Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method

Javitch, Jonathan A.; Fu, Ding-Yi; Chen, Jiayun; Karlin, Arthur

doi:10.1016/0896-6273(95)90226-0

Cited by 169 publications

(146 citation statements)

References 26 publications

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“…The rate constants for the reaction of MTSEA with I184C and N186C, 160 and 140 M Ϫ1 ⅐s Ϫ1 , respectively, were comparable to the fastest rate constants we have determined in the TMD of the D2R, for which the range was 0.9 to 290 M Ϫ1 ⅐s Ϫ1 (10,14,15,(27)(28)(29)(30)(31). Thus, these two residues are as exposed as any in the binding-site crevice but not more so.…”

Section: Discussionsupporting

confidence: 54%

The second extracellular loop of the dopamine D₂receptor lines the binding-site crevice

Shi

Javitch²

2004

Proc. Natl. Acad. Sci. U.S.A.

200

184

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The binding site of the dopamine D2 receptor (D2R), like those of homologous rhodopsin-like G protein-coupled receptors (GPCRs) that bind small molecules, is contained within a water-accessible crevice formed among its seven transmembrane segments (TMs). The highresolution structure of bovine rhodopsin, however, revealed that the second extracellular loop (E2), which connects TM4 and TM5, folds down into the transmembrane domain and forms part of the ligandbinding surface for retinal. Whether E2 plays a related role in other rhodopsin-like GPCRs is unclear. To address this issue, we have now mutated to cysteine, one at a time, 10 consecutive residues in E2 of D2R. The reaction of five of these mutants with sulfhydryl reagents inhibited antagonist binding, and bound antagonist protected two, I184C and N186C, from reaction. The pattern of accessibility in E2 is consistent with a structure similar to that of bovine rhodopsin, in which the region C-terminal to the conserved disulfide bond is deeper in the binding-site crevice than is the N-terminal part of E2. Thus, E2 likely contributes to the binding site in the D2R and probably in other aminergic GPCRs as well. Knowledge of its detailed positioning and interactions with ligand would benefit GPCR molecular modeling and facilitate the design of novel drugs.T he extracellular loops are important in ligand binding in G protein-coupled receptors (GPCRs) with large molecular weight ligands, such as peptides (1). The role of these loops in aminergic GPCRs that bind small ligands has received much less attention, and it is widely believed that the transmembrane domain (TMD) is sufficient for ligand binding in these receptors (1). In the high-resolution bovine rhodopsin structure, however, the second extracellular loop (E2) folds down into the binding-site crevice to form a lid over retinal (2). It is unknown in aminergic GPCRs whether the E2 structure is similar to that of rhodopsin or whether E2 plays a role in ligand binding.For nearly all rhodopsin-like GPCRs, the disulfide bond between Cys 3.25 [Cys-107 in dopamine D 2 receptor (D2R)] and the conserved Cys in E2 (Cys e2, Cys-182 in D2R) connects E2 with the TMD § (Fig. 1A), and this disulfide bond (SS-E2) is crucial to the structural integrity and function of many GPCRs. The removal of SS-E2 by mutagenesis severely disrupts ligand binding to muscarinic acetylcholine receptors (3, 4) and destabilizes the high-affinity state of the ␤ 2 adrenergic receptor (AR) (5). Moreover, antagonist protected the ␤ 2 AR from the effects of reduction by DTT (6). Thus, SS-E2 is protected by a conformational change or steric block within the binding site.Although it had been argued that the presence of E2 within the TMD may be a feature unique to rhodopsin (7,8), several reports implicate E2 in ligand specificity in aminergic and other small molecule-ligand GPCRs (reviewed in ref. 9 and Discussion). Our studies of transmembrane segment 4 (TM4) of the D2R also suggested a role for the extracellular end of TM4 and the Nterminal part of E...

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Section: Discussionsupporting

confidence: 54%

The second extracellular loop of the dopamine D₂receptor lines the binding-site crevice

Shi

Javitch²

2004

Proc. Natl. Acad. Sci. U.S.A.

