b ing site for positively charged dopamine agonists and antagonists. Furthermore, both agobistsand antagonists of the. D2 receptor protected against irreversible ...
Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10355-10359, October 1994
Biochemsstry
A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice (surface mappng/mutagenesl/s
ydryl/methanelou ft/caehlamlne receptor)
JONATHAN A. JAVITCH*ti§, XIAOCHUAN LI*, JOSHUA KABACK*, AND ARTHUR KARLIN*¶ *Center for Molecular Recognition and tDepartment of Psychiatry and Departments of Biochemistry and Molecular Biophysics, Neurology, and Physiology and Cellular Biophysics, Columbia University, New York, NY 10032; and New York State Psychiatric Institute, New York, NY 10032
Communicated by H. Ronald Kaback, July 20, 1994
ABSTRACT The binding site in G-protein-linked neurotramsmitter receptors is formed among their membranepning segments. Because the binding site is in the plane of the bilayer and is accessible to charged, water-soluble agonists, it must lie in a crevice open to the extracellular, aqueous medium. Information about the structure of these receptors can be obtained by identifying the residues in the membranesnning segments which face this water-filed crevice. Human D2 dopa receptor was expressed in human embryonic kidney 293 cells. Small, charged, sulihydryl-specific methanethiosulfonate (MTS) derivatives irreversibly inhibited the bnding of the D2-specific antaoit [3H]YM-09151-2 to these cells. The highly polar MITS derivatives should react with cysteine sulfhydryl groups only at the water-accessible surface of the receptor, which indudes the surface of the binding-site crevice. In contrast, these reagents will have little access to sulfhydryls facing the lipid bilayer or buried in the protein interior. Positively charged MITS reagents irreversibly inhibited binding several hundredfold faster than a negatively charged MTS reagent, consistent with the affinity of the b ing site for positively charged dopamine agonists and antagonists. Furthermore, both agobists and antagonists of the D2 receptor protected against irreversible inbition by the MTS reagents. To identify the susceptible cysteine, we mutated, one at a time, five transmembrane and two extracellular cysteine redues to serine. Only the mutation of Cys11s to serine decreased the ssceptibity of antagonist binding to irreversible Inhibition by the MTS reagents. Thus, Cys118, a residue in the middle of the third membrane-spning segment, is exposed in the D2 receptor binding-site crevice.
Our understanding of the molecular mechanisms of membrane receptor function depends on our knowledge of the molecular structures of receptors and on the assignment of functional roles to structural domains. High-resolution structures of membrane receptors are not yet available, but local structural information obtained by biochemical approaches, such as affinity labeling and mutagenesis, in conjunction with low-resolution structural information obtained by biophysical approaches, has provided some insight into receptor mechanisms and the potential functional roles of specific residues and regions of receptors. Affinity labeling has the advantage that the residues labeled can be inferred to be at the surface, near the residues involved in binding; however, only a few of the residues forming a binding site are likely to be affinity labeled. Mutagenesis has the advantage that any residue can be altered; however, the mutation of residues outside of a binding site can alter binding by long-range perturbation of receptor structure, thus confounding the identification of binding-site residues.
