Pronounced Conformational Changes following Agonist Activation of the M3 Muscarinic Acetylcholine Receptor
2005; Elsevier BV; Volume: 280; Issue: 26 Linguagem: Inglês
10.1074/jbc.m500379200
ISSN1083-351X
AutoresSung‐Jun Han, Fadi F. Hamdan, Soo‐Kyung Kim, Kenneth A. Jacobson, Lars Brichta, Lanh M. Bloodworth, Jian H. Li, Jürgen Wess,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M3 muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M3 muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M3 receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M3 receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M3 receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs. The conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M3 muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M3 muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M3 receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M3 receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M3 receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs. The superfamily of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; [3H]NMS, N-[3H]methylscopolamine; i3 loop, the third intracellular loop of G protein-coupled receptors; IP1, inositol monophosphate; TM I–VII, the seven transmembrane domains of G protein-coupled receptors. represents the largest group of cell surface receptors found in nature (1Fredriksson R. Lagerström M.C. Lundin L.G. Schiöth H.B. Mol. Pharmacol. 2003; 63: 1256-1272Crossref PubMed Scopus (2201) Google Scholar, 2Foord S.M. Curr. Opin. Pharmacol. 2002; 2: 561-566Crossref PubMed Scopus (46) Google Scholar, 3Takeda S. Kadowaki S. Haga T. Takaesu H. Mitaku S. FEBS Lett. 2002; 520: 97-101Crossref PubMed Scopus (321) Google Scholar). A structural hallmark of all GPCRs is the presence of a bundle of seven transmembrane helices (TM I–VII) that are connected by alternating intracellular and extracellular loops (4Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (511) Google Scholar, 5Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1235) Google Scholar, 6Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar) (Fig. 1). The structural elements determining ligand binding and G protein recognition have been studied in considerable detail, at least for some members of the GPCR superfamily (5Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1235) Google Scholar, 6Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 7Ji T.H. Grossmann M. Ji I. J. Biol. Chem. 1998; 273: 17299-17302Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 8Wess J. Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (371) Google Scholar). In contrast, the conformational changes that activating ligands induce in their target receptors are still not well understood at present. The currently available evidence suggests that GPCR activation opens a cleft on the intracellular side of the receptor that promotes the recognition and activation of specific G protein heterotrimers (4Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (511) Google Scholar, 6Gether U. Endocr. Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar, 9Meng E.C. Bourne H.R. Trends. Pharmacol. Sci. 2001; 22: 587-593Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 10Hubbell W.L. Altenbach C. Hubbell C.M. Khorana H.G. Adv. Protein Chem. 2003; 63: 243-290Crossref PubMed Scopus (342) Google Scholar, 34Gether U. Kobilka B.K. J. Biol. Chem. 1998; 273: 17979-17982Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar). At present, bovine rhodopsin (in its inactive state) is the only GPCR for which high resolution structural information is currently available (11Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5059) Google Scholar). Most GPCRs share a considerable degree of structural homology with bovine rhodopsin (12Ballesteros J.A. Shi L. Javitch J.A. Mol. Pharmacol. 