title: 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.
abstract: 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.
authors.affiliation: Department of Cellular Biology, Centre de Recherche Pierre Fabre, CASTRES Cédex, France. peter.pauwels@pierre-fabre.com
authors.forename: P J
authors.initials: PJ
authors.lastname: Pauwels
authors.forename: S
authors.initials: S
authors.lastname: Tardif
authors.forename: T
authors.initials: T
authors.lastname: Wurch
authors.forename: F C
authors.initials: FC
authors.lastname: Colpaert
mesh_terms: D000322:Adrenergic Agonists / Q000494:pharmacology*; D018674:Adrenergic Antagonists / Q000494:pharmacology*; D058669:Adrenergic alpha-2 Receptor Antagonists; D000818:Animals; D016466:CHO Cells; D019976:Cloning, Organism; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007120:Immunochemistry; D066298:In Vitro Techniques; D008024:Ligands; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects* / Q000378:metabolism; D011994:Recombinant Proteins; D013997:Time Factors; D014162:Transfection
chemical_list: D000322:Adrenergic Agonists; D018674:Adrenergic Antagonists; D058669:Adrenergic alpha-2 Receptor Antagonists; D008024:Ligands; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins