Disparate ligand-mediated Ca2+ responses by wild-type, mutant Ser200Ala and Ser204Ala α(2A)-adrenoceptor: G(α15) fusion proteins: Evidence for multiple ligand-activation binding sites

Abstract

1. Ligand:receptor interactions were analysed at wt, mutant Ser200Ala and Ser204Ala α(2A) ARs by measuring Ca2+ responses in CHO-K1 cells either by co-expression with a G(α15) protein or at a receptor:G(α15) protein stoichiometry of 1.0 using fusion proteins. 2. The magnitude of the UK 14304-mediated Ca2+ response as elicited by a G(α15) protein was largest with both mutant Ser200Ala and Ser204Ala α(2A) ARs compared to the wt α(2A) AR in the co-expression and fusion protein experiments. 3. The activation profiles of the wt and both mutant α(2A) ARs as analysed by a series of α2 AR agonists differed. d-Medetomidine and clonidine appeared most efficacious at the Ser204Ala α(2A) AR, whereas oxymetazoline was also partially active at the Ser200Ala α(2A) AR. Talipexole was silent at both mutant α(2A) ARs. The intrinsic activity of (-)-adrenaline was either absent or partial at the Ser204Ala and Ser200Ala α(2A) AR, respectively. This latter observation is related to its lower binding affinity for both mutant α(2A) ARs. 4. Ligands characterized as antagonists at wt and Ser200Ala α(2A), ARs demonstrated either no intrinsic activity (i.e., RX 811059) or positive efficacy with a different rank order of maximal response at the Ser204Ala α(2A) AR (atipamezole = SKF 86466 = idazoxan > dexefaroxan) than Asp79Asn α(2A) AR (atipamezole > idazoxan ≃ SKF 86466 > dexefaroxan) and Thr373Lys α(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 α(2A) AR, and that their activation may be affected in different ways by the mutations being investigated.