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 r eagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfo nate (MTSEA) and the alpha(2)-adrenergic receptor (alpha(2)-AR) agonist chl oroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human alph a(2A)-AR (H alpha 2A). The second-order rate constants for the reaction of the receptor with MTSEA and CEC were determined with the wild type H alpha 2A (cysteine at position 201) and receptor mutants containing accessible cy steines 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 Cy s-200, however, was more than 5 times that of MTSEA, suggesting that these compounds may interact with two different receptor conformations. MTSEA, ha ving no recognition specificity for the receptor, likely reacts with the pr edominant inactive receptor conformation (R), whereas the agonist CEC may s tabilize and react preferentially with the active receptor conformation (R* ), This hypothesis was consistent with three-dimensional receptor-ligand mo dels, which further suggest that alpha(2A)-AR activation may involve the cl ockwise rotation of transmembrane domain 5.