Use of a Disulfide Cross-linking Strategy to Study Muscarinic Receptor Structure and Mechanisms of Activation*

Fu-Yue Zeng, Amanda Hopp, Andrea Soldner, and Jürgen WessDagger

From the Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

G protein-coupled receptors (GPCRs)1 form one of the largest protein families found in nature, and current estimates are that approximately one thousand different such receptors exist in mammals (1). Despite the remarkable structural diversity of their activating ligands, all GPCRs are predicted to share a common molecular architecture consisting of seven transmembrane helices (TM I-VII) linked by alternating intracellular (i1-i3) and extracellular (o2-o4) loops (Fig. 1). Whereas residues located on the extracellular receptor surface are known to be involved in ligand binding, the cytoplasmic receptor surface is critical for G protein recognition and activation (2-6).

At present, high resolution structural information is not available for any GPCR, primarily due to the difficulties of studying integral membrane proteins by biophysical techniques such as NMR spectroscopy or x-ray crystallography. However, guided by low-resolution projection density maps of bovine (7) and frog (8) rhodopsin obtained by electron cryo-microscopy and structural information gathered from the analysis of approximately 500 different GPCRs, Baldwin and coworkers (9, 10) have generated a model for the alpha -carbon positions in TM I-VII of GPCRs of the rhodopsin family. This model is consistent with the results of a great number of mutagenesis and biochemical studies (10-12).

Whereas the relative positions of the amino acids located within the transmembrane receptor core of GPCRs can be predicted with a relatively high degree of certainty, much less is known about the structural characteristics of the intracellular receptor surface predicted to be involved in G protein coupling. To gain insight into the molecular architecture of the cytoplasmic receptor regions, NMR and circular dichroism studies have been carried out with peptides derived from distinct intracellular GPCR domains (13-17). Such studies have shown, for example, that the i1-i4 regions of the photoreceptor, rhodopsin, can adopt defined structures in solution (13-15). It remains unclear, however, whether the isolated peptides fold in a fashion similar to that found in the native receptor and how the different intracellular domains are arranged relative to each other in the full-length receptor. Thus, additional experimental strategies need to be applied to gain deeper insight into the three-dimensional arrangement of the cytoplasmic GPCR domains.

One such approach, referred to as "site-directed disulfide mapping," involves the ability of two Cys residues that are adjacent to each other in the three-dimensional structure of a protein to form disulfide bonds, either spontaneously or upon application of oxidizing conditions. This strategy has been successfully employed, for example, to elucidate key structural features of several bacterial chemoreceptors (18-21). Since these proteins form homodimers, disulfide cross-linked homodimers could be detected by their specific migration patterns on SDS gels (non-reducing conditions). Recently, a similar strategy has also been used to study the assembly of the photoreceptor, rhodopsin (22-26). To be able to monitor the formation of disulfide bonds between two proximal Cys residues, Oprian and co-workers (22, 27) took advantage of the observation that rhodopsin, as well as other GPCRs (28-33), can be properly assembled from two coexpressed receptor fragments. Cross-linking between juxtaposed Cys residues located on different receptor fragments (following site-specific Cys mutagenesis) was again monitored via SDS-PAGE (22, 23). Moreover, Khorana and coworkers (24-26) carried out disulfide cross-linking studies using full-length rhodopsin molecules, following systematic Cys-scanning mutagenesis. These investigators took advantage of two native protease cleavage sites present in the i3 and i4 regions of rhodopsin, allowing the monitoring of disulfide bond formation by studying changes in the electrophoretic mobility of oxidized, protease-treated receptors on SDS gels (24-26).

At present, rhodopsin is the only GPCR that has been subjected to site-directed disulfide cross-linking studies, primarily due to the fact that rhodopsin can be expressed at very high levels in cultured cells, can be readily purified with high yields, and can be assayed functionally by spectrophotometry in a straightforward manner. To examine whether the results of disulfide cross-linking studies obtained with the photoreceptor, rhodopsin, are of broad general relevance, we decided to develop a site-directed disulfide mapping approach that can also be applied to other classes of GPCRs. In the present study, we used a member of the muscarinic acetylcholine receptor family, the rat m3 muscarinic receptor subtype (34, 35), as a model system.

To facilitate the interpretation of disulfide cross-linking data, we initially generated a modified version of the m3 muscarinic receptor (m3'(3C)) in which most native Cys residues (except for Cys-140, Cys-220, and Cys-532) were deleted (Cys residues located within the central portion of the i3 loop) or replaced with Ala or Ser (Fig. 1 and Table I). In the next step, a protease (factor Xa) cleavage site was introduced into the i3 loop of the m3'(3C) construct, yielding the m3'(3C)-Xa mutant receptor. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional.

As mentioned above, our knowledge about the structural organization of the intracellular surface of GPCRs is still rather limited. We therefore decided to focus our disulfide scanning analysis on the arrangement of the intracellular regions of the m3'(3C)-Xa mutant receptor. This initial study was designed to define the spatial relationship between residues located at the N terminus of the i2 loop and the C-terminal segment of the i3 domain, two receptor regions known to be critically involved in receptor/G protein recognition and activation (2-6, 34).

By using the m3'(3C)-Xa mutant receptor as a template, we generated a total of 10 double Cys mutants. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the i2 loop (Fig. 1). A second Cys residue was introduced within a receptor segment located at the junction between the i3 loop and TM VI (Fig. 1), by individually replacing amino acids 484-493 with Cys. The mutant receptors were expressed in COS-7 cells, and the ability of juxtaposed Cys residues to form disulfide bonds was then studied using a combined immunoprecipitation/immunoblotting strategy, involving the use of the oxidizing agent Cu(II)-(1,10-phenanthroline)3 (hereafter Cu(II)-phenanthroline) and factor Xa protease. In addition, the functional consequences of disulfide cross-linking were examined in [35S]GTPgamma S binding studies.

Our results are consistent with the notion that the intracellular surface of the m3 muscarinic receptor is highly dynamic and that this high degree of conformational flexibility is essential for efficient receptor/G protein coupling.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Carbamoylcholine chloride (carbachol), acetylcholine chloride, atropine sulfate, copper sulfate, 1,10-phenanthroline, and CNBr-activated Sepharose 4B were purchased from Sigma. N-[3H]Methylscopolamine ([3H]NMS; 79 Ci/mmol), myo-[3H]inositol (20 Ci/mmol), and [35S]GTPgamma S (1300 Ci/mmol) were from NEN Life Science Products. All enzymes used for molecular cloning were obtained from New England Biolabs. Other sources of reagents were as follows: 12CA5 monoclonal antibody (Roche Molecular Biochemicals), factor Xa (Roche Molecular Biochemicals), anti-mouse or anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Amersham Pharmacia Biotech), and enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech). Nitrocellulose membranes (0.2 µm) were obtained from Schleicher & Schuell. All other chemicals used for SDS-PAGE and Western blotting were purchased from Bio-Rad.

Construction of Mutant m3 Muscarinic Receptor Genes-- All mutations were introduced into Rm3pcD-N-HA (also referred to as m3(wt); Table I), a mammalian expression plasmid coding for the rat m3 muscarinic receptor (35) containing a 9-amino acid hemagglutinin (HA) epitope tag (YPYDVPDYA) at the N terminus (30). Most native Cys residues were replaced, in a sequential fashion, with either Ala or Ser, by using standard polymerase chain reaction (PCR) mutagenesis techniques (36) (Fig. 1 and Table I). To eliminate the three Cys residues (Cys-289, Cys-310, and Cys-419) located within the i3 loop, the central portion of the i3 loop (amino acids Ala-274 to Lys-469) was deleted, as described by Maggio et al. (37). In addition, in two of the mutant receptors (m3'(3C) and m3'(3C)-Xa; see Table I), the five N-terminal Asn residues that are predicted to serve as targets for N-linked glycosylation (Asn-6, Asn-15, Asn-41, Asn-48, and Asn-52; Fig. 1) were simultaneously replaced with Gln residues, using a two-step polymerase chain reaction procedure.

