Electrostatic and Aromatic Microdomains within the Binding-Site Crevice of the D2 Receptor: Contributions of the Second Membrane-Spanning Segment † Jonathan A. Javitch,* ,‡,§ Juan A. Ballesteros, | Jiayun Chen, ‡ Victor Chiappa, ‡ and Merrill M. Simpson ‡ Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, College of Physicians and Surgeons, Columbia UniVersity, 630 West 168th Street, New York, New York 10032, and Department of Physiology and Biophysics, Mount Sinai School of Medicine, New York, New York 10029 ReceiVed March 8, 1999; ReVised Manuscript ReceiVed April 30, 1999 ABSTRACT: The binding-site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. Using the substituted cysteine accessibility method (SCAM), we previously mapped the residues in the third, fifth, sixth, and seventh membrane-spanning segments that contribute to the surface of this binding-site crevice. We have now mutated to cysteine, one at a time, 22 consecutive residues in the second membrane-spanning segment (M2) and expressed the mutant receptors in HEK 293 cells. Eleven of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and 9 of these 11 were protected from reaction by a reversible dopamine antagonist, sulpiride. We infer that the side chains of the residues at the 11 reactive loci (D80, L81, V83, V87, P89, W90, V91, V92, L94, E95, V96) are on the water-accessible surface of the binding-site crevice and that 9 of these are occluded by bound antagonist. The pattern of accessibility suggests an R-helical conformation. A broadening of the angle of accessibility near the binding site is consistent with the presence of a kink at Pro89. On the basis of the enhanced rates of reaction of positively charged sulfhydryl reagents, we infer the presence of an electrostatic microdomain composed of three acidic residues in M2 and the adjacent M3 that could attract and orient cationic ligands. Furthermore, based on the enhanced reactivity of the hydrophobic cation-containing reagent, we infer the presence of an aromatic microdomain formed between M2, M3, and M7. The dopamine receptors, like the homologous receptors for the other biogenic amines, bind neurotransmitters present in the extracellular medium and couple this binding to the activation of intracellular G-proteins (1, 2). The binding sites of these receptors are formed among their seven, mostly hydrophobic, membrane-spanning segments (2, 3) and are accessible to charged, water-soluble agonists, like dopamine. Thus, for each of these receptors, the binding site is contained within a water-accessible crevice, the binding-site crevice, extending from the extracellular surface of the receptor into the transmembrane domain. The surface of this crevice is formed by residues that can contact specific agonists and/or antagonists and by other residues that may play a structural role and affect binding indirectly. To identify the residues that form the surface of the binding-site crevice in the human D2 receptor, we have used the substituted-cysteine accessibility method (SCAM) 1 (4- 10). Consecutive residues in the membrane-spanning seg- ments are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. If ligand binding to a cysteine substitution mutant is near normal, we assume that the structure of the mutant receptor, especially around the binding site, is similar to that of wild-type and that the substituted cysteine lies in a similar orientation to that of the wild-type residue. In the membrane-spanning segments, the sulfhydryl of a cysteine facing into the binding- site crevice should react much faster with charged sulfhydryl- specific reagents than should sulfhydryls facing into the interior of the protein or into the lipid bilayer. For such reagents, we use derivatives of methanethiosulfonate (MTS): positively charged MTS ethylammonium (MTSEA) and MTS ethyltrimethylammonium (MTSET), and negatively charged MTS ethylsulfonate (MTSES) (11). These reagents are about the same size as dopamine, with maximum dimensions of approximately 10 Å by 6 Å. They form mixed disulfides with the cysteine sulfhydryl, covalently linking -SCH 2 CH 2 X, where X is NH 3 + , N(CH 3 ) 3 + , or SO 3 - . We use two criteria for identifying an engineered cysteine as forming the surface of the binding-site crevice: (i) the reaction with an MTS reagent alters binding irreversibly; (ii) this reaction is retarded by the presence of ligand. † This work was supported in part by NIH Grants MH57324 and MH54137, by the G. Harold & Leila Y. Mathers Charitable Trust and by the Lebovitz Trust. * To whom correspondence should be addressed, Columbia Uni- versity, P&S 11-401, Box 7, 630 W. 168th St., New York, NY 10032. E-mail: jaj2@columbia.edu. Phone: 212-305-7308. Fax: 212-305- 5594. ‡ Center for Molecular Recognition. § Departments of Psychiatry and Pharmacology. | Department of Physiology and Biophysics at Mt. Sinai. 1 Abbreviations: SCAM, substituted-cysteine accessibility method; Mn, the nth membrane-spanning segment; MTS, methanethiosulfonate; MTSEA, MTS ethylammonium; MTSET, MTS ethyltrimethylammo- nium; MTSES, MTS ethylsulfonate; MMTS, methylmethanethiosul- fonate; GPCRs, G-protein-coupled receptors; WT, wild-type. 7961 Biochemistry 1999, 38, 7961-7968 10.1021/bi9905314 CCC: $18.00 © 1999 American Chemical Society Published on Web 06/03/1999