15) protein ratio of 1.