2001; 60: 1-19Crossref PubMed Scopus (405) Google Scholar) and are therefore also referred to as rhodopsin-like or family A GPCRs. However, whereas the endogenous ligand of rhodopsin, 11-cis-retinal, is covalently bound to the receptor protein, all other family A GPCRs known to date are activated by diffusible ligands. The possibility therefore exists that the precise structural mechanisms involved in receptor activation may not be identical between rhodopsin and other class A GPCRs. During the past decade, considerable progress has been made in elucidating the light-induced conformational changes in bovine rhodopsin (9Meng E.C. Bourne H.R. Trends. Pharmacol. Sci. 2001; 22: 587-593Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 10Hubbell W.L. Altenbach C. Hubbell C.M. Khorana H.G. Adv. Protein Chem. 2003; 63: 243-290Crossref PubMed Scopus (342) Google Scholar, 13Sakmar T.P. Menon S.T. Marin E.P. Awad E.S. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 443-484Crossref PubMed Scopus (208) Google Scholar). Biophysical and biochemical studies suggest that rhodopsin activation triggers a reorientation of the cytoplasmic end of TM VI and changes in the relative disposition of TM VI and III, along with smaller movements involving several other TM helices (10Hubbell W.L. Altenbach C. Hubbell C.M. Khorana H.G. Adv. Protein Chem. 2003; 63: 243-290Crossref PubMed Scopus (342) Google Scholar, 14Lin S.W. Sakmar T.P. Biochemistry. 1996; 35: 11149-11159Crossref PubMed Scopus (225) Google Scholar, 15Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1117) Google Scholar, 16Sheikh S.P. Zvyaga T.A. Lichtarge O. Sakmar T.P. Bourne H.R. Nature. 1996; 383: 347-350Crossref PubMed Scopus (399) Google Scholar). Considerable evidence indicates that a similar movement (reorientation of the cytoplasmic end of TM VI versus that of TM III) occurs in other GPCRs, including the β2-adrenergic receptor (17Gether U. Lin S. Ghanouni P. Ballesteros J.A. Weinstein H. Kobilka B.K. EMBO J. 1997; 16: 6737-6747Crossref PubMed Google Scholar, 18Javitch J.A. Fu D. Liapakis G. Chen J. J. Biol. Chem. 1997; 272: 18546-18549Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 19Rasmussen S.G. Jensen A.D. Liapakis G. Ghanouni P. Javitch J.A. Gether U. Mol. Pharmacol. 1999; 56: 175-184Crossref PubMed Scopus (197) Google Scholar, 20Sheikh S.P. Vilardarga J-P. Baranski T.J. Lichtarge O. Iiri T. Meng E.C. Nissenson R.A. Bourne H.R. J. Biol. Chem. 1999; 274: 17033-17041Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 21Jensen A.D. Guarnieri F. Rasmussen S.G. Asmar F. Ballesteros J.A. Gether U. J. Biol. Chem. 2001; 276: 9279-9290Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). More specifically, site-directed spin labeling studies (15Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1117) Google Scholar) suggested that rhodopsin activation involves a rigid body movement of the cytoplasmic end of TM VI (away from the C terminus of TM III) that is accompanied by a rotational movement of ∼30° (clockwise as viewed from the cytoplasm). The pioneering biophysical and biochemical studies carried out with bovine rhodopsin have led to important new insights into the structural mechanisms involved in rhodopsin activation. However, the vast majority of these studies were carried out with mutant versions of rhodopsin in the solution state (receptor proteins were solubilized in dodecyl maltoside micelles), and some data suggest that the structural and dynamic properties of rhodopsin present in solution may not be identical with those found in native disk membranes (10Hubbell W.L. Altenbach C. Hubbell C.M. Khorana H.G. Adv. Protein Chem. 2003; 63: 243-290Crossref PubMed Scopus (342) Google Scholar). Thus, the development of techniques that would allow the monitoring of agonist-induced conformational changes in GPCRs present in their native membrane environment would be highly desirable. To address this issue, we recently described a novel in situ disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M3 muscarinic acetylcholine receptors present in their native membrane environment (22Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23Hamdan F.F. Ward S.D. Siddiqui N.A. Bloodworth L.M. Wess J. Biochemistry. 2002; 41: 7647-7658Crossref PubMed Scopus (34) Google Scholar). The M3 muscarinic receptor is a prototypical class A GPCR that preferentially interacts with G proteins of the Gq family (24Wess J. Crit. Rev. Neurobiol. 1996; 10: 69-99Crossref PubMed Scopus (425) Google Scholar). Agonist binding to the M3 muscarinic receptor and most other class A GPCRs involves, among other sites of contact, several key residues present within the exofacial portion of TM VII (24Wess J. Crit. Rev. Neurobiol. 