200

184

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“…There are several assumptions implicit to this approach (15)(16)(17)(18)(19)(20). First, it is assumed that following mutation of a given residue to Cys, if significant 1,2,3-benzenetricarboxylate (i.e.…”

Section: Resultsmentioning

confidence: 99%

“…Specifically, we sought to examine the accessibility of sequential residues in this domain to the charged, membrane impermeant, cysteine-specific MTS reagents to deduce information regarding both the secondary structure of this domain, and its contribution of residues to the substrate translocation pathway through the CTP. Whereas this general approach has been applied to a variety of channel and receptor proteins (15)(16)(17)(18)(19)(20)23), to our knowledge the present study represents the first application of this approach to the mitochondrial carrier protein family.…”

Section: Discussionmentioning

confidence: 99%

“…These reagents are small, charged, water soluble, and cysteine-specific (15). They form a mixed disulfide via addition of -SCH 2 CH 2 X to the reduced sulfhydryl of cysteine where X is SO 3 Ϫ , N(CH 3 ) 3 ϩ , or NH 3 ϩ for MTSES, MT-SET, and MTSEA, respectively (15)(16)(17)(18)(19)(20). Importantly, similar to the findings reported by Holmgren et al (21), we observed that when employing liposomes loaded with 5,5Ј-dithio-bis-(2-nitrobenzoic acid) and dithiothreitol (in the absence of added protein), MTSES and MTSET do not significantly permeate the lipid bilayer, whereas in marked contrast MTSEA is quite permeable (data not shown).…”

Section: Accessibility Of Single Cys Ctp Mutants To Mtsesmentioning

confidence: 99%

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The Yeast Mitochondrial Citrate Transport Protein

Kaplan¹,

Mayor²,

Brauer³

et al. 2000

Journal of Biological Chemistry

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The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional properties served as the starting template. The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based on the pattern of accessibility we conclude that residues 177-193 exist as an ␣-helix. The citrate transport protein (i.e. CTP) 1 from mammalian mitochondria catalyzes an electroneutral exchange across the inner mitochondrial membrane of a tricarboxylate (i.e. citrate, isocitrate, cis-aconitate) plus a proton, for either another tricarboxylate-H ϩ , a dicarboxylate (i.e. malate, succinate), or phosphoenolpyruvate (1). Following the efflux of citrate from the mitochondrial matrix via the CTP, cytoplasmic citrate serves as both a carbon precursor for the fatty acid and the sterol biosynthetic pathways, as well as a source of NAD ϩ (via the concerted actions of ATP-citrate lyase and malate dehydrogenase) for the glycolytic pathway (2-5). Because of the central role of the CTP in higher eukaryotic metabolism, the transporter has been extensively studied. Thus, it has been purified (6, 7), kinetically characterized (8), cloned (9), and overexpressed (10). More recently we have extended our studies of the CTP to the yeast homologue of the higher eukaryotic protein (11). An advantage afforded by the yeast mitochondrial CTP is that following overexpression in Escherichia coli and subsequent isolation, CTP function can be reconstituted with high specific activity. Thus, the yeast CTP represents ideal material with which to conduct a comprehensive structure/function analysis. With this goal in mind, we recently constructed a Cys-less CTP that displays near native functional properties. Moreover, we have demonstrated that upon isolation, both the isolated wild-type and Cys-less CTPs exist as functional dimers.3 Based on hydropathy analysis, each monomer of the homodimer is predicted to contain six membrane-spanning domains (11).Within the CTP, transmembrane domain IV (i.e. TMDIV) is of particular interest for several reasons. First, this domain contains two arginine residues (Arg-181 and -189). We have previously demonstrated that CTP function requires the presence of positive charge at both sites.2 Second, TMDIV can be modeled as an ␣-helix with both arginines residing on the same face of the helix, thereby providing a polar domain, which might conceivably be accessible to citrate and provide a partial neutra...