Three factor Xa cleavage sites ((IEGR)3, triple direct repeat) were introduced into the i3 loop, between Glu-273 and Thr-470, of the m3'(3C) mutant receptor in which Cys-140, Cys-220, and Cys-532 were the only remaining Cys residues (Table I and Fig. 1). The resulting construct was referred to as m3'(3C)-Xa throughout this study. Single Cys residues were reintroduced into m3'(3C)-Xa at positions Ile-169 and Lys-484 to Ser-493, by using the QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Double Cys mutants (in the m3'(3C)-Xa background) were obtained by subcloning a 0.4-kilobase pair BstxI-SacI fragment derived from the mutant m3'(3C)-Xa constructs containing a Cys substitution within the Lys-484 to Ser-493 sequence into the m3'(3C)-Xa construct containing the additional I169C point mutation. The identity of all mutant constructs was verified by DNA sequencing.

Transient Expression of Receptor Constructs-- COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/ml penicillin, and 100 units/ml streptomycin, at 37 °C in a humidified 5% CO2 incubator. For transfections, 1 × 106 cells were seeded into 100-mm dishes. About 24 h later, cells were transfected with 4 µg of plasmid DNA/dish by using the LipofectAMINETM Plus kit (Life Technologies, Inc.) according to the manufacturer's instructions.

Receptor Binding Assays-- For radioligand binding studies, transfected cells were harvested about 70 h after transfections. Binding assays were carried out with membrane homogenates prepared from transfected cells essentially as described (38). Incubations were carried out for 2-3 h at room temperature in a 0.5-ml volume. Binding buffer consisted of 25 mM sodium phosphate (pH 7.4) and 5 mM MgCl2. In [3H]NMS saturation binding assays, six to eight different concentrations (15-2,000 pM) of the radioligand were employed. In competition binding studies, a fixed concentration of [3H]NMS (200 pM) and seven different concentrations (0.01-10 mM) of the muscarinic agonist, carbachol, were used. Nonspecific binding was determined in the presence of 1 µM atropine.

Binding data were analyzed by nonlinear least square curve-fitting procedures, using the computer program LIGAND (saturation binding data; Ref. 39) or KALEIDAGRAPH (competition binding data; Synergy software), respectively.

Phosphatidylinositol (PI) Assays-- Transfected COS-7 cells were transferred into 6-well plates (approximately 0.5 × 105 cells/well) about 24 h after transfections, and 3 µCi/ml myo-[3H]inositol was added to the growth medium. On the following day (20-24 h later), the labeled cells were washed once with 2 ml of Hank's balanced salt solution containing 20 mM HEPES and then incubated for 20 min (at room temperature) with 1 ml of the same medium containing 10 mM LiCl. Subsequently, different concentrations of carbachol were added, and cells were incubated for 1 h at 37 °C. After removal of the medium, reactions were terminated by addition of 0.75 ml of 20 mM formic acid, followed by a 30-min incubation at 4 °C. Samples were then neutralized with 0.25 ml of 60 mM ammonium hydroxide, and the inositol monophosphate (IP1) fraction was isolated by anion exchange chromatography as described (40) and counted on an LKB liquid scintillation counter.

Preparation of Membrane Extracts-- COS-7 cells were scraped into ice-cold phosphate-buffered saline (PBS) and collected by centrifugation about 70 h after transfections. Cell pellets were resuspended in 0.2% digitonin in PBS, followed by a 20-min incubation on ice. After centrifugation at 2,000 × g for 10 min, supernatants containing soluble as well as peripheral membrane proteins were discarded. Cell pellets were treated with lysis buffer (1% digitonin in 50 mM Tris-HCl (pH 8.0), containing 100 mM NaCl and 1 mM CaCl2) at 4 °C for 1 h. Cell lysates were then centrifuged in a refrigerated Eppendorf 5417R microcentrifuge at maximal speed for 30 min. Supernatants containing solubilized receptors were used either directly for factor Xa digestion or stored at -80 °C.

Factor Xa Digestion-- Membrane extracts (100 µl/100-mm dish) were incubated with 5 µg of factor Xa at room temperature (22 °C) for up to 16 h. Reactions were terminated by addition of 1 mM phenylmethylsulfonyl fluoride (final concentration).

Cross-linking with Cu(II)-Phenanthroline-- Factor Xa-digested membrane extracts were incubated with Cu(II)-(1,10-phenanthroline)3 complex (final concentration, 50 µM), by adding a freshly prepared aqueous mixture of equal volumes of 1 mM CuSO4 and 3 mM 1,10-phenanthroline. Reactions were carried out on ice for 30 min in a 0.1-ml volume and terminated by the addition of 10 mM EDTA and 10 mM NEM (final concentrations) for further experiments (see below) or by adding an equal volume of 2-fold Laemmli sample buffer containing 25 mM NEM and 20 mM EDTA for SDS-PAGE.

Immunoprecipitation-- To monitor the formation of intramolecular disulfide bonds in the different double Cys mutant receptors, factor Xa- and Cu(II)-phenanthroline-treated membrane lysates were subjected to immunoprecipitation by the anti-HA 12CA5 monoclonal antibody coupled to CNBr-activated Sepharose 4B. To eliminate non-covalent interactions between the N- and C-terminal receptor fragments generated by digestion of the solubilized receptors with factor Xa (referred to as m3-trunk and m3-tail, respectively), samples were incubated with 2.0% SDS at 37 °C for 1 h prior to immunoprecipitation. This step ensured that m3-tail fragments were coimmunoprecipitated by the anti-HA antibody only when cross-linked to m3-trunk fragments via disulfide bonds. Following the addition of PBS (containing 0.2% BSA) to reduce the SDS concentration to 0.1%, samples were incubated with the anti-HA antibody under rotation for at least 2 h at 4 °C. Immunoprecipitates were washed three times with PBS containing 0.2% digitonin and once with PBS, incubated with Laemmli sample buffer containing 50 mM dithiothreitol (DTT) at room temperature for 30 min, and then subjected to SDS-PAGE and Western blotting using the anti-C-m3 polyclonal antibody (Fig. 1) to detect the presence of the m3-tail fragment.

Western Blotting-- SDS-PAGE was performed essentially as described by Laemmli (41). Samples were mixed with an equal volume of 2-fold concentrated Laemmli sample buffer (composition, 125 mM Tris-HCl (pH 6.8), 20% glycerol, 100 mM DTT (only if indicated), 4% SDS, and 0.01% (w/v) bromphenol blue) and incubated at room temperature for 30 min. Subsequently, proteins were resolved on 12% (w/v) acrylamide slab gels in the presence of 0.1% SDS. Proteins were electroblotted onto nitrocellulose membranes (0.2 µm) as described (42). Membranes were blocked with 5% BSA in PBS and incubated with primary antibody (1 µg/ml) for 1 h at room temperature in PBS containing 0.075% Tween 20 and 1% BSA (PBS-T/BSA). Bound antibody was then probed with a secondary antibody conjugated to horseradish peroxidase (goat anti-mouse IgG or monkey anti-rabbit IgG antibody (Amersham Pharmacia Biotech); final dilution in PBS-T/BSA, 1:3,000) for 1 h at room temperature. After extensive washing of blots with PBS containing 0.075% Tween 20, proteins were visualized using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).