1996; 10: 69-99Crossref PubMed Scopus (425) Google Scholar, 25Lu Z.L. Saldanha J.W. Hulme E.C. Trends Pharmacol. Sci. 2002; 23: 140-146Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Moreover, the endofacial segment of TM VII contains the highly conserved NPXXY motif (corresponding to Asn-539 to Tyr-543 in the rat M3 receptor sequence) (Fig. 1), which may provide a point of flexibility for agonist-induced structural changes. We therefore tested the hypothesis that diffusible ligands may induce conformational changes within the cytoplasmic segment of TM VII (the region located C-terminal of the NPXXY motif), using our previously developed in situ disulfide cross-linking strategy (22Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23Hamdan F.F. Ward S.D. Siddiqui N.A. Bloodworth L.M. Wess J. Biochemistry. 2002; 41: 7647-7658Crossref PubMed Scopus (34) Google Scholar). Since the C-terminal segment of TM VII is predicted to be located in the vicinity of the C-terminal portion of TM I (11Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5059) Google Scholar), we generated 30 double Cys mutant M3 muscarinic receptors, all of which contained one Cys substitution within the C-terminal portion of TM VII (Val-5417.51–Ser-5467.56) and another Cys substitution within the C-terminal segment of TM I (Val-881.53–Phe-921.57) (the superscripts indicate amino acid positions according to the nomenclature proposed by Ballesteros and Weinstein (36Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Crossref Scopus (2512) Google Scholar)). All Cys mutations were introduced into a modified version of the rat M3 muscarinic receptor (M3′(3C)-Xa) (22Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 26Zeng F.Y. Hopp A. Soldner A. Wess J. J. Biol. Chem. 1999; 274: 16629-16640Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) that lacked most native Cys residues and contained a factor Xa cleavage site within the third intracellular loop (i3 loop) (Fig. 1). Disulfide cross-linking experiments, carried out in the absence or the presence of a muscarinic agonist (carbachol), led to the identification of three double Cys mutant M3 muscarinic receptors (V88C1.53/Y543C7.53, A91C1.56/L545C7.55, and A91C1.56/S546C7.56) that showed agonist-promoted disulfide bond formation. The observed cross-linking pattern, in combination with a newly established three-dimensional model of the rat M3 muscarinic receptor, strongly suggested that receptor activation leads to a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the C-terminal ends of TM I and VII. Given the high degree of structural homology found among all class A GPCRs, our findings should be of broad general relevance. Materials—Copper sulfate (CuSO4), 1,10-phenanthroline, N-ethylmaleimide, carbamylcholine chloride (carbachol), atropine sulfate, and mammalian protease inhibitor mixture were purchased from Sigma. N-[3H]Methylscopolamine ([3H]NMS; 79–83 Ci/mmol) and myo-[3H]inositol (20 Ci/mmol) were from PerkinElmer Life Sciences. Factor Xa protease and digitonin were obtained from Roche Applied Science. Precast Novex Tris-glycine polyacrylamide gels and SeeBlue Plus 2 prestained molecular mass standards were from Invitrogen. Hybond™ ECL™ nitrocellulose membranes, anti-rabbit IgG antibody conjugated to horseradish peroxidase, ECL™ detection reagents, and Hyperfilm™ ECL™ chemiluminescence film were from Amersham Biosciences. Laemmli loading buffer was from Bio-Rad. All other reagents used were of the highest grade commercially available. Generation of Cys-substituted Mutant M3 Muscarinic Receptor Constructs—All Cys substitutions were introduced into a pCD-based expression plasmid coding for a modified version of the rat M3 muscarinic receptor, previously referred to as M3′(3C)-Xa receptor (Fig. 1). The generation of the M3′(3C)-Xa expression plasmid has been described previously (26Zeng F.Y. Hopp A. Soldner A. Wess J. J. Biol. Chem. 1999; 274: 16629-16640Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The M3′(3C)-Xa receptor contains an N-terminal hemagglutinin epitope tag and lacks all five potential N-terminal N-glycosylation sites and most endogenous Cys residues, except for Cys140, Cys220, and Cys532. Importantly, the central portion of the i3 loop (Ala274–Lys469) was replaced