Analysis of ligand activation of α2-adrenoceptor subtypes under conditions of equal Gα protein stoichiometry

Variations in the measurement of ligand's intrinsic activity between receptor subtypes is a common consequence of unequal receptor:G protein density ratios. We have investigated ligand activation at the alpha (2)-adrenoceptor (alpha (2)-AR) (alpha (2)-AR) subtypes under defined expression conditions of one receptor molecule for one G(alpha) protein molecule using fusion proteins. Fusion between either a wt alpha (2C) AR or a mutant Thr(381)Lys alpha (2C) AR and G(alphaq/il) protein displayed robust, transient (-)-adrenaline-mediated Ca2+ responses with similar potencies (pEC(50): 7.78 and 7.66) and kinetic properties. A comparison of the intrinsic activities of alpha (2) AR agonists found d-medetomidine to be the only compound with an efficacy similar to that of (-)-adrenaline. The Ca2+ responses as mediated by UK 14304, oxymetazoline and clonidine became more potent and efficacious at the Thr(381)Lys alpha (2C) AR, whereas the response as mediated by talipexole displayed a higher potency with an unaltered maximal response. Whereas only small differences in ligand's intrinsic activities between the wt alpha (2A), alpha (2B) and alpha (2C) AR fusion proteins were observed with most ligands, oxymetazoline was virtually silent at the alpha (2A) AR while active as a partial and apparently full agonist at the alpha (2C) AR and alpha (2B) AR, respectively, The mutant alpha (2) AR subtypes could be differentiated using the apparent parent positive efficacy of ligands that used to be defined as antagonists. The following rank order of maximal responses was observed for the Thr(381)Lys alpha (2C) similar or equal to SKF 86466 > atipamezole > > dexefaroxan; Thr(373)Lys alpha (2A) AR: SKF 86466 > idazoxan = atipamezole > dexefaroxan; and Thr(373)Lys alpha (2B) AR: atipamezole > idazoxan similar or equal to dexefaroxan. RX 811059 (10 muM) was the only compound to be completely silent at both the wt and mutant probably rare in these specified alpha (2). AR PLB systems. Most antagonists may actually possess partial agonist properties at the alpha (2) AR subtypes, which are facilitated by the same mutation in the distal portion of their third intracellular loop.