[35S]GTPgamma S Binding Assays-- [35S]GTPgamma S binding assays were performed essentially as described by Lazareno et al. (43). COS-7 cells grown in 100-mm plates were harvested about 70 h after transfections, suspended in 20 mM HEPES buffer (pH 7.0) containing 10 mM EDTA, and homogenized using a Polytron homogenizer for 30 s, followed by a 10-min centrifugation at 1,000 × g (4 °C). COS-7 cell membranes were then collected by spinning supernatants in a refrigerated Eppendorf 5417R microcentrifuge at maximal speed for 45 min. Membranes (about 50 µg of protein) were incubated with 50 µM Cu(II)-phenanthroline in PBS on ice for 30 min. Following the addition of 10 mM EDTA (final concentration), membranes were washed with 20 mM HEPES binding buffer (pH 7.0) containing 100 mM NaCl and 10 mM MgCl2. Subsequently, membranes (about 50 µg of protein) were incubated in binding buffer with 200 pM [35S]GTPgamma S and 1 µM GDP, in the presence or absence of the agonist, carbachol (0.5 mM). Incubations were carried out for 30 min at 30 °C in a 0.25-ml volume. Nonspecific binding was measured in the presence of 1 µM unlabeled GTPgamma S. In a set of control experiments (reducing conditions), membranes prepared from transfected cells were preincubated in PBS containing 5 mM DTT (30 min at room temperature), and [35S]GTPgamma S binding reactions were carried out in the presence of 1 mM DTT to prevent the formation of disulfide bonds. Binding reactions were terminated via filtration through Whatman GF/C filters using a Brandell cell harvester.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To develop a generally applicable disulfide cross-linking strategy useful for studying GPCR structure, we used the rat m3 muscarinic receptor as a model system. All wild-type and mutant muscarinic receptors analyzed in this study contained an N-terminal HA epitope tag. It is known from previous work that the presence of the HA epitope tag does not interfere with m3 muscarinic receptor function (30).

Construction of a Functional Mutant m3 Muscarinic Receptor Lacking Most Native Cys Residues-- The rat m3 muscarinic receptor contains 13 native Cys residues (Fig. 1), 4 within the extracellular loops (Cys-140, Cys-220, Cys-516, and Cys-519), 5 on the intracellular receptor surface (Cys-289, Cys-310, Cys-419, Cys-560, and Cys-562), 1 within TM II (Cys-111), and 3 within TM VII (Cys-532, Cys-542, and Cys-546). To facilitate the interpretation of disulfide cross-linking data, our first goal was to generate a functional mutant m3 muscarinic receptor that lacked most native Cys residues. The structures of selected mutant receptors lacking one or more native Cys residues are given in Table I (note that the different mutant receptors were named after the number of remaining Cys residues). All mutant receptors were transiently expressed in COS-7 cells and then tested for their ability to bind the muscarinic antagonist, [3H]NMS, and the agonist, carbachol, respectively (Table II). Moreover, to examine whether the different mutant receptors were still capable of coupling to G proteins, carbachol-induced increases in intracellular inositol phosphate levels were also determined (Table II).


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Fig. 1.   Transmembrane topology and structural modification of the rat m3 muscarinic receptor. Native Cys residues are shown boxed. To generate the m3'(3C)-Xa mutant receptor, the rat m3 muscarinic receptor was modified as follows. The five potential N-glycosylation sites present in the N-terminal portion of the receptor protein (Asn-6, Asn-15, Asn-41, Asn-48, and Asn-52; shown circled) were replaced with Gln residues. In addition, the central portion of the i3 loop (amino acids Ala-274 to Lys-469) containing Cys-289, Cys-310, and Cys-419 was replaced with a string of three factor Xa sites ((IEGR)3; double underlined). Except for Cys-140, Cys-220, and Cys-532 (highlighted in black), all remaining Cys residues were replaced with Ser or Ala (see Table I). As indicated, Cys-140 and Cys-220 are predicted to be linked via a disulfide bridge (44-46). Pairs of Cys residues were reintroduced into the m3'(3C)-Xa mutant receptor, one at position 169 (arrow) at the beginning of the i2 loop and the other one within the C-terminal portion of the i3 loop (positions 484-493; underlined). As a result, 10 different m3'(3C)-Xa-derived double Cys mutant receptors were obtained. In addition, all receptor constructs contained an N-terminal HA epitope tag. Cleavage of the m3'(3C)-Xa mutant receptor by factor Xa is predicted to yield two receptor fragments that are referred to as m3-trunk and m3-tail throughout this study. A rabbit polyclonal antibody (referred to as anti-C-m3) was raised against the indicated C-terminal receptor sequence. Numbers refer to amino acid positions in the rat m3 muscarinic receptor sequence (35).

                              
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Table I
Structure of mutant m3 muscarinic receptors investigated in this study
See text and Fig. 1 for further details.

                              
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Table II
Ligand binding and functional properties of mutant m3 muscarinic receptors expressed in COS-7 cells
Radioligand binding studies and PI assays were carried out as described under "Experimental Procedures" (for receptor structures, see Table I and Fig. 1). Kapp values were calculated from IC50 values using the Cheng-Prusoff equation and can be considered approximations of Ki values. PI data were analyzed by a nonlinear least squares curve-fitting procedure, using the computer program KALEIDAGRAPH (Synergy software). Data are presented as means ± S.E. of three independent experiments, each performed in duplicate.

Initially, individual Cys residues were replaced, in a sequential fashion, with either Ser or Ala residues. Analysis of the ligand binding and G protein-coupling properties of the m3(12C), m3(11C), m3(10C-a), m3(10C-b), and m3(9C) mutant receptors indicated that Cys-111, Cys-542, Cys-546, Cys-560, and Cys-562 were not required for proper m3 receptor function (Tables I and II). Interestingly, the m3(10C-b) and m3(9C) mutant receptors displayed pronounced increases (>30-fold) in carbachol binding affinities, as compared with the wild-type receptor (Fig. 2 and Table II). A similar increase in binding affinities was also observed for the agonist, acetylcholine (Kapp values in µM: m3(wt), 70.4 ± 2.5; m3(10C-b), 1.8 ± 0.5; m3(9C), 1.6 ± 0.4) (see also Fig. 2).


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Fig. 2.   Displacement of specific [3H]NMS binding to wild-type and mutant m3 muscarinic receptors by the agonists, carbachol and acetylcholine. Competition binding studies were carried out with membranes prepared from COS-7 cells transfected with the indicated m3 receptor constructs, as described under "Experimental Procedures" (for receptor structures, see Fig. 1 and Table I). In all experiments, the concentration of the radioligand, [3H]NMS, was 200 pM. Data are expressed as percentage of total binding determined in the absence of the cold inhibitors (100%). Generally, carbachol and acetylcholine binding curves were somewhat flatter in the case of the m3(10C-b) and m3(9C) mutant receptors (as compared with m3(wt)). Hill coefficients ranged from 0.9 to 1.0 for m3(wt) and from 0.65 to 0.75 for the two mutant receptors. Each point is the mean of two or three independent experiments, each carried out in duplicate.

To eliminate the three Cys residues located within the i3 loop (Cys-289, Cys-310, and Cys-419), the central portion of the i3 loop (amino acids Ala-274 to Lys-469) was deleted in the m3(6C), m3(4C), m3(3C), and m3(2C) mutant receptors (Table I). The m3(3C) mutant receptor in which Cys-140, Cys-220, and Cys-532 were the only remaining Cys residues (Table I), as well as the m3(6C) and m3(4C) constructs, displayed ligand binding affinities and G protein-coupling properties similar to those of the wild-type receptor. As shown in Table II, the [3H]NMS and carbachol binding affinities of the m3(3C) mutant receptor differed from those found with the wild-type receptor by less than 1.5-fold (note, however, that Bmax values were reduced by a factor of about 3). Moreover, the m3(3C) mutant receptor did not differ significantly from the wild-type receptor in its ability to stimulate carbachol-dependent increases in inositol phosphate production (Table II), indicating that 10 of the 13 native Cys residues are not essential for proper m3 receptor function.

