Facilitation of constitutive alpha(2A)-adrenoceptor activity by both single amino acid mutation (Thr(373)Lys) and g(alphao) protein coexpression: evidence for inverse agonism.

The recombinant human alpha(2A)-adrenoceptor (alpha(2A)-AR, RC 2.1. ADR.A2A) can be transformed into a constitutively activated form in CHO-K1 cells by coexpression with a rat G(alphao) protein. Constitutive activity could be enhanced more by both mutation of Thr(373) of the alpha(2A)-AR to a Lys and Cys(351) of the G(alphao) protein by an Ile. The basal [(35)S]GTPgammaS binding response displayed a constitutive alpha(2A)-AR activity that amounted to 21% of the maximal receptor activation as obtained with 10 microM (-)-adrenaline. UK 14304, BHT 920, d-medetomidine, oxymetazoline, and clonidine acted as efficacious agonists. The enhancement of basal activity was entirely blocked (-50 +/- 3%) by ligands that thus appeared to act as inverse agonists (i.e., RX 811059 and its (+)-enantiomer, (+)-RX 821002, RS 15385, and yohimbine); the potencies of the ligands corresponded with their binding affinities for the alpha(2A)-AR. Fluparoxan and WB 4101 displayed partial inverse agonism. Atipamezole and dexefaroxan at 10 microM were virtually free of intrinsic activity and thus acted as neutral antagonists; idazoxan displayed potent partial agonist properties as observed with BRL 44408 and SKF 86466. The inverse agonist activity induced by (+)-RX 811059 could be reversed by atipamezole with a pK(B) value (8.73 +/- 0.07) that was similar to that required for blockade of the UK 14304-mediated response. Constitutive alpha(2A)-AR activation was mainly observed with the G(alphao) Cys(351)Ile protein compared with the pertussis toxin-resistant mutants of the G(alphai) protein subtypes. The observed spectrum of intrinsic activities for the various ligands suggests that pure, neutral antagonists are rather uncommon in this specified alpha(2A)-AR system.