DIFFERENTIATING DOPAMINE D-2 LIGANDS BY THEIR SENSITIVITIES TO MODIFICATION OF THE CYSTEINE EXPOSED IN THE BINDING-SITE CREVICE

Cys(118), in the third membrane-spanning segment of the dopamine D-2 r eceptor, is exposed in the binding-site crevice. Cys(118) reacts with the highly polar, sulfhydryl-specific reagents methanethiosulfonate et hylammonium (MTSEA) and methanethiosulfonate ethyltrimethylammonium (M TSET), and this reaction is retarded by the presence of antagonists an d agonists. The reaction of MTSEA covalently attaches -SCH2CH2NH3+ to the cysteine sulfhydryl, producing a lysine-like side chain. The react ion of MTSEA with Cys(118) decreased the affinity of substituted-benza mide antagonists, such as YM-09151-2, by 50-2800-fold, whereas the aff inities of other antagonists, such as N-methyl-spiperone, were decreas ed less than or equal to 6-fold. Agonist affinities were decreased 3-1 2,000-fold. Mutation of Cys(118) to Lys had effects similar to that of the reaction of Cys(118) with MTSEA. In contrast, mutation to the unc harged Met, the side-chain volume of which is similar to that of Lys, had much lesser effects on binding. All of the agonists and antagonist s contain a positively charged nitrogen that is thought to interact wi th the side chain of Asp(114), located one alpha-helical turn above Cy s(118). If this nitrogen is close to Asp(114), then in the substituted -benzamides, the group on the nitrogen or the pyrrolidine ring itself could extend toward Cys(118). Modification of Cys(118) would then inte rfere with binding. The reaction of MTSET with Cys(118) covalently att aches -SCH2CH2N(CH3)(3)(+), which is bulkier and similar to 2 Angstrom longer than the -SCH2CH2NH3+ added by MTSEA. In contrast to MTSEA, MT SET had equally large effects on the binding of YM-09151-2 and N-methy l-spiperone. Therefore, the effect on binding depends on both the size and the charge of the side chain substituted for that of Cys(118).