PT - JOURNAL ARTICLE AU - Petrus J. Pauwels AU - Stéphanie Tardif AU - Thierry Wurch AU - Francis C. Colpaert TI - Facilitation of Constitutive α<sub>2A</sub>-Adrenoceptor Activity by Both Single Amino Acid Mutation (Thr<sup>373</sup>Lys) and G<sub>αo</sub> Protein Coexpression: Evidence for Inverse Agonism DP - 2000 Feb 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 654--663 VI - 292 IP - 2 4099 - http://jpet.aspetjournals.org/content/292/2/654.short 4100 - http://jpet.aspetjournals.org/content/292/2/654.full SO - J Pharmacol Exp Ther2000 Feb 01; 292 AB - The recombinant human α2A-adrenoceptor (α2A-AR, RC 2.1.ADR.A2A) can be transformed into a constitutively activated form in CHO-K1 cells by coexpression with a rat Gαo protein. Constitutive activity could be enhanced more by both mutation of Thr373 of the α2A-AR to a Lys and Cys351 of the Gαo protein by an Ile. The basal [35S]GTPγS binding response displayed a constitutive α2A-AR activity that amounted to 21% of the maximal receptor activation as obtained with 10 μM (−)-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 α2A-AR. Fluparoxan and WB 4101 displayed partial inverse agonism. Atipamezole and dexefaroxan at 10 μM 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 pKB value (8.73 ± 0.07) that was similar to that required for blockade of the UK 14304-mediated response. Constitutive α2A-AR activation was mainly observed with the Gαo Cys351Ile protein compared with the pertussis toxin-resistant mutants of the Gαi protein subtypes. The observed spectrum of intrinsic activities for the various ligands suggests that pure, neutral antagonists are rather uncommon in this specified α2A-AR system. The American Society for Pharmacology and Experimental Therapeutics