title: Modulation of ligand responses by coupling of alpha(2A)-adrenoceptors to diverse G(alpha)-proteins.
abstract: The hypothesis that different signalling may be mediated via a single alpha(2A)-adrenoceptor (alpha(2A) AR) subtype was investigated by challenging alpha(2) AR ligands in combination with diverse recombinant wt, mutant, and chimeric G(alpha)-proteins. Possible coupling of alpha(2A) AR to endogenous G(alphai/o)-proteins in CHO-K1 cells was excluded by measuring pertussis toxin (PTX)-resistant [(35)S]GTPgammaS-binding responses as a common functional response to alpha(2A) AR activation. (-)-Adrenaline (10 microM) displayed the highest magnitude of [(35)S]GTPgammaS-binding response in the co-presence of a PTX-resistant G(alphao)Cys(351)Ile protein, whereas a decreased response was obtained with the mutant G(alphai1/2)-proteins. Replacement of the last six amino acids at the C-terminal portion of the G(alphao)-protein by the corresponding amino acid region of either the G(alphaz)-, G(alphas)-, G(alphaq)-, or G(alpha15)-protein and co-expression with the alpha(2A) AR resulted in similar maximal (-)-adrenaline-mediated [(35)S]GTPgammaS-binding responses with these chimeric G(alphao)-proteins. The ligands D-medetomidine, BHT 920 (6-allyl-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin-2-ylamine) and (+)-RX 811059 (2-(2-ethoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole) were weakly active or virtually inactive at the chimeric G(alphao/s)-, G(alphao/q)-, and G(alphao/15)-proteins in contrast to the G(alphao/z)-protein. Furthermore, combining the constitutively active mutant Thr(373)Lys alpha(2A) AR with these chimeric G(alphao)-proteins enhanced the apparent intrinsic activity of d-medetomidine and BHT 920. A similar observation was made using the corresponding fusion proteins, where the stoichiometry of the mutant alpha(2A) AR to the chimeric G(alphao)-protein was fixed at 1.0. These data indicate that a single ligand may display different magnitudes of activation at the alpha(2A) AR subtype coupled to chimeric G(alphao) proteins under controlled conditions of alpha(2A) AR: G(alphao)-protein expression.
authors: Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17 Avenue Jean Moulin, F-81106, Castres Cédex, France. peter.pauwels@pierre-fabre.com
authors: P J
authors: PJ
authors: Pauwels
authors: S
authors: S
authors: Tardif
authors: F C
authors: FC
authors: Colpaert
authors: T
authors: T
authors: Wurch
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020926:Medetomidine / Q000494:pharmacology; D016296:Mutagenesis; D011869:Radioligand Assay; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013462:Sulfur Radioisotopes
chemical_list: D000316:Adrenergic alpha-Agonists; D001381:Azepines; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C024763:talipexole; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D020926:Medetomidine