by two factor Xa cleavage sites. Cys residues were reintroduced into the M3′(3C)-Xa construct at positions Val-881.53–Phe-921.57 and Val-5417.51–Ser-5467.56, by using the QuikChange™ site-directed mutagenesis kit (Stratagene), according to the manufacturer's instructions. Double Cys mutant receptors were obtained by subcloning a 1.7-kb BglII-NdeI fragment derived from the mutant M3′(3C)-Xa constructs containing single Cys substitutions at positions Val-541 to Val-546 into the M3′(3C)-Xa constructs containing single Cys substitutions at positions Val-88 to Phe-92. The identity of all mutant constructs was verified by DNA sequencing. Expression of Receptor Constructs in Mammalian Cells—All mutant M3 muscarinic receptors were transiently expressed in COS-7 cells. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 incubator. Approximately 24 h prior to transfections, 1 × 106 cells were seeded into 100-mm dishes. Cells were transfected with 4 μg of receptor plasmid DNA/dish using the Lipofectamine™ Plus kit (Invitrogen), according to the manufacturer's recommendations. In order to increase muscarinic receptor expression levels, 1 μm atropine was routinely added to the incubation medium for the last 24 h of culture. Preparations of Membranes from Transfected COS-7 Cells—Transfected cells were harvested ∼48 h after transfections. To ensure complete removal of atropine that was present in the incubation medium during the last 24 h of culture, cells were washed twice (10 min each wash) with 10 ml of ice-cold phosphate-buffered saline (pH 7.4). Subsequently, 2 ml of ice-cold buffer A (25 mm sodium phosphate and 5 mm MgCl2, pH 7.4) was added to each 100-mm dish, followed by a 15-min incubation at 4 °C. Cells were then scraped off of the plates and homogenized using a Polytron tissue homogenizer (setting 5; 20 s), followed by a 15-min centrifugation at 20,000 × g at 4 °C. The membrane pellets were then resuspended in buffer A (1 ml/100-mm dish), rehomogenized, frozen on dry ice, and stored at -70 °C until needed. Protein concentrations were measured using the Micro BCA protein assay reagent kit with bovine serum albumin as a standard. Radioligand Binding Studies—Radioligand binding assays were carried out using membrane homogenates prepared from transfected COS-7 cells essentially as described previously (22Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In brief, all incubations were carried out in 1 ml of buffer A (∼10–20 μg of membrane protein/tube) for 2 h at room temperature (22 °C). In saturation binding assays, six different concentrations (ranging from 20 to 3,000 pm) of [3H]NMS were used. In competition binding assays, a fixed concentration of [3H]NMS (500 pm) was employed in the presence of 10 different concentrations (13 pm to 10 μm) of the cold competitor, carbachol, a muscarinic agonist. Reactions were terminated by rapid filtration over GF/C Brandel filters followed by three washes (∼4 ml each) with ice-cold distilled water. In all assays, nonspecific binding was defined as the binding remaining in the presence of 1 μm atropine. The amount of bound radioactivity was determined by liquid scintillation spectrometry. Binding data were analyzed using the nonlinear curve-fitting program Prism 3.0 (GraphPad). Agonist-induced Stimulation of Phosphatidylinositol Hydrolysis— The ability of the muscarinic agonist, carbachol, to stimulate increases in intracellular inositol monophosphate (IP1) levels was determined using transfected COS-7 cells grown in 6-well plates, as described previously (26Zeng F.Y. Hopp A. Soldner A. Wess J. J. Biol. Chem. 1999; 274: 16629-16640Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). After labeling of cells for 20–24 h with myo-[3H]inositol (3 μCi/ml), cells were incubated in the presence of 10 mm LiCl for 1 h at 37 °C with increasing concentrations of carbachol. The IP1 fraction was isolated and quantitated as described (26Zeng F.Y. Hopp A. Soldner A. Wess J. J. Biol. Chem. 1999; 274: 16629-16640Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Carbachol concentration-response curves were analyzed using the nonlinear curve-fitting program Prism 3.0 (GraphPad). Oxidation, Solubilization, and Factor Xa Treatment of Mutant M3 Muscarinic Receptors—Membrane preparations obtained from transfected