Anna-Marja Hoffrén

CHLOROETHYLCLONIDINE AND 2-AMINOETHYL METHANETHIOSULFONATE RECOGNIZE TWO DIFFERENT CONFORMATIONS OF THE HUMAN ALPHA 2A-ADRENERGIC RECEPTOR

The substituted cysteine-accessibility method and two sulfhydryl-specific reagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the α2-adrenergic receptor (α2-AR) agonist chloroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human α2A-AR (Hα2A). The second-order rate constants for the reaction of the receptor with MTSEA and CEC were determined with the wild type Hα2A (cysteine at position 201) and receptor mutants containing accessible cysteines at other positions within the binding-site crevice (positions 197, 200, and 204). The rate of reaction of CEC was similar to that of MTSEA at residues Cys-197, Cys-201, and Cys-204. The rate of reaction of CEC with Cys-200, however, was more than 5 times that of MTSEA, suggesting that these compounds may interact with two different receptor conformations. MTSEA, having no recognition specificity for the receptor, likely reacts with the predominant inactive receptor conformation (R), whereas the agonist CEC may stabilize and react preferentially with the active receptor conformation (R*). This hypothesis was consistent with three-dimensional receptor-ligand models, which further suggest that α2A-AR activation may involve the clockwise rotation of transmembrane domain 5.

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

The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 α2‐adrenergic agonists and a panel of mutated human α2A‐adrenoceptors. The α2ASer201 mutant had a Cys → Ser201 (position 5.43) amino‐acid substitution, and α2ASer201Cys200 and α2ASer201Cys204 had Ser → Cys200 (5.42) and Ser → Cys204 (5.46) substitutions, respectively, in addition to the Cys → Ser201 substitution. Automated docking methods were used to predict the receptor interactions of the ligands. Radioligand‐binding assays and functional [35S]GTPγS‐binding assays were performed using transfected Chinese hamster ovary cells to experimentally corroborate the predicted binding modes. The hydroxyl groups of phenethylamines were found to have different effects on ligand affinity towards the activated and resting forms of the wild‐type α2A‐adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the α2A‐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.