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“…The substituted-cysteine-accessibility method (SCAM) is an approach to the characterization of channel structure (51)(52)(53) and binding-site structure (54)(55)(56) that probes the environment of any residue by mutating it to Cys and by characterizing the reaction of the Cys with sulfhydryl-specific reagents. Both because of the polarity of the methanethiosulfonates used (51,54) and because these reagents react at least 10 orders of magnitude faster with ionized thiolates than with unionized thiols (57), the reactions are directed to Cys at the water-accessible surface of the protein.…”

mentioning

confidence: 99%

Inaugural Article: Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method

Wilson¹

2001

Proceedings of the National Academy of Sciences

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The nicotinic acetylcholine (ACh) receptors cycle among classes of nonconducting resting states, conducting open states, and nonconducting desensitized states. We previously probed the structure of the mouse-muscle ACh receptor channel in the resting state obtained in the absence of agonist and in the open states obtained after brief exposure to ACh. We now have probed the structure in the stable desensitized state obtained after many minutes of exposure to ACh. Muscle-type receptor has the subunit composition ␣2␤␥␦. Each subunit has four membrane-spanning segments, M1-M4. The channel lumen in the membrane domain is lined largely by M2 and to a lesser extent by M1 from each of the subunits. We determined the rates of reaction of a small, sulfhydryl-specific, charged reagent, 2-aminoethyl methanethiosulfonate with cysteines substituted for residues in ␣M2 and the ␣M1-M2 loop in the desensitized state and compared these rates to rates previously obtained in the resting and open states. The reaction rates of the substituted cysteines are different in the three functional states of the receptor, indicating significant structural differences. By comparing the rates of reaction of extracellularly and intracellularly added 2-aminoethyl methanethiosulfonate, we previously located the closed gate in the resting state between ␣G240 and ␣T244, in the predicted M1-M2 loop at the intracellular end of M2. Now, we have located the closed gate in the stable desensitized state between ␣G240 and ␣L251. The gate in the desensitized state includes the resting state gate and an extension further into M2.T he nicotinic acetylcholine (ACh) receptors cycle among three classes of functional states: resting, open, and desensitized (1). The receptor is conducting in the open state and nonconducting in the resting and desensitized states. The resting state is the most stable state when no agonist is bound, whereas the desensitized state is the most stable state when agonist is bound. Muscle-type ACh receptors have two ACh binding sites corresponding to the two ␣ subunits in the pentameric complex (Fig. 1, refs. 2 and 3). In the prevailing cycle of transitions, receptors in the resting state bind two molecules of ACh, isomerize to the open state, and, in the continued presence of ACh eventually desensitize. After the removal of free ACh and its dissociation from the binding sites, receptors in the desensitized state predominantly isomerize directly to the resting state. This cycle of activation, desensitization, and recovery has been extensively studied electrophysiologically (1, 4-7). The role of desensitization in cholinergic neurotransmission under normal physiological conditions is uncertain but is evident under some pathological conditions and in neurotransmission by other neurotransmitters (8).Desensitization occurs in stages (9). Receptor isomerizes to a transient, fast-onset desensitized state on the 0.1-to 10-s time scale and to a stable, slow-onset desensitized state on the 10-to 100-s time scale (10-17). The ACh affinities of the...

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Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method

Cited by 169 publications

References 26 publications

The second extracellular loop of the dopamine D₂receptor lines the binding-site crevice

The second extracellular loop of the dopamine D₂receptor lines the binding-site crevice

The Yeast Mitochondrial Citrate Transport Protein

Inaugural Article: Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method

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Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method

Cited by 169 publications

References 26 publications

The second extracellular loop of the dopamine D2receptor lines the binding-site crevice

The second extracellular loop of the dopamine D2receptor lines the binding-site crevice

The Yeast Mitochondrial Citrate Transport Protein

Inaugural Article: Acetylcholine receptor channel structure in the resting, open, and desensitized states probed with the substituted-cysteine-accessibility method

Contact Info

Product

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The second extracellular loop of the dopamine D₂receptor lines the binding-site crevice

The second extracellular loop of the dopamine D₂receptor lines the binding-site crevice