COS-7 cells were thawed at room temperature and rehomogenized as described under “Preparations of Membranes from Transfected COS-7 Cells.” Membranes from one 100-mm dish (∼1 mg of protein present in a 1-ml volume) were incubated in microcentrifuge tubes with end-over-end rotation (30 rpm; 10 min at room temperature) with the oxidizing agent, Cu(II)-phenanthroline (2.5 μm), either in the absence or the presence of different concentrations of the muscarinic agonist, carbachol, or the antagonist, atropine. Reactions were terminated by the addition of EDTA and N-ethylmaleimide (10 mm each), followed by a 10-min incubation on ice. To obtain membrane lysates (22Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), samples were then centrifuged at 8,000 × g for 10 min at 4 °C. The resulting membrane pellets were incubated with 250 μl of 0.2% digitonin in phosphate-buffered saline (pH 7.4) for 20 min on ice (to remove peripheral membrane proteins). Following another centrifugation step (8,000 × g for 10 min at 4 °C), membrane pellets were incubated with 1.2% digitonin in buffer B (50 mm Tris-HCl, pH 8, 100 mm NaCl, and 1 mm CaCl2) for 90–120 min at 4 °C with end-over-end rotation (30 rpm). After another centrifugation step (same conditions as above), the supernatants (membrane lysates containing solubilized mutant M3 muscarinic receptors) were transferred to fresh microcentrifuge tubes. Membrane lysates (∼15 μg of protein) were then incubated with factor Xa protease (final concentration, 0.1 μg/μl) at room temperature for 16–20 h (final volume, 50 μl). The reactions were then terminated by incubation for 30 min at room temperature with a mammalian protease inhibitor mixture (1:25 dilution; Sigma). Samples were then used directly for SDS-PAGE or stored at -70 °C until use. Western Blot Analysis—SDS-PAGE was performed essentially as described (22Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Samples were incubated for 30 min at 37 °C with Laemmli loading buffer (nonreducing conditions) and then loaded onto 10–20% Tris-glycine polyacrylamide gels, which were run at 125 V in the presence of 0.1% SDS. Western blotting studies were carried out essentially as described by Ward et al. (22Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), using the anti-M3 antibody directed against the C-terminal 18 amino acids of the M3 receptor protein (27Zeng F.Y. Soldner A. Schöneberg T. Wess J. J. Neurochem. 1999; 72: 2404-2414Crossref PubMed Scopus (61) Google Scholar). Receptor proteins were visualized by using ECL detection reagents and autoradiography. The intensities of immunoreactive bands were quantitated by scanning densitometry using the program ImageQuant TL (Amersham Biosciences). A Three-dimensional Model of the Rat M3 Muscarinic Receptor—A three-dimensional model of the TM core of the rat M3 muscarinic receptor (including the various loop regions and helix 8) was built using homology modeling based on the high resolution (2.8 Å) x-ray structure of the inactive state of bovine rhodopsin (11Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5059) Google Scholar). All calculations were performed on a Silicon Graphics Octane work station (300-MHz MIPS R12000 (IP30) processor) using the SYBYL 6.9 program (Sybyl Molecular Modeling System, version 6.9; Tripos Inc., St. Louis, MO). For the conformational refinement of the initial M3 muscarinic receptor model, the optimized structures were used as the starting point for subsequent molecular dynamics studies. Overall, the M3 receptor model showed high structural similarity with that of the rhodopsin template, especially within the regions endowed with secondary structure (for details, see Supplemental Data). Generation of 30 Double Cys Mutant M3 Muscarinic Receptors—This study was designed to monitor agonist-induced conformational changes in the M3 muscarinic receptor with the receptor being present in its native membrane environment. Our major goal was to detect potential activity-dependent structural changes occurring at the cytoplasmic end of TM VII. Toward this aim, we used an in situ disulfide cross-linking strategy to monitor the positions of six consecutive amino acids located at the C terminus of TM VII, relative to a string of residues located at the C terminus of TM I. Altogether, we