Facilitation of constitutive alpha(2A)-adrenoceptor activity by both single amino acid mutation (Thr(373)Lys) and G(alpha o) 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(alpha o) protein. Constitutive activity could be enhanced more by both mutation of Thr(373) of the alpha(2A)-AR to a Lys an d Cys(351) of the G(alpha o) protein by an Ile. The basal [S-35]GTP gamma S binding response displayed a constitutive alpha(2A)-AR activity that amoun ted to 21% of the maximal receptor activation as obtained with 10 mu M (-)- adrenaline. UK 14304, BHT 920, d-medetomidine, oxymetazoline, and clonidine acted as efficacious agonists. The enhancement of basal activity was entir ely blocked (-50 +/- 3%) by ligands that thus appeared to act as inverse ag onists (i.e., RX 811059 and its (+)-enantiomer, (+)-RX 821002, RS 15385, an d yohimbine); the potencies of the ligands corresponded with their binding affinities for the alpha(2A)-AR. Fluparoxan and WB 4101 displayed partial i nverse agonism. Atipamezole and dexefaroxan at 10 mM were virtually free of intrinsic activity and thus acted as neutral antagonists; idazoxan display ed potent partial agonist properties as observed with BRL 44408 and SKF 864 66. 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 alph a(2A)-AR activation was mainly observed with the Gao Cys(351)Ile protein co mpared with the pertussis toxin-resistant mutants of the G(alpha i) protein subtypes. The observed spectrum of intrinsic activities for the various li gands suggests that pure, neutral antagonists are rather uncommon in this s pecified alpha(2A)-AR system.