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

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Abstract
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 α2A 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 α2A AR, and that their activation may be affected in different ways by the mutations being investigated. British Journal of Pharmacology (2000) 130, 1505–1512; doi:10.1038/sj.bjp.0703455

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