Agonist efficacy at the alpha2A-adrenoceptor:G alpha15 fusion protein: an analysis based on Ca2+ responses.

A fusion protein was constructed between the recombinant human alpha2A-adrenoceptor and a mouse G alpha15 protein to measure the efficacy of agonist-induced Ca2+ responses at a receptor:G alpha15 protein stoichiometry of 1. Activation of this fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50: 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 alpha2A-adrenoceptor binding sites as estimated by [3H]RX 821002 binding. Whereas UK 14304 was as efficacious as (-)-adrenaline, the following ligands displayed partial agonist properties [Emax 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 alpha2A-adrenoceptor with increasing amounts of the G alpha15 protein. In conclusion, the fusion protein approach opens perspectives to quantify intrinsic activities of ligands under controlled experimental conditions of a fixed receptor:G alpha15 protein ratio of 1.