Cys-140 and Cys-220, which, besides Cys-532, were still present in the m3(3C) construct, are predicted to be linked via a disulfide bond (Refs. 44-46; Fig. 1) that is thought to be critical for proper receptor folding (46-51). Consistent with this notion, mutational modification of Cys-140 or Cys-220 in the wild-type m3 receptor resulted in a strong reduction (>50-fold) in carbachol and [3H]NMS binding affinities and the number of detectable [3H]NMS binding sites.2 Moreover, replacement of Cys-140 or Cys-220 by Ala in the m3(3C) construct (yielding m3(2C-b) and m3(2C-c), respectively; Table I) resulted in a complete loss of [3H]NMS binding activity and functional coupling (Table II). Similarly, introduction of a C532A point mutation (location, TM VII) into the m3(3C) mutant receptor (yielding m3(2C); Table I) resulted in a complete loss of radioligand binding activity (highest [3H]NMS concentration tested,10 nM) (Table II). Similar results were obtained when Cys-532 was replaced with Ser, Val, Met, Phe, Tyr, Gly, Ile, or Leu (data not shown). These findings indicated that the presence of Cys-140, Cys-220, and Cys-532 was essential for the proper folding and/or function of the m3(3C) receptor.

Finally, the five potential N-glycosylation sites present within the o1 region of the m3(3C) mutant receptor (Fig. 1) were eliminated by site-directed mutagenesis (Asn-6, Asn-15, Ans-41, Asn-48, Asn-52 right-arrow Gln), yielding the m3'(3C) construct (Table I). This modification was introduced to prevent the appearance of multiple immunoreactive m3 receptor species on immunoblots caused by heterogeneous glycosylation.2 As shown in Table II, the ligand binding and G protein-coupling properties of the m3'(3C) mutant receptor did not differ significantly from those of the m3(3C) construct (see also Fig. 3), consistent with a previous study analyzing the pharmacologic properties of a glycosylation-defective mutant m2 muscarinic receptor (52). Based on these observations, the m3'(3C) mutant receptor was chosen as a template for further mutagenesis studies.


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Fig. 3.   Carbachol-induced PI hydrolysis mediated by the m3'(3C) and m3'(3C)-Xa mutant m3 muscarinic receptors. COS-7 cells were transfected with 4 µg of m3(wt) DNA or the m3'(3C) and m3'(3C)-Xa mutant receptor constructs (for receptor structures, see Table I and Fig. 1). PI assays were carried out in 6-well plates as described under "Experimental Procedures." Data (mean values) are presented as fold increase in IP1 accumulation above basal levels in the absence of carbachol. Each curve is representative of three independent experiments, each carried out in duplicate (for carbachol EC50 values, see Table II).

Introduction of a Factor Xa Cleavage Site into the m3'(3C) Mutant Receptor-- To facilitate the interpretation of disulfide cross-linking data (see below), a string of three factor Xa recognition sites ((IEGR)3) was introduced into the shortened i3 loop of the m3'(3C) construct, between Glu-273 and Thr-470 (numbering of amino acids is based on the wild-type receptor sequence), yielding the m3'(3C)-Xa mutant receptor (Fig. 1). Table II and Fig. 3 show that this modification had little effect on the ligand binding affinities and G protein-coupling properties of the m3'(3C) mutant receptor.

We next wanted to examine whether the m3'(3C)-Xa mutant receptor could be cleaved efficiently by factor Xa. Preliminary studies showed that receptor solubilization was required for efficient factor Xa cleavage (data not shown). Thus, membrane extracts were prepared from m3'(3C)-Xa-expressing COS-7 cells as described under "Experimental Procedures." Subsequently, samples (100 µl containing about 500 µg of protein) were incubated at room temperature (22 °C) with or without factor Xa (5 µg/100 µl) for up to 16 h. Consistently, virtually complete cleavage (>95%) of the m3'(3C)-Xa mutant receptor was obtained after a 16-h incubation (see below). Reactions were terminated by a 30-min incubation of samples with SDS-PAGE sample buffer (at room temperature), in the absence (non-reducing conditions) or presence of 50 mM DTT (reducing conditions). Samples were then subjected to 12% SDS-PAGE and Western blotting analysis. Receptor-specific immunoreactive bands were detected using the anti-HA monoclonal antibody (Fig. 4, A and B) or the anti-C-m3 polyclonal antibody (Fig. 4, C and D) which specifically recognizes the C-terminal 18 amino acids of the rat m3 muscarinic receptor (Fig. 1; Ref. 30).


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Fig. 4.   Complete cleavage of the m3'(3C)-Xa mutant receptor by factor Xa. A-D, COS-7 cells were transiently transfected with the m3'(3C)-Xa receptor construct (lanes 1 and 2) or with vector (V) DNA (pcD-PS) as a control. Membrane extracts were incubated with or without factor Xa for 16 h at 22 °C, as described under "Experimental Procedures." Subsequently, samples (containing about 20 µg of protein) were incubated with SDS sample buffer in the absence or presence of 50 mM DTT (room temperature, 30 min) and then subjected to 12% SDS-PAGE. Receptors/receptor fragments were visualized via Western blotting using the anti-HA antibody (A and B) that recognizes the N-terminal HA epitope tag or the anti-C-m3 antibody (C and D) which is directed against the C terminus of the rat m3 receptor. Note that the anti-HA antibody also detected an endogenous COS-7 cell protein migrating at about 80 kDa (A and B, lane V). Protein molecular mass standards (in kDa) are indicated. Two additional experiments gave similar results.

When immunoblotting experiments were performed under non-reducing conditions with control samples (no factor Xa treatment), both antibodies detected several immunoreactive bands (Fig. 4, A and C, lane 1) as follows: a major band migrating at around 45 kDa corresponding in size to the putative m3'(3C)-Xa receptor monomer, as well as several higher molecular mass forms of >80 kDa. These high molecular mass immunoreactive species are likely to represent disulfide-linked m3 receptor dimers or oligomers, since the 45-kDa receptor monomer represented the only specific immunoreactive band when Western blotting experiments were performed under reducing conditions (Fig. 4, B and D, lane 1). It should be noted that the anti-HA antibody also detected a cross-reacting endogenous COS-7 cell protein of about 80 kDa in size (Fig. 4, A and B, lane V).

When immunoblotting studies were carried out with factor Xa-treated samples (16 h, room temperature) under reducing or non-reducing conditions, the putative 45-kDa m3'(3C)-Xa receptor monomer could no longer be observed (Fig. 4, A-D, lane 2). However, in this case, the anti-HA antibody detected an immunoreactive species of about 30 kDa (Fig. 4, A and B, lane 2), corresponding in size to the N-terminal receptor fragment (referred to as m3-trunk) predicted to be generated by cleavage of the m3'(3C)-Xa mutant receptor within the i3 loop. The 30-kDa band was the only detectable immunoreactive species when SDS-PAGE was performed under reducing conditions (Fig. 4B, lane 2) (note that the nonspecific 80-kDa band that is usually detected by the anti-HA antibody is no longer seen with factor Xa-treated samples, probably due to factor-Xa-dependent proteolytic degradation). In contrast, several additional bands ranging in size from about 60 to >120 kDa were observed when Western blotting experiments were carried out under non-reducing conditions (Fig. 4A, lane 2), consistent with the presence of disulfide-linked m3-trunk dimers or oligomers. Analysis of factor Xa-treated samples with the anti-C-m3 antibody revealed a single band of approximately 13 kDa, under both non-reducing and reducing conditions (Fig. 4, C and D, lane 2). This band corresponded in size to the C-terminal receptor polypeptide, referred to as m3-tail, predicted to be released by factor Xa-dependent cleavage of the m3'(3C)-Xa mutant receptor.