The substitution by mutagenesis of cysteines for other residues is generally well tolerated. The characteristics of the environment surrounding a native or engineered cysteine can be inferred from the relative rates of reaction of sulfhydrylspecific reagents that differ in polarity, charge, or size. For example, hydrophilic, lipophobic reagents will react much faster with cysteines at the water-accessible surface of the protein than at the lipid/protein interface or in the protein interior. The electrostatic potential and steric constraints around cysteines can be probed with reagents of different charges and sizes. A particularly convenient arena for this approach is in ion channels, which are formed by membrane-spanning segments ofthe proteins. Among the residues in the membrane-spanning segments, only those which face the channel lumen should be water accessible. Cysteines substituted for residues in these surfaces should be accessible to small, highly polar reagents, while cysteines substituted for residues facing away from these surfaces, toward the interior of the protein or toward the lipid bilayer, should be inaccessible to such reagents. By determining the functional effects of charged methanethiosulfonate (MTS) derivatives on cysteine-substitution mutants, residues lining the channels of the nicotinic acetylcholine receptor (1), GABAA y-raminobutyrate receptor (2), and the cystic fibrosis transmembrane conductance regulator (3) have been identified. Covalent modification of substituted cysteines has also been used to study the structures ofaspartate receptor (4, 5), colicin El (6, 7), bacteriorhodopsin (8, 9), and lactose permease (10). The binding site in most G-protein-linked receptors is formed among their membrane-spanning segments (11-13). Because the binding site is in the plane of the bilayer and is accessible to charged, water-soluble agonists, it is in a crevice open to the extracellular, aqueous medium. Thus, as in the ion channels, the residues in membrane-spanning segments which form the surface of this binding-site crevice can be mapped by determining the accessibility of substituted cysteines to hydrophilic reagents. The dopamine receptors belong to the superfamily of G-protein-coupled receptors and are targets for drugs used in the treatment of schizophrenia and other psychoses. The D2, D3, and D4 dopamine receptors have different affinities for these drugs (14-18), and these differences may be therapeutically significant. The structural bases for the differences in pharmacological specificity among the dopamine receptor types-and, in fact, among all the catecholamine receptorsare unknown. Site-directed mutagenesis of the D2 receptor has implicated Asp8O, Asp114, and Ser193 and Ser97 (in memAbbreviations: MTS, methanethiosulfonate; MTSEA+, ethylammonium MTS; MTSET+, trimethylammonium MTS; MTSES-, ethylsulfonate MTS; HEK, human embryonic kidney. §To whom reprint requests should be addressed at: Center for Molecular Recognition, Columbia University, P&S 11401, 630 West 168th Street, New York, NY 10032.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10355
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brane-spanning segments II, III, and V) as possible contributors to the binding site (19-21). These four residues are conserved among all catecholamine receptors. Differences in binding specificity among these receptors, however, must be due to nonconserved residues. Before determining the accessibility of substituted cysteines in the D2 dopamine receptor, we needed to determine the accessibility of wild-type cysteines to polar reagents. It had been previously found- that antagonist binding to 12 receptor was inhibited by sulfhydryl reagents (22-26). Although most of the reagents used were hydrophobic and could have reacted with cysteines anywhere in the protein, the polar reagents Hg2+ and 5,5'-dithio(bis-2-nitrobenzoate) also inhibited binding to the D2 receptor (25, 26). We have used highly polar, sulfhydryl-specific reagents to determine whether binding is sensitive to modification of cysteines at the surface of the protein and whether any of these cysteines are in the binding-site crevice. There are 12 cysteine residues in the human D2 dopaniine receptor sequence (Fig. 1A). By analogy with the homologous P-adrenergic receptor (28) and rhodopsin (29, 30), two completely conserved'extracellular cysteines (Cys1'0 and Cys112) are likely to form a disuffide bond and would not react with sulfhydryl reagents. According to the predicted topology of this protein in the membrane, two additional cysteines (Cys4m and Cys4) are extracellular, three cysteines (Cys244, Cys253, and Cys444) are cytoplasmic, and five (Cys-6, Cys118, Cys126, Cysla, and Cys3") are in membrane-spanning segments (Fig. 1A). We have used the following criteria to identify cysteines in the binding-site crevice of the D2 receptor. (i) Binding should be irreversibly inhibited by hydrophilic sulfhydryl-specific reagents added extracellularly. (ii) Binding should be proA
B
FIG. 1. Predicted structure of the D2 dopamine receptor. (A) Topology of the receptor in the membrane, showing the approximate positions of the 12 cysteines. Stippled regions represent the membrane domain. The boxes labeled I-VII represent the putative membrane-spanning segments. EX, extracellular; IN, intracellular. (B) Relative positions of the seven helices in the mid-plane of the membrane and their orientation toward the binding-site crevice as predicted on the basis of sequence analysis by Baldwin (27). Predicted orientations of the transmembrane cysteines and of potential binding-site residues previously identified in the D2 receptor are shown. View is from the extracellular side.
