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

摘要

class="enumerated" style="list-style-type:decimal">Ligand : receptor interactions were analysed at wt, mutant Ser²⁰⁰Ala and Ser²⁰⁴Ala α2A ARs by measuring Ca²⁺ responses in CHO-K1 cells either by co-expression with a Gα15 protein or at a receptor : Gα15 protein stoichiometry of 1.0 using fusion proteins.The magnitude of the UK 14304-mediated Ca²⁺ response as elicited by a Gα15 protein was largest with both mutant Ser²⁰⁰Ala and Ser²⁰⁴Ala α2AARs compared to the wt α2A AR in the co-expression and fusion protein experiments.The activation profiles of the wt and both mutant α2A ARs as analysed by a series of α2 AR agonists differed. d-Medetomidine and clonidine appeared most efficacious at the Ser²⁰⁴Ala α2A AR, whereas oxymetazoline was also partially active at the Ser²⁰⁰Ala α2A AR. Talipexole was silent at both mutant α2A ARs. The intrinsic activity of (−)-adrenaline was either absent or partial at the Ser²⁰⁴Ala and Ser²⁰⁰Ala α2A AR, respectively. This latter observation is related to its lower binding affinity for both mutant α2A ARs.Ligands characterized as antagonists at wt and Ser²⁰⁰Ala α2A ARs demonstrated either no intrinsic activity (i.e., RX 811059) or positive efficacy with a different rank order of maximal response at the Ser²⁰⁴Ala α2A AR (atipamezole=SKF 86466=idazoxan>dexefaroxan) than Asp⁷⁹Asn α2A AR (atipamezole>idazoxan≃SKF 86466>dexefaroxan) and Thr³⁷³Lys α_2A AR (SKF 86466>atipamezole≃idazoxan>dexefaroxan). These effects were only observed in the co-expression experiments at concentrations in line with their binding affinities.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.