The pattern of bands observed in Fig. 4 also indicates that no significant degradation of the m3'(3C)-Xa mutant receptor (other than cleavage by factor Xa) occurred during factor Xa incubation and the preparation of samples for SDS-PAGE.

Generation and Functional Analysis of m3'(3C)-Xa-derived Double Cys Mutant Receptors-- Mutational analysis of different classes of GPCRs has shown that residues at the N terminus of the i2 loop and at the C terminus of the i3 domain are critically involved in G protein recognition and activation (2-6, 34). However, the three-dimensional orientation of these residues relative to each other remains to be elucidated. Thus, our initial goal was to develop a disulfide cross-linking strategy to define proximities between residues located in these intracellular domains, using the m3'(3C)-Xa mutant receptor as a tool.

Specifically, we generated 10 double Cys mutant receptors by reintroducing pairs of Cys residues into the m3'(3C)-Xa construct. All double Cys mutant receptors contained an I169C point mutation at the beginning of the i2 loop (Fig. 1). The second Cys residue was introduced at the C terminus of the i3 loop, at positions 484-493 (Fig. 1).

Radioligand binding studies showed that all 10 double Cys mutants were able to bind the antagonist, [3H]NMS, and the agonist, carbachol, with high affinity (Table III). With only few exceptions, affinity estimates as well as receptor expression levels (Bmax) differed from the corresponding values determined with the m3'(3C)-Xa mutant receptor by <2.5-fold. In PI assays, all double Cys mutant receptors displayed high carbachol potencies with EC50 values ranging from 6.5 to 75.5 nM (m3'(3C)-Xa, EC50 = 33.8 ± 7.5 nM) (Table III). With the exception of the I169C-E485C construct which showed considerably reduced coupling efficiency, all mutant receptors retained robust efficacy in the PI assays, displaying Emax values that ranged from about 40 to 90% (m3'(3C)-Xa, Emax = 100%). These results suggested that virtually all double Cys mutant receptors were properly folded.

                              
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Table III
Ligand binding and functional properties of Cys-substituted m3'(3C)-Xa-derived mutant m3 muscarinic receptors expressed in COS-7 cells
Radioligand binding and PI assays were carried out as described under "Experimental Procedures." The indicated single and double Cys substitutions were introduced into the m3'(3C)-Xa mutant receptor (for receptor structure, see Fig. 1 and Table I). Kapp values were calculated from IC50 values using the Cheng-Prusoff equation and can be considered approximations of Ki values. PI data were analyzed by a nonlinear least squares curve-fitting procedure, using the computer program KALEIDAGRAPH (Synergy software). The maximum stimulation of IP1 production by the m3'(3C)-Xa receptor was defined as 100%. Maximum stimulation of the m3'(3C)-Xa receptor led to 6-8-fold increases in IP1 accumulation above basal levels. Data are presented as means ± S.E. of three independent experiments, each performed in duplicate.

Disulfide Cross-linking Studies with m3'(3C)-Xa-derived Double Cys Mutants-- To induce and monitor the formation of disulfide bonds in the double Cys mutant receptors, we initially employed the following experimental protocol. Membrane extracts prepared from COS-7 cells individually expressing the different mutant receptors were treated with factor Xa as described above, followed by a 30-min incubation on ice with the oxidizing agent, Cu(II)-phenanthroline (final concentration, 50 µM). Factor Xa- and Cu(II)-phenanthroline-treated samples were then subjected to SDS-PAGE, under reducing or non-reducing conditions, followed by Western blotting analysis using the anti-C-m3 antibody. Under reducing conditions, the 13-kDa m3-tail fragment was the only detectable immunoreactive species (shown for the I169C-A489C mutant receptor in Fig. 5, lane 3), indicating that cleavage of the mutant receptors by factor Xa was virtually complete. We speculated that the formation of intramolecular disulfide bonds in the factor Xa-treated double Cys mutant receptors should lead to the appearance of full-length receptor bands (due to covalent cross-linking of the m3-trunk and m3-tail fragments) when samples were processed for SDS-PAGE under non-reducing conditions. Consistent with this notion, non-reducing SDS-PAGE resulted in the appearance of a 45-kDa band corresponding in size to a putative receptor monomer in the case of most double Cys mutant receptors studied (shown for the I169C/A489C mutant receptor in Fig. 5, lane 2), except for the I169C/L492C and I169C/S493C constructs. However, besides this putative receptor monomer band, a great number of additional immunoreactive species, ranging in size from about 30 to >120 kDa, were observed. Since these bands were not observed under reducing conditions (Fig. 5, lane 3), they probably resulted from additional intermolecular disulfide cross-links, leading, for example, to the formation of dimeric and oligomeric forms of cross-linked receptors or receptor fragments (see also Fig. 3). Moreover, the possibility cannot be excluded that at least some of the observed bands were caused by cross-linking of the m3 receptor or m3 receptor fragments to other receptor-associated membrane proteins.


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Fig. 5.   Detection of intramolecular disulfide cross-linking in an m3'(3C)-Xa-derived mutant m3 muscarinic receptor via Western blot analysis. Membrane extracts were prepared from COS-7 cells transfected with the m3'(3C)-Xa-derived I169C/A489C double Cys mutant construct (for receptor structure, see Table I and Fig. 1), digested with factor Xa, and then incubated on ice for 30 min in the absence (lane 1) or presence (lanes 2 and 3) of the oxidizing agent, Cu(II)-phenanthroline (Cu-Phen) (50 µM), as described under "Experimental Procedures." Subsequently, samples were mixed with SDS sample buffer in the absence (lane 1 and 2) or presence (lane 3) of the reducing agent, DTT (50 mM), and subjected to 12% SDS-PAGE. Receptor proteins/fragments were detected via Western blotting using the anti-C-m3 antibody. Protein molecular mass standards (in kDa) are indicated. Two additional experiments gave similar results.

Clearly, this complex pattern of observed immunoreactive bands greatly complicated the interpretation of the disulfide cross-linking experiments. We therefore developed an alternative experimental strategy, involving an immunoprecipitation step, to monitor the formation of intramolecular disulfide bonds in a more straightforward manner. The basic steps involved in this protocol are summarized in Scheme 1 (for details, see "Experimental Procedures"). Initially, membrane extracts prepared from COS-7 cells expressing the different double Cys mutant receptors were treated with factor Xa as described above. Western blot analysis confirmed that all mutant receptors could be cleaved efficiently by factor Xa, as judged by the appearance of intense 13-kDa m3-tail bands on immunoblots probed with the anti-C-m3 antibody (Fig. 6A).


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Scheme 1.   Detection of intramolecular disulfide bonds in m3'(3C)-Xa-derived mutant m3 muscarinic receptors using a combined immunoprecipitation/immunoblotting strategy.


