Disparate ligand-mediated Ca2+ responses by wild-type, mutant Ser200Ala and Ser204Ala α2A-adrenoceptor:: Gα15 fusion proteins:: evidence for multiple ligand-activation binding sites

1 Ligand:receptor interactions were analysed at wt, mutant Ser(200)Ala and Ser(204)Ala alpha(2A) ARs by measuring Ca2+ responses in CHO-K1 cells either by co-expression with a G(alpha 15) protein or at a receptor:G(alpha 15) protein stoichiometry of 1.0 using fusion proteins. 2 The magnitude of the UK 14304-mediated Ca2+ response as elicited by a G(alpha 15) protein was largest with both mutant Ser(200)Ala and Ser204Ala alpha(2A)ARs compared to the wt alpha(2A) AR in the co-expression and fusion protein experiments. 3 The activation profiles of the wt and both mutant alpha(2A) ARs as analysed by a series of alpha(2) AR agonists differed. d-Medetomidine and clonidine appeared most efficacious at the Ser(204)Ala alpha(2A) AR, whereas oxymetazoline was also partially active at the Ser(200)Ala alpha(2A) AR. Talipexole was silent at both mutant alpha(2A) ARs. The intrinsic activity of (-)-adrenaline was either absent or partial at the Ser204Ala and Ser200Ala alpha(2A) AR, respectively. This latter observation is related to its lower binding affinity for both mutant alpha(2A) ARs. 4 Ligands characterized as antagonists at wt and Ser(200)Ala alpha(2A) ARs demonstrated either no intrinsic activity (i.e., RX 811059) or positive efficacy with a different rank order of maximal response at the Ser(204)Ala alpha(2A) AR (atipamezole = SKF 86466 = idazoxan > dexefaroxan) than Asp(79)Asn alpha(2A) AR (atipamezole > idazoxan similar or equal to SKF 86466 > dexefaroxan) and Thr(373)Lys alpha(2A) AR (SKF 86466 > atipamezole - idazoxan > dexefaroxan). These effects were only observed in the coexpression experiments at concentrations in line with their binding affinities. 5 In conclusion, these Ca2+ data suggest that multiple activation binding sites exist for these ligands at the alpha(2A) AR, and that their activation may be affected in different ways by the mutations being investigated.