Proc. Natl. Acad. Sci. USA 91 (1994)
tected from such reagents by D2 agonists and antagonists. (iii) Mutation of a cysteine in the binding-site crevice to serine should markedly decrease the sensitivity of binding to these sulfhydryl reagents. [Mutation of a cysteine to a serine is usually conservative, and is likely, in itself, to have modest effects on binding (31).] We used small, highly water-soluble, charged, sulflhydrylspecific reagents which are derivatives of MTS: ethylammonium MTS, CH3SO2SCH2CH2NH' (MTSEA+); trimethylammonium MTS, CH3SO2SCH2CH2N(CH3)3+ (MTSET+); and ethylsulfonate MTS, CH3SO2SCH2CH2SO3 (MTSES-) (1-3, 32). These reagents form mixed disulfides, covalently linking the -SCH2CH2X moiety to the cysteine sulfhydryl, where X is NH', N(CH3)+, or SO3-. These hydrophilic reagents should react only with sulfhydryl groups at the water-accessible surface of the protein and not with sulfhydryls buried in the protein or facing the lipid bilayer. If added extracellularly to intact cells, the reagents will have little access to intracellular cysteines. The reagents do not react with disulfide-bonded cysteines. We found that antagonist binding to the dopamine receptor is blocked irreversibly by the MTS reagents and that D2 receptor agonists and antagonists prevented this block. To identify the sensitive cysteine, we mutated, one at a time, the five transmembrane and the two extracellular cysteine residues to serine and tested for loss of sensitivity of antagonist binding to MTS reagents. Antagonist binding was 100-fold less sensitive to MTS reagents in the mutant in which Cys118 was replaced with serine.
EXPERIMENTAL PROCEDURES Materials. Human D2 dopamine receptor cDNA (DRD2; ref. 33) was provided by 0. Civelli (Vollum Institute, Portland, OR), and pRSVTag (34) was provided by J. Nathans (Johns Hopkins University, Baltimore). pcDNAl/Amp was from Invitrogen and pAlter-1 was from Promega. [3H]YM09151-2 (87 Ci/mmol; 1 Ci = 37 GBq) was obtained from DuPont/NEN. (+)-Butaclamol, (+)- and (-)-sulpiride, and quinpirole were from Research Biochemicals (Natick, MA). The MTS reagents were synthesized according to Stauffer and Karlin (32). The neutral compound methyl MTS was from Aldrich. Human embryonic kidney (HEK) 293 cells (34) were provided by T. Livelli (Columbia University). Preparation of Plasmids and Ofligonucleotide-Mediated Mutagenesis. The coding region of the D2 receptor cDNA was subcloned at the EcoRI site into pcDNA1/Amp, yielding the plasmid pcD2, and into pAlter-1, yielding pAlterD2. Oligonucleotides were synthesized to generate the appropriate serine mutation and to introduce simultaneously a silent restriction site. The Altered Sites mutagenesis system (Promega) was used to introduce the desired mutation. Mutations were identified by restriction mapping and confirmed by DNA sequencing. An appropriate cassette containing the mutation was sequenced in its entirety and was subcloned into pcD2 to generate the appropriate mutant expression plasmid. Mutants are named as (wild-type residue)(residue number)(mutant residue), where the residues are given in the single-letter code. Stable Transfection of Cells. Cells were grown in Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (1:1), containing glucose at 3.15 mg/ml (Specialty Media, Lavellette, NJ) with 10%1 bovine calf serum (HyClone) at 37°C and 5% CO2. On a 100-mm culture plate, 20 .g of pcD2 and 200 ng of pRSVneo were cotransfected into HEK 293 cells by calcium phosphate precipitation (34). After selection with Geneticin (GIBCO) at 700 ug/ml, single colonies were isolated. Cells derived from these individual colonies were screened for [3H]YM-09151-2 binding. One cell line stably expressed -10 pmol of binding sites per mg of membrane