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Fig. 6.   Detection of intramolecular disulfide cross-linking in m3'(3C)-Xa-derived mutant m3 muscarinic receptors using an immunoprecipitation (IP) strategy. Membrane extracts (100 µl) prepared from COS-7 cells transfected with m3'(3C)-Xa-derived mutant constructs containing the indicated single or double Cys substitutions (for receptor structures, see Table I and Fig. 1) were incubated with factor Xa as described under "Experimental Procedures." Aliquots of the factor Xa-digested samples (containing about 10 µg protein) were mixed directly with SDS sample buffer containing 50 mM DTT and subjected to 12% SDS-PAGE and Western blotting analysis using the anti-C-m3 antibody (A). The remainder of the samples (containing about 100 µg of protein) were incubated on ice for 30 min in the absence (B) or presence (C) of the oxidizing agent, Cu(II)-phenanthroline (Cu-Phen) (50 µM), followed by the addition of SDS (final concentration, 2%) and a 1-h incubation at 37 °C. Subsequently, samples were diluted with PBS containing 0.2% BSA to reduce the SDS concentration to 0.1%. In the next step, disulfide cross-linked m3-trunk-3-tail complexes and/or m3-trunk fragments were immunoprecipitated with the anti-HA antibody as described under "Experimental Procedures" (B and C). Immunoprecipitates were mixed with SDS ample buffer containing 50 mM DTT and subjected to 12% SDS-PAGE. Finally, m3-tail fragments (predicted molecular mass,13 kDa) were detected via Western blotting analysis using the anti-C-m3 antibody. The increase in intensities of the m3-tail bands seen with Cu(II)-phenanthroline-treated samples (C), as compared with those found with samples not treated with the oxidizing agent (B), represents a direct measure of intramolecular disulfide cross-linking in the double Cys mutants.

Factor Xa-treated samples were then incubated on ice for 30 min, in the absence or presence of Cu(II)-phenanthroline (50 µM). Subsequently, samples were treated with 2.0% SDS (37 °C, 1 h) to disrupt non-covalent interactions between the m3-trunk and m3-tail polypeptides generated by factor Xa cleavage of the mutant receptors. Following dilution of samples with PBS (containing 0.2% BSA) to reduce the SDS concentration to 0.1%, receptors/receptor fragments were subjected to immunoprecipitation using the anti-HA monoclonal antibody coupled to Sepharose 4B. This antibody is predicted to immunoprecipitate disulfide cross-linked m3-trunk-m3-tail complexes, m3-trunk fragments, and, potentially, a small proportion of full-length receptors that escaped cleavage by factor Xa. Immunoprecipitates were eluted with SDS-PAGE sample buffer containing 50 mM DTT (incubation at room temperature for 30 min). This step ensured that m3-tail polypeptides were released from disulfide cross-linked m3-trunk-m3-tail complexes (note that m3-tail polypeptides can only be generated from factor Xa-digested receptors). Subsequently, samples were subjected to SDS-PAGE and Western blotting analysis using the anti-C-m3 polyclonal antibody to detect the presence of the 13-kDa m3-tail fragment. Under the chosen experimental conditions, the appearance of the m3-tail band can be considered a direct measure for the formation of intramolecular disulfide bonds.

For control purposes, immunoblotting studies were also carried out with factor Xa-treated, immunoprecipitated receptors that had not been subjected to incubation with the oxidizing agent, Cu(II)-phenanthroline (Fig. 6B). In this case, m3-tail bands were either undetectable or only very faint, indicating that m3-tail fragments generated by cleavage of receptors with factor Xa (see Fig. 6A) did not coprecipitate with the m3-trunk fragment under the chosen experimental conditions (treatment of samples with 2% SDS, see above). A possibility is that the very faint m3-tail bands observed in Fig. 6B were due to spontaneous formation of intramolecular disulfide bonds.

A completely different pattern emerged when factor Xa-digested samples that had been treated with Cu(II)-phenanthroline were analyzed (Fig. 6C). As expected, no m3-tail signals were observed with the m3'(3C)-Xa and I169C mutant receptors, indicative of the lack of intramolecular disulfide bonds in these constructs. In contrast, very intense m3-tail bands were obtained with 5 of the 10 double Cys mutants (I169C/K486C, I169C/K487C, I169C/A488C, I169C/A489C, and I169C/Q490C), indicating that Cys-169 was able to efficiently form disulfide bonds with Cys residues present at positions 486-490. The m3-tail signals were reduced but still quite prominent in the mutant receptors harboring the I169C/K484C, I169C/E485C, and I169C/T491C substitutions but were only very faint in the case of the I169C/L492C and I169C/S493C double Cys constructs (Fig. 6C).

Functional Consequences of Disulfide Cross-linking-- We next carried out [35S]GTPgamma S binding assays to examine the effects of disulfide cross-linking on the ability of the different double Cys mutant receptors to interact with G proteins. Toward this aim, membranes prepared from COS-7 cells expressing the different mutant receptors were first treated with 50 µM Cu(II)-phenanthroline or 5 mM DTT as described under "Experimental Procedures." Subsequently, samples were incubated with the muscarinic agonist, carbachol (0.5 mM), for 30 min at 30 °C, and carbachol-dependent increases in [35S]GTPgamma S binding were determined by employing a standard filtration binding assay. Analysis of DTT-treated control samples showed that all studied mutant receptors (m3'(3C)-Xa, m3'(3C)-Xa(I169C), and the 10 m3'(3C)-Xa-derived double Cys mutants) were able to mediate small but significant increases in [35S]GTPgamma S binding (10-25% above basal values determined in the absence of carbachol) (Fig. 7). Treatment of membranes with the oxidizing agent, Cu(II)-phenanthroline, had little or no effect on the ability of the m3'(3C)-Xa and m3'(3C)-Xa(I169C) constructs as well as of the I169C/L492C and I169C/S493C double Cys mutant receptors to induce agonist-dependent [35S]GTPgamma S binding activity. In contrast, following Cu(II)-phenanthroline treatment, carbachol-mediated increases in [35S]GTPgamma S binding were either drastically reduced (I169C/K484C, I169C/E485C, I169C/K486C, and I169C/Q490C) or completely abolished (I169C/K487C, I169C/A488C, I169C/A489C, and I169C/T491C) in the remaining double Cys mutant constructs (Fig. 7).


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Fig. 7.   Effect of disulfide cross-linking on receptor-mediated G protein activation. COS-7 cells were transfected with 4 µg of the indicated m3'(3C)-Xa-derived mutant constructs containing the indicated single or double Cys substitutions (for receptor structures, see Table I and Fig. 1). To study the ability of the different mutant receptors to activate G proteins in an agonist-dependent fashion, [35S]GTPgamma S binding assays were carried out using membranes prepared from transfected cells as described under "Experimental Procedures." Basal [35S]GTPgamma S binding was determined with samples pretreated with DTT (5 mM, 30 min at room temperature; reducing conditions) or Cu(II)-phenanthroline (50 µM, 30 min on ice; oxidizing conditions), in the absence of the agonist, carbachol. Basal [35S]GTPgamma S counts were similar for all mutant receptor constructs (m3'(3C)-Xa, 7710 ± 350 cpm/sample). The ability of the agonist, carbachol (0.5 mM), to promote [35S]GTPgamma S binding was studied under both reducing and oxidizing conditions. Results are expressed as carbachol-dependent percent increase in [35S]GTPgamma S binding above basal levels. Data (mean values) shown here are taken from a representative experiment, carried out in duplicate (S.E. <10% for each bar). Two additional experiments gave similar results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we used a site-directed disulfide mapping approach to gain insight into the molecular architecture of the intracellular surface of the m3 muscarinic receptor. Moreover, the effect of constraining the conformational flexibility of the intracellular receptor domains via formation of disulfide bridges was examined in functional assays. Disulfide cross-linking approaches have been used previously to examine structural features of the photoreceptor, rhodopsin (22-26). However, this is the first report describing the application of a disulfide mapping strategy to the structural analysis of a hormone-activated GPCR.

