Differentiating dopamine D2 ligands by their sensitivities to modification of the cysteine exposed in the binding-site crevice.
Cys118, in the third membrane-spanning segment of the dopamine D2 receptor, is exposed in the binding-site crevice. Cys118 reacts with the highly polar, sulfhydryl-specific reagents methanethiosulfonate ethylammonium (MTSEA) and methanethiosulfonate ethyltrimethylammonium (MTSET), and this reaction is retarded by the presence of antagonists and agonists. The reaction of MTSEA covalently attaches-SCH2CH2NH3+ to the cysteine sulfhydryl, producing a lysine-like side chain. The reaction of MTSEA with Cys118 decreased the affinity of substituted-benzamide antagonists, such as YM-09151-2, by 50-2800-fold, whereas the affinities of other antagonists, such as N-methyl-spiperone, were decreased < / = 6-fold. Agonist affinities were decreased 3-12,000-fold. Mutation of Cys118 to Lys had effects similar to that of the reaction of Cys118 with MTSEA. In contrast, mutation to the uncharged Met, the side-chain volume of which is similar to that of Lys, had much lesser effects on binding. All of the agonists and antagonists contain a positively charged nitrogen that is thought to interact with the side chain of Asp114, located one alpha-helical turn above Cys118. If this nitrogen is close to Asp114, then in the substituted-benzamides, the group on the nitrogen or the pyrrolidine ring itself could extend toward Cys118. Modification of Cys118 would then interfere with binding. The reaction of MTSET with Cys118 covalently attaches-SCH2CH2N(CH3)3+, which is bulkier and approximately 2 angstroms longer than the -SCH2CH2NH3+ added by MTSEA. In contrast to MTSEA, MTSET had equally large effects on the binding of YM-09151-2 and N-methyl-spiperone. Therefore, the effect on binding depends on both the size and the charge of the side chain substituted for that of Cys118.
Javitch
J A
JA
Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York 10032, USA.
Fu
D
D
Chen
J
J
D001549:Benzamides / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017448:Receptors, Dopamine D2 / Q000378:metabolism*; D013134:Spiperone / Q000031:analogs & derivatives / Q000378:metabolism; D013329:Structure-Activity Relationship
D001549:Benzamides; D017448:Receptors, Dopamine D2; D013134:Spiperone; C039167:3-N-methylspiperone; D003545:Cysteine; C030265:nemonapride