generated 30 double Cys mutant receptors, all of which contained one Cys substitution within the C-terminal segment of TM I (Val-881.53–Phe-921.57) and another Cys substitution within the C-terminal portion of TM VII (Val-5417.51–Ser-5467.56) (Fig. 1). All Cys mutations were introduced into a modified version of the rat M3 muscarinic receptor (M3′(3C)-Xa) (26Zeng F.Y. Hopp A. Soldner A. Wess J. J. Biol. Chem. 1999; 274: 16629-16640Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) that lacked most native Cys residues and contained a factor Xa cleavage site within the i3 loop (Fig. 1). Transient Expression of Mutant M3 Muscarinic Receptors and Radioligand Binding Studies—All 30 double Cys mutant M3 receptor constructs, along with the M3′(3C)-Xa “background” receptor, were transiently expressed in COS-7 cells and initially examined for their ability to bind the muscarinic radioligand, [3H]NMS. To increase receptor expression levels, transfected cells were incubated with atropine (1 μm) for the last 24 h of culture. We previously demonstrated that this strategy leads to a pronounced increase in the density of the M3′(3C)-Xa receptor and all Cys-substituted mutant receptors derived from this construct (22Ward S.D. Hamdan F.F. Bloodworth L.M. Wess J. J. Biol. Chem. 2002; 277: 2247-2257Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23Hamdan F.F. Ward S.D. Siddiqui N.A. Bloodworth L.M. Wess J. Biochemistry. 2002; 41: 7647-7658Crossref PubMed Scopus (34) Google Scholar). Saturation binding studies with the muscarinic antagonist, [3H]NMS, showed that the M3′(3C)-Xa receptor was expressed at a density of 3.54 ± 0.01 pmol/mg protein (Bmax). The expression levels of the majority of the 30 double Cys mutant receptors differed from this value by less than 2-fold (Table I). Interestingly, several mutant receptors yielded Bmax values that were significantly higher than that observed with the M3′(3C)-Xa construct (Table I). The mutant receptors displaying the highest receptor densities were V90C1.55/V541C7.51 and F92C1.57/L545C7.55, which exhibited Bmax values of 19.06 ± 0.53 and 16.42 ± 0.26 pmol/mg, respectively. All 30 double Cys mutant receptors were able to bind [3H]NMS with high affinity (Table I). The [3H]NMS KD values displayed by these receptors differed from the KD value determined for the M3′(3C)-Xa “base mutant” (KD = 240 ± 45 pm) by less than 2-fold.Table ILigand binding properties of double Cys mutant M3 muscarinic receptors The indicated mutant M3 muscarinic receptors were transiently expressed in COS-7 cells. All double Cys mutant receptors were derived from the M3′(3C)-Xa construct. Bmax and KD values for [3H]NMS were determined from saturation binding experiments using membrane homogenates prepared from transfected COS-7 cells. Carbachol binding affinities (Ki) were determined in [3H]NMS competition binding assays (nH = Hill coefficient). Carbachol binding data were corrected for the Cheng-Prusoff shift. Binding data were analyzed using the nonlinear curve-fitting program Prism 3.0 (GraphPad). Data are given as means ± S.E. from 2–5 independent experiments, each performed in duplicate.Receptor[3H]NMS bindingCarbachol bindingKDBmaxKinHpmpmol/mg proteinμmM3′(3C)-Xa240 ± 453.54 ± 0.0115.2 ± 1.70.55 ± 0.02V88C/V541C254 ± 1102.15 ± 0.4912.1 ± 0.30.54 ± 0.02V88C/A542C300 ± 735.30 ± 0.3738.1 ± 7.70.61 ± 0.05V88C/Y543C364 ± 1201.92 ± 0.529.4 ± 1.30.49 ± 0.03V88C/A544C343 ± 433.86 ± 0.0122.6 ± 4.80.55 ± 0.01V88C/L545C288 ± 713.33 ± 0.6312.1 ± 0.30.53 ± 0.02V88C/S546C267 ± 12.53 ± 1.0526.3 ± 10.30.51 ± 0.06I89C/V541C277 ± 832.04 ± 0.3014.5 ± 6.00.43 ± 0.01I89C/A542C241 ± 664.55 ± 0.6739.5 ± 4.20.54 ± 0.04I89C/Y543C341 ± 1851.48 ± 0.545.4 ± 1.80.41 ± 0.08I89C/A544C260 ± 882.47 ± 0.5322.2 ± 0.20.44 ± 0.01I89C/L545C280 ± 683.01 ± 0.6215.2 ± 1.60.51 ± 0.03I89C/S546C405 ± 1694.23 ± 0.39102.8 ± 49.00.78 ± 0.12V90C/V541C334 ± 8519.06 ± 0.5343.6 ± 11.50.62 ± 0.06V90C/A542C334 ± 1088.84 ± 0.4077.2 ± 9.30.64 ± 0.02V90C/Y543C365 ± 714.00 ± 0.6012.3 ± 2.40.48 ± 0.02V90C/A544C280 ± 874.80 ± 0.6556.0 ± 23.00.61 ± 0.07V90C/L545C238 ± 1145.61 ± 1.3227.4 ± 4.30.63 ± 0.04V90C/S546C223 ± 203.76 ± 0.6111.4 ± 1.60.72 ± 0.07A91C/V541C282 ± 1215.01 ± 1.436.7 ± 1.90.50 ± 0.02A91C/A542C280 ± 375.
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