To facilitate the proper interpretation of disulfide cross-linking data, our initial goal was to generate a modified version of the m3 muscarinic receptor that lacked most native Cys residues. The rat m3 muscarinic receptor contains 13 native Cys residues (Fig. 1), 9 of which (Cys-111, Cys-140, Cys-220, Cys-516, Cys-519, Cys-532, Cys-542, Cys-546, and Cys-560) are conserved among all muscarinic receptor subtypes (m1-m5) (34, 46). The three non-conserved Cys residues located within the i3 loop (Cys-289, Cys-310, and Cys-419) were deleted by replacing the central portion of the i3 loop (amino acids Ala-274 to Lys-469) with three factor Xa recognition sites ((IEGR)3). Consistent with previous studies using i3 loop-shortened versions of various muscarinic receptor subtypes (37, 53-56), this modification had no significant effect on the ligand binding and functional properties of the m3 muscarinic receptor. The remaining Cys residues were replaced, in a sequential fashion, with either Ser or Ala residues (Table I). Radioligand binding and functional assays showed that a mutant receptor, referred to as m3'(3C)-Xa (Table I), in which Cys-140, Cys-220, and Cys-532 were the only remaining Cys residues displayed ligand binding affinities and G protein-coupling properties similar to those of the wild-type receptor (Table II and Fig. 3). This observation suggests that 10 of the 13 Cys residues present in the m3 muscarinic receptor are not critically involved in ligand binding and agonist-induced receptor activation. However, individual replacement of Cys-140, Cys-220, or Cys-532 in the m3(3C) mutant receptor background with either Ala or Ser resulted in a complete loss of radioligand ([3H]NMS) binding activity. Cys-140 and Cys-220, which are located on the extracellular receptor surface and are conserved among most GPCRs, are predicted to be linked via a disulfide bond (Refs. 44-46; Fig. 1). Several studies have shown that this disulfide bond is critical for correct GPCR folding (46-51), providing an explanation for our observation that the presence of Cys-140 and Cys-220 is required for proper m3 receptor function.

Cys-532, which is located within the N-terminal segment of TM VII, is predicted to face TM III within the interior of the transmembrane receptor core (10). Our observation that Cys-532 is essential for proper m3 receptor function, at least in the m3(3C) mutant receptor background, suggests that this conserved Cys residue plays an important structural role in m3 receptor folding and/or function. Consistent with this notion, mutational modification of Cys-407 in the wild-type m1 muscarinic receptor (which is homologous to Cys-532 in the rat m3 muscarinic receptor) also resulted in a pronounced impairment of receptor function (46).

Interestingly, two of the mutant receptors that were generated during the preparation of the m3'(3C)-Xa construct, m3(10C-b) and m3(9C), showed marked increases (>30-fold) in agonist binding affinities, as compared with the wild-type receptor (Fig. 2 and Table II). These two mutant receptors differ from the m3(11C) and m3(10C-a) constructs only in the presence of the C542S substitution. In agreement with this observation, replacement of the homologous Cys residue in the wild-type m1 muscarinic receptor (C417S) also resulted in a severalfold increase in agonist binding affinities (46). One possible explanation for these findings is that this conserved Cys residue modulates access to the agonist-binding site, either through direct or indirect structural effects.

Based on its pharmacologic and functional profile, the m3'(3C)-Xa mutant receptor was chosen as a template for further mutagenesis studies. Since our plan was to introduce pairs of Cys residues into distinct intracellular domains and the three Cys residues remaining in the m3'(3C)-Xa construct were located either extracellularly or within the extracellular segment of TM VII, it appeared very unlikely that the introduced Cys residues might be able to interact with any of three remaining native Cys residues.

After having demonstrated that the m3'(3C)-Xa mutant receptor can be efficiently cleaved by factor Xa (Fig. 4), we reintroduced pairs of Cys residues into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the i2 loop and a second one within the C-terminal portion of the i3 loop, at positions 484-493. Radioligand binding studies and PI assays showed that all 10 double Cys mutants were properly expressed and able to activate G proteins in an agonist-dependent fashion (Table III), indicating that the different mutations did not interfere with proper receptor folding.

Site-directed mutagenesis and biophysical studies with several different GPCRs suggest that the C-terminal as well as the N-terminal segments of the i3 loop form amphipathic alpha -helices and that the hydrophobic sides of these helical domains play key roles in G protein recognition and activation (5, 6, 11, 12). As discussed above, we found that individual replacement of residues 484-493 with Cys (in the m3'(3C)-Xa background) did not lead to major impairments in receptor/G protein coupling (Table III). This observation suggests that individual Cys substitutions in this region do not severely disrupt its overall structure. Similar findings have been obtained when the homologous segment of bovine rhodopsin was subjected to Cys-scanning mutagenesis (57). These data are also consistent with the concept that receptor/G protein coupling involves many different contact sites on the receptor protein (5, 6, 11, 12).

A great number of mutagenesis studies suggest that residues within the N-terminal segment of the i2 loop and the C-terminal portion of the i3 domain play key roles in proper receptor/G protein recognition and agonist-dependent G protein activation (2-6, 34). However, other cytoplasmic receptor regions, such as the N-terminal segments of the i3 and i4 domains, are also known to be involved in receptor/G protein coupling (2-6, 34). Electron cryo-microscopic studies of bovine (7) and frog (8) rhodopsin suggest that the N terminus of the i2 loop (which can be considered an extension of TM III) and the C terminus of the i3 loop (which can be considered an extension of TM VI) are located adjacent to each other on the intracellular receptor surface (10). This notion is also supported by biochemical and biophysical analysis of bovine rhodopsin using different experimental approaches, including histidine mutagenesis to generate metal ion-binding sites (58), Cys cross-linking experiments (24), and site-directed spin labeling (SDSL) studies (24). In agreement with predictions made based on mutagenesis studies (5, 6), SDSL studies (59, 60) also suggest that the C-terminal portion of the i3 domain and the N-terminal segment of the i2 loop are likely to be alpha -helically arranged.

To gain deeper insight into the molecular architecture of the G protein binding surface of the m3 muscarinic receptor, the 10 m3'(3C)-Xa double Cys mutant receptors were analyzed for their ability to form intramolecular disulfide bonds. Since the maximum distance found in proteins between alpha -carbons linked by disulfide bonds is about 7 Å (61), such cross-linking experiments should identify amino acids that are located close to each other in the properly folded receptor protein.

To detect the formation of intramolecular disulfide bonds in the double Cys mutant receptors, we initially employed a strategy that is based on the altered electrophoretic mobility of the cross-linked proteins on SDS gels. Specifically, solubilized factor Xa-digested mutant receptors were incubated with a low concentration of the oxidizing agent Cu(II)-phenanthroline and then subjected to reducing and non-reducing SDS-PAGE, followed by Western blot analysis using the anti-C-m3 antibody. By using this procedure, non-reducing SDS-PAGE resulted in a very complex pattern of bands (shown for the I169C/A489C construct in Fig. 5). Most double Cys mutant receptors (except for the I169C/L492C and I169C/S493C double Cys constructs) yielded a clearly visible DTT-sensitive 45-kDa band corresponding in size to a disulfide cross-linked m3-trunk-m3-tail complex, as well as a great number of additional immunoreactive receptor species, ranging in size from about 30 to >120 kDa, as detected by the use of the anti-C-m3 antibody (Fig. 5, lane 2). These extra bands, like the 45-kDa band, were not observed when immunoblotting studies were carried out in the presence of DTT (reducing conditions), suggesting that they were probably caused by intermolecular disulfide cross-links involving two or more receptor/receptor fragment molecules or other receptor-associated membrane proteins. Indeed, we recently obtained evidence that the m3 muscarinic receptor, like other GPCRs (62-64), is able to form disulfide-linked dimers and oligomers in vivo (Fig. 4, A and C, lane 1).2 Preliminary studies suggest that the formation of m3 receptor dimers/multimers is dependent on the presence of Cys-140 and Cys-220.2

