Disparate ligand‐mediated Ca 2+ responses by wild‐type, mutant Ser 200 Ala and Ser 204 Ala α 2A ‐adrenoceptor : G α15 fusion proteins: evidence for multiple ligand‐activation binding sites

Ligand : receptor interactions were analysed at wt, mutant Ser 200 Ala and Ser 204 Ala α 2A ARs by measuring Ca 2+ 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. The magnitude of the UK 14304‐mediated Ca 2+ response as elicited by a G α15 protein was largest with both mutant Ser 200 Ala and Ser 204 Ala α 2A ARs compared to the wt α 2A AR in the co‐expression and fusion protein experiments. The activation profiles of the wt and both mutant α 2A ARs as analysed by a series of α 2 AR agonists differed. d‐Medetomidine and clonidine appeared most efficacious at the Ser 204 Ala α 2A AR, whereas oxymetazoline was also partially active at the Ser 200 Ala α 2A AR. Talipexole was silent at both mutant α 2A ARs. The intrinsic activity of (−)‐adrenaline was either absent or partial at the Ser 204 Ala and Ser 200 Ala α 2A AR, respectively. This latter observation is related to its lower binding affinity for both mutant α 2A ARs. Ligands characterized as antagonists at wt and Ser 200 Ala 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 Ser 204 Ala α 2A AR (atipamezole=SKF 86466=idazoxan>dexefaroxan) than Asp 79 Asn α 2A AR (atipamezole>idazoxan≃SKF 86466>dexefaroxan) and Thr 373 Lys 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 Ca 2+ 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.British Journal of Pharmacology (2000) 130 , 1505–1512; doi: 10.1038/sj.bjp.0703455