Proc. Natl. Acad. Sci. USA 91 (1994)
Biochemistry: Javitch et al. protein, and this line was used for experiments requiring stably transfected cells. Transient Transfection of Cels. Sixty-millimeter dishes of HEK 293 cells at 60-80% confluence were cotransfected with 2 pg of pcD% or mutant pcD2 and 0.4 pg ofpRSVTag by using 15 p4 of LipofectAMINE (GIBCO) and 3 ml of OptiMEM (GIBCO). Five hours after transfection, the plates were diluted with 3 ml of medium containing 20% bovine calf serum. Twenty-four hours after transfection the medium was changed, and 48 hr after transfection, cells were harvested for binding assay (see below). The same methods were used to transfect cells in 35-mm plates, with volumes adjusted for the surface areas of the plates. Treatment with Sulihydryl Reagents. Cells were washed with phosphate-buffered saline (PBS: 8.1 mM NaH2PO4/1.5 mM KH2PO4/138 mM NaCl/2.7 mM KCI, pH 7.2), briefly treated with enzyme-free dissociation buffer (Specialty Media, Lavellette, NJ), and then dissociated in PBS. Cells were pelleted at 1000 x g for 5 min at 40C, and cells from a 60-mm plate were suspended in 1 ml of PBS, pH 7.4, containing 0.9 mM CaCl2 and 0.5 mM MgCl2 (PBS/Ca2+/Mg2+). Aliquots (100 p4) of cell suspension were incubated with sulfhydryl reagents at the stated concentrations at room temperature for 15 min. Cell suspensions were then diluted with 600 p4 of PBS containing 0.1% bovine serum albumin (Calbiochem), and 100-pd aliquots were used to assay for [3H]YM-09151-2 binding. In some experiments cells were diluted 60-fold with PBS/Ca2+/Mg2+ and 900-pd aliquots were used to assay for [3H]YM-09151-2 binding. Both techniques gave equivalent results. Binding of [3H]YM-09151-2. [3H]YM-09151-2 binding was determined by a modification of reported procedures (35, 36). Triplicate borosilicate tubes contained 100 pM [3H]YM09151-2 in binding buffer (20 mM Hepes/120 mM NaCl/10 mM EDTA, pH 7.4) with cell suspension in a final volume of 2 ml. In some experiments PBS/Ca2+/Mg2+ was used as binding buffer, with identical results. The mixture was incubated at room temperature for 60 min and then filtered with a Brandel cell harvester through a Whatman GF/B filter
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pretreated with 2% polyethyleneimine (Sigma). The filter was washed twice with 5 ml of 10 mM Tris-HCI/120 mM NaCa, pH 7.4, at room temperature. Specific [3H]YM-09151-2 binding was defined as total binding less nonspecific binding in the presence of 1 puM (+)-butaclamol.
RESULTS The positively charged MTSEA+ and MTSET+ maximally abolished >90% of specific [3H]YM-09151-2 binding (Fig. 2A). MTSEA+ and MTSET+ inhibited 50%1 of binding at about 50 pM and 250 pM, respectively. The inhibition of specific binding was caused by a decrease in the maximal number of binding sites, without a significant change in binding affinity (Fig. 2B). The inhibition could not be reversed by removal ofthe MTS reagents but could be reversed partially by 100 mM 2-mercaptoethanol (data not shown), which is capable of reducing the disulfide bond formed between the reagent and the cysteine. In the presence of sulpiride, an antagonist, or quinpirole, an agonist, the receptor was protected against reaction with MTSET+ (Fig. 2C) or MTSEA+ (data not shown). (-)-Sulpiride protected more potently than did (+)-sulpiride, consistent with the relative affinities of the compounds at the D2 receptor binding site
(37).
In contrast to the positively charged reagents, the negatively charged MTSES- was ineffective at inhibiting binding: 100 mM MTSES- inhibited specific binding by
