G‐protein activation by putative antagonists at mutant Thr 373 Lys α 2A adrenergic receptors

Replacement of a threonine by a lysine at position 373 in the C‐terminal portion of the third intracellular loop of the human α 2A ‐adrenergic receptor (α 2A AR) has been reported to generate a constitutively active mutant receptor in analogy with similar mutations in the α 1B and β 2 AR ( Ren et al ., 1993 ). In the present study, the mutant Thr 373 Lys α 2A AR receptor was investigated by measuring the formation of inositol phosphates in either the absence or presence of mouse G α15 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 α 2A AR: G‐protein state. The basal inositol phosphates response was similar at the wild‐type (wt) and mutant α 2A AR, but was enhanced at the mutant α 2A AR upon co‐expression with the mouse G α15 protein. This enhanced response could not be attenuated in the presence of any of the tested α 2 AR antagonists (10 μ M ), suggesting that inverse agonist activity did not occur at the mutant α 2A AR. Ligands that so far have been identified as antagonists at the wt α 2A AR demonstrated either no intrinsic activity (MK 912, WB 4101, RS 15385, RX 811059 and RX 821002) or positive efficacy [E max , % vs. 1 μ M UK 14304: dexefaroxan (27±7), idazoxan (34±9), atipamezole (27±4), BRL 44408 (59±5) and SKF 86466 (54±9)] at the mutant α 2A AR, but only in the presence of the mouse G α15 protein. The ligand potencies corresponded with their respective p K i values at the mutant α 2A 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 G αi1 protein. It was virtually absent in the presence of 10 μ M RS 15385. SKF 86466 was without intrinsic activity upon co‐expression of the mouse G αq protein. Some putative α 2 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 α 2A AR.British Journal of Pharmacology (1999) 126 , 939–948; doi: 10.1038/sj.bjp.0702379