Molecular mechanisms of ligand-receptor interactions in transmembrane domain V of the α2A-adrenoceptor

1 The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 alpha(2)-adrenergic agonists and a panel of mutated human alpha(2A)-adrenoceptors. The alpha(2A)Ser201 mutant had a Cys-->Ser201 (position 5.43) amino-acid substitution, and alpha(2A)Ser201Cys200 and alpha(2A)Ser201Cys204 had Ser-->Cys200 (5.42) and Ser-->Cys204 (5.46) substitutions, respectively, in addition to the Cys-->Ser201 substitution. 2 Automated docking methods were used to predict the receptor interactions of the ligands. Radioligand-binding assays and functional [S-35] GTPgammaS-binding assays were performed using transfected Chinese hamster ovary cells to experimentally corroborate the predicted binding modes. 3 The hydroxyl groups of phenethylamines were found to have different effects on ligand affinity towards the activated and resting forms of the wild-type alpha(2A)-adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the alpha(2A)-adrenoceptor. The findings indicate that (i) Cys201 plays a significant role in the binding of catecholamine ligands and UK14,304 (for the latter, by a hydrophobic interaction), but Cys201 is not essential for receptor activation; (ii) Ser200 interacts with the meta-hydroxyl group of phenethylamine ligands, affecting both catecholamine binding and receptor activation; while (iii) substituting Ser204 with a cysteine interferes both with the binding of catecholamine ligands and with receptor activation, due to an interaction between Ser204 and the para-hydroxyl group of the catecholic ring.