G‐protein activation by putative antagonists at mutant Thr373Lys α2A adrenergic receptors

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Abstract
Replacement of a threonine by a lysine at position 373 in the C‐terminal portion of the third intracellular loop of the human α2A‐adrenergic receptor (α2A AR) has been reported to generate a constitutively active mutant receptor in analogy with similar mutations in the α1B and β2 AR (Ren et al., 1993). In the present study, the mutant Thr373Lys α2A AR receptor was investigated by measuring the formation of inositol phosphates in either the absence or presence of mouse Gα15 protein in Cos‐7 cells. Increased affinity, potency and/or efficacy for the agonists [(−)‐adrenaline, UK 14304, clonidine, guanabenz and oxymetazoline] was observed, consistent with a precoupled mutant α2A AR: G‐protein state. The basal inositol phosphates response was similar at the wild‐type (wt) and mutant α2A AR, but was enhanced at the mutant α2A AR upon co‐expression with the mouse Gα15 protein. This enhanced response could not be attenuated in the presence of any of the tested α2 AR antagonists (10 μM), suggesting that inverse agonist activity did not occur at the mutant α2A AR. Ligands that so far have been identified as antagonists at the wt α2A AR demonstrated either no intrinsic activity (MK 912, WB 4101, RS 15385, RX 811059 and RX 821002) or positive efficacy [Emax, % vs. 1 μM UK 14304: dexefaroxan (27±7), idazoxan (34±9), atipamezole (27±4), BRL 44408 (59±5) and SKF 86466 (54±9)] at the mutant α2A AR, but only in the presence of the mouse Gα15 protein. The ligand potencies corresponded with their respective pKi values at the mutant α2A AR receptor. The partial agonist effect of SKF 86466 was resistant to pertussis toxin treatment (100 ng ml−1) and not affected by co‐expression of the rat Gαi1 protein. It was virtually absent in the presence of 10 μM RS 15385. SKF 86466 was without intrinsic activity upon co‐expression of the mouse Gαq protein. Some putative α2 AR antagonists exerted a partial agonist activity that was highly dependent on the presence of specific G‐protein α‐subunits, suggesting that these ligands cause selective G‐protein activation at the mutant α2A AR. British Journal of Pharmacology (1999) 126, 939–948; doi:10.1038/sj.bjp.0702379