Since the formation of intermolecular disulfide bonds greatly complicated the interpretation of the disulfide cross-linking experiments, we developed an alternative experimental approach to circumvent these difficulties (Scheme 1). In this case, samples containing solubilized mutant receptors were treated with factor Xa and oxidizing agent, followed by an incubation with a high concentration of SDS (2%) to dissociate non-disulfide linked m3-trunk-m3-tail complexes. Subsequently, disulfide-linked m3-trunk-m3-tail complexes as well as non-cross-linked m3-trunk polypeptides were immunoprecipitated with the anti-HA monoclonal antibody that recognizes the HA epitope tag present at the N terminus of all mutant receptors. Immunoprecipitated proteins were then treated with DTT to release cross-linked m3-tail fragments that were subsequently detected via SDS-PAGE and Western blotting analysis using the anti-C-m3 antibody (Fig. 6). Control experiments showed that factor Xa-digested mutant receptors that were not subjected to incubation with the oxidizing agent, Cu(II)-phenanthroline, gave only very faint or no m3-tail signals at all (Fig. 6B), indicating that intramolecular disulfide bonds did not form spontaneously to a significant degree. A completely different pattern was obtained when factor Xa-digested samples that had been treated with the oxidizing agent were analyzed (Fig. 6C). As expected, the m3'(3C)-Xa mutant receptor, into which the different double Cys substitutions had been introduced, did not yield a detectable m3-tail band, indicative of the absence of intramolecular disulfide bonds linking the N- and C-terminal receptor portions. On the other hand, 8 of the 10 examined double Cys mutant receptors containing Cys substitutions at positions 484-491 gave rather intense m3-tail bands. In contrast, m3-tail signals were only very faint in the case of the I169C/L492C and I169C/S493C double Cys constructs (Fig. 6C). This observation suggests that the Cys residue at position 169 can form disulfide bonds with Cys residues introduced at any position within the Lys-484 to Thr-491 sequence.

As discussed above, disulfide bond formation requires that the two cross-linked Cys residues are located in close proximity to each other (<7 Å; Ref. 61). Thus, besides confirming that the N terminus of the i2 loop is located adjacent to the C terminus of the i3 domain, our disulfide cross-linking data strongly suggest that the intracellular surface of the m3 muscarinic receptor is highly dynamic, probably due to extensive backbone fluctuations. As a consequence, even relatively short-lived low probability conformations, which may deviate considerably from the average structure and are difficult to detect by other biochemical or biophysical techniques, can be trapped by disulfide cross-linking (18, 65). The proposed high degree of conformational flexibility would explain that even Cys residues that are not directly juxtaposed in the average three-dimensional structure of the receptor are able to collide and form disulfide bonds. Consistent with this notion, electron cryo-microscopic studies of two-dimensional crystals of bovine (7) and frog (8) rhodopsin suggested that the intracellular receptor surface is not well ordered. Moreover, Cys cross-linking experiments carried out with mutant versions of bovine rhodopsin containing Cys substitutions at positions similar to those reported here also revealed highly promiscuous disulfide cross-linking (24). Interestingly, disulfide cross-linking studies have also shown that the cytoplasmic domain of the transmembrane aspartate receptor, a bacterial chemoreceptor, is also characterized by a highly dynamic structure (66). These observations suggest that extensive backbone fluctuations may be a general property of the intracellular signaling domains of cell-surface receptors.

To examine the functional consequences of the formation of intramolecular disulfide bonds on the ability of the different double Cys mutant receptors to interact with G proteins, [35S]GTPgamma S binding assays were carried out using membrane preparations prepared from COS-7 cells individually expressing the different mutant receptors (Fig. 7). Specifically, we examined the ability of the muscarinic agonist, carbachol, to promote [35S]GTPgamma S binding, either under reducing or oxidizing conditions, using samples pretreated with DTT or Cu(II)-phenanthroline, respectively. Under reducing conditions, all studied mutant receptors were able to mediate small but significant agonist-dependent increases in [35S]GTPgamma S binding activity. However, when assays were carried out under oxidizing conditions, the eight mutant receptors that displayed pronounced intramolecular disulfide cross-linking (see above) showed no or only residual functional activity (Fig. 7). On the other hand, the m3'(3C)-Xa mutant receptor and the two double Cys constructs (I169C/L492C and I169C/S493C) that showed no or little evidence of intramolecular disulfide formation retained strong functional activity in the [35S]GTPgamma S binding assays. These results indicate that constraining the conformational freedom of the intracellular receptor surface via disulfide cross-links prevents agonist-dependent m3 receptor activation.

In an analogous fashion, biochemical studies with Cys- or His-substituted mutant versions of bovine rhodopsin have demonstrated that cross-linking of the cytoplasmic ends of TM III and VI, either via disulfide bond formation (24) or via binding of metal ions (58), respectively, disrupts productive rhodopsin/transducin coupling. Consistent with these findings, SDSL studies have shown that rhodopsin activation is accompanied by "outward" movements of the cytoplasmic portions of TM III (60, 67) and, particularly, TM VI (24), accompanied by a clockwise rotation of the cytoplasmic segment of TM VI by approximately 30o, as viewed from the cell interior (24). These findings are also supported by two recent studies on the structural and dynamic properties of a series of mutant beta 2-adrenergic receptors, employing either Cys-specific fluorescence marker molecules (68) or the "substituted cysteine accessibility method" (69), respectively.

As indicated above, [35S]GTPgamma S binding assays were carried out with oxidized membrane preparations that had not been treated with factor Xa. In contrast, the formation of intramolecular disulfide cross-links in the different double Cys mutant receptors was monitored using factor-Xa-treated membrane lysates. However, as outlined above, we observed an excellent correlation between the results obtained in the [35S]GTPgamma S binding and the disulfide cross-linking assays. It is therefore highly unlikely that the pattern of promiscuous disulfide cross-linking observed with the factor Xa-treated membrane lysates was caused artifactually by partial receptor unfolding during the preparation of membrane lysates and the factor Xa incubation step.

In conclusion, this is the first study using a disulfide cross-linking strategy to examine the molecular architecture of a hormone-activated GPCR. Our cross-linking data indicate that the intracellular surface of the m3 muscarinic receptor is characterized by a high degree of conformational flexibility which appears to be a prerequisite for efficient receptor/G protein coupling. Given the fact that all GPCRs share a high degree of structural homology, the approach outlined here, which does not involve a receptor purification step, should also be applicable to other classes of GPCRs.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: NIDDK, Laboratory of Bioorganic Chemistry, Bldg. 8A, Rm. B1A-05, Bethesda, Maryland 20892. Tel.: 301-402-3589; Fax: 301-402-4182; E-mail:jwess{at}helix.nih.gov.

2 F.-Y. Zeng and J. Wess, unpublished results.

    ABBREVIATIONS

The abbreviations used are: GPCRs, G protein-coupled receptors; BSA, bovine serum albumin; DTT, dithiothreitol; HA tag, hemagglutinin epitope tag; i1-i4, the four intracellular domains of GPCRs; IP1, inositol monophosphate; NEM, N-ethylmaleimide; NMS, N-methylscopolamine; o1-o4, the four extracellular domains of GPCRs; PBS, phosphate-buffered saline; PI, phosphatidylinositol; SDSL, site-directed spin labeling; TM I-VII, the seven transmembrane domains of GPCRs; PAGE, polyacrylamide gel electrophoresis; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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