Heterogeneous ligand-mediated Ca++ responses at wt and mutant alpha(2A)-adrenoceptors suggest multiple ligand activation binding sites at the alpha(2A)-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-dependen t Ca++ response with the following magnitude: wt alpha(2A) AR>Thr(373)Lys a lpha(2A) AR>Asp(79)Asn alpha(2A) AR. The maximal amplitude of activation by d-medetomidine and clonidine versus that of (-)-adrenaline was not affecte d by the Asp(79)Asn mutation, whereas it was significantly lower for both U K 14304 (-42%) and oxymetazoline (-35%). BHT 920 induced a higher Ca++ resp onse (+19%) at the Asp79Asn alpha(2A) AR. Some (atipamezole>BRL 44408=idazo xan similar or equal to SKF 86466>dexefaroxan) but not all (RX 811059 and R S 15385) of the putative alpha(2)AR antagonists tested also displayed parti al agonist properties at the Asp(79)Asn alpha(2A) AR. At the Thr(373)Lys al pha(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 obse rved heterogeneity of Ca++ responses produced by differ ent ligands at wt a nd mutant alpha(2A) AR may be explained by assuming the existence of multip le ligand activation binding sites at the alpha(2A) AR. (C) 2000 Elsevier S cience Ltd. All rights reserved.