Chloroethylclonidine and 2-aminoethyl methanethiosulfonate recognize two different conformations of the human alpha(2A)-adrenergic receptor

Anne Marjamäki, Heini Frang, Marjo Pihlavisto, Anna-Marja Hoffrén, Tiina Salminen, Mark S Johnson, Jaana Kallio, Jonathan A. Javitch, Mika Scheinin

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Abstract

The substituted cysteine-accessibility method and two sulfhydryl-specific reagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the alpha(2)-adrenergic receptor (alpha(2)-AR) agonist chloroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human alpha(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 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 alpha(2A)-AR activation may involve the clockwise rotation of transmembrane domain 5.
Original languageUndefined/Unknown
Pages (from-to)21867–21872
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number31
DOIs
Publication statusPublished - 1999
MoE publication typeA1 Journal article-refereed

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