Heterogeneous ligand-mediated Ca++ responses at wt and mutant α2A-adrenoceptors suggest multiple ligand activation binding sites at the α2A-adrenoceptor

Ligand:receptor interactions were analysed at wild-type, Asp(79)Asn and Th(373)Lys alpha(2A) AR by measuring Ca++ responses in the co-presence of a G(alpha 15) protein in CHO-K1 cells. (-)-Adrenaline displayed a time-dependent Ca++ response with the following magnitude: wt alpha(2A) AR>Thr(373)Lys alpha(2A) AR>Asp(79)Asn alpha(2A) AR. The maximal amplitude of activation by d-medetomidine and clonidine versus that of (-)-adrenaline was not affected by the Asp(79)Asn mutation, whereas it was significantly lower for both UK 14304 (-42%) and oxymetazoline (-35%). BHT 920 induced a higher Ca++ response (+19%) at the Asp79Asn alpha(2A) AR. Some (atipamezole>BRL 44408=idazoxan similar or equal to SKF 86466>dexefaroxan) but not all (RX 811059 and RS 15385) of the putative alpha(2)AR antagonists tested also displayed partial agonist properties at the Asp(79)Asn alpha(2A) AR. At the Thr(373)Lys alpha(2A) AR, high-efficacy responses were produced by each of the agonists, whereas the putative antagonists showed the following rank order of maximal responses: BRL 44408 >SKF 86466>Btipamezole-idazoxan>dexefaroxan. The observed heterogeneity of Ca++ responses produced by differ ent ligands at wt and mutant alpha(2A) AR may be explained by assuming the existence of multiple ligand activation binding sites at the alpha(2A) AR. (C) 2000 Elsevier Science Ltd. All rights reserved.