Section 9
Chapter 8,477

Differentiating dopamine D2 ligands by their sensitivities to modification of the cysteine exposed in the binding-site crevice

Javitch, J.A.; Fu, D.; Chen, J.

Molecular Pharmacology 49(4): 692-698


ISSN/ISBN: 0026-895X
PMID: 8609898
Accession: 008476032

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Cys-118, in the third membrane-spanning segment of the dopamine D-2 receptor, is exposed in the binding-site crevice. Cys-118 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 -SCH-2CH-2NH-3+ to the cysteine sulfhydryl, producing a lysine-like side chain. The reaction of MTSEA with Cys-118 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 ltoreq 6-fold. Agonist affinities were decreased 3-12,000-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 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 Asp-114, located one alpha-helical turn above Cys-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 interfere with binding. The reaction of MTSET with Cys-118 covalently attaches -SCH-2CH-2N(CH-3)-3+, which is bulkier and apprx 2 ANG longer than the -SCH-2CH-2NH-3+ 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 Cys-118.

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