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Cys-140 Is Critical for Metabotropic Glutamate Receptor-1 Dimerization

2000; Elsevier BV; Volume: 275; Issue: 44 Linguagem: Inglês

10.1074/jbc.m005581200

1083-351X

Kausik K. Ray, Benjamin C. Hauschild,

Receptor Mechanisms and Signaling

Metabotropic glutamate receptor 1 (mGluR1) expresses at the cell surface as disulfide-linked dimers and can be reduced to monomers with sulfhydryl reagents. To identify the dimerization domain, we transiently expressed in HEK-293 cells a truncated version of mGluR1 (RhodC-R1) devoid of the extracellular domain (ECD). RhodC-R1 was a monomer in the absence or presence of the reducing agents, suggesting that dimerization occurs via the ECD. To identify cysteine residues involved in dimerization within the ECD, cysteine to serine point mutations were made at three cysteines within the amino-terminal half of the ECD. A mutation at positions Cys-67, Cys-109, and Cys-140 all resulted in significant amounts of monomers in the absence of reducing agents. The monomeric C67S and C109S mutants were not properly glycosylated, failed to reach the cell surface, and showed no glutamate response, indicating that these mutant receptors were improperly folded and/or processed and thus retained intracellularly. In contrast, the monomeric C140S mutant was properly glycosylated, processed, and expressed at the cell surface. Phosphoinositide hydrolysis assay showed that the glutamate response of the C140S mutant receptor was similar to the wild type receptor. Substitution of a cysteine for Ser-129, Lys-134, Asp-143, and Thr-146 on the C140S mutant background restored receptor dimerization. Taken together, the results suggest that Cys-140 contributes to intermolecular disulfide-linked dimerization of mGluR1. Metabotropic glutamate receptor 1 (mGluR1) expresses at the cell surface as disulfide-linked dimers and can be reduced to monomers with sulfhydryl reagents. To identify the dimerization domain, we transiently expressed in HEK-293 cells a truncated version of mGluR1 (RhodC-R1) devoid of the extracellular domain (ECD). RhodC-R1 was a monomer in the absence or presence of the reducing agents, suggesting that dimerization occurs via the ECD. To identify cysteine residues involved in dimerization within the ECD, cysteine to serine point mutations were made at three cysteines within the amino-terminal half of the ECD. A mutation at positions Cys-67, Cys-109, and Cys-140 all resulted in significant amounts of monomers in the absence of reducing agents. The monomeric C67S and C109S mutants were not properly glycosylated, failed to reach the cell surface, and showed no glutamate response, indicating that these mutant receptors were improperly folded and/or processed and thus retained intracellularly. In contrast, the monomeric C140S mutant was properly glycosylated, processed, and expressed at the cell surface. Phosphoinositide hydrolysis assay showed that the glutamate response of the C140S mutant receptor was similar to the wild type receptor. Substitution of a cysteine for Ser-129, Lys-134, Asp-143, and Thr-146 on the C140S mutant background restored receptor dimerization. Taken together, the results suggest that Cys-140 contributes to intermolecular disulfide-linked dimerization of mGluR1. metabotropic glutamate receptor G-protein-coupled receptor phosphoinositide vomeronasal organ receptor (Type 2) taste receptor (Type 1) extracellular domain human embryonic kidney 293 cells polyacrylamide gel electrophoresis peptide N-glycosidase F endo-β-N-acetylglucosaminidase H d-biotinoyl-ε-aminocaproic acid-N-hydroxysuccinimide ester 1,4-piperazinediethanesulfonic acid polymerase chain reaction peroxidase-conjugated γ-aminobutyric acid The metabotropic glutamate receptors (mGluRs)1 are a family of neurotransmitter receptors that mediate a variety of physiological functions in the central nervous system (1Conn P.J. Pin J.-P. Ann. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2716) Google Scholar). The mGluRs are members of the superfamily of G-protein-coupled receptors (GPCR) and belong to the subfamily (family 3 (2Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1220) Google Scholar)) that includes Ca2+receptor (3Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2351) Google Scholar), putative pheromone receptors in the vomeronasal organ (V2Rs) (4Ryba N.J. Tirindelli R. Neuron. 1997; 19: 371-379Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 5Herrada G. Dulac C. Cell. 1997; 90: 763-773Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 6Matsunami H. Buck L.B. Cell. 1997; 90: 775-784Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar), putative taste receptors (T1Rs) (7Hoon M.A. Adler E. Lindemeier J. Battey J.F. Ryba N.J.P. Zuker C.S. Cell. 1999; 96: 541-551Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar), and GABAB receptors (8Kaupmann K. Huggel K. Heid J. Flor P.J. Bischoff S. Mickel S.J. McMaster G. Angst C. Bittiger H. Froestl W. Bettler B. Nature. 1997; 386: 239-246Crossref PubMed Scopus (874) Google Scholar). The eight members of the mGluR family (mGluR1–8) of receptors have been divided into three subgroups based on sequence homology, signal transduction properties, and pharmacological profile (1Conn P.J. Pin J.-P. Ann. Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2716) Google Scholar). The mGluR1 and mGluR5 receptors and their splice variants make up the group 1 mGluRs, which are coupled to the stimulation of phosphoinositol turnover via the Gq subfamily of G-proteins. The mGluRs and other family 3 GPCRs are characterized by a very large (approximately ∼600 residues) extracellular amino-terminal domain (ECD). The mGluR1 ECD is the glutamate binding domain and believed to be structurally related to the bilobed "venus flytrap" structure of bacterial periplasmic binding proteins (9O'Hara P.J. Sheppard P.O. Thogersen H. Venezia D. Haldeman B.A. McGrane V. Houamed K.M. Thomsen C. Gilbert T.L. Mulvihill E.R. Neuron. 1993; 11: 41-52Abstract Full Text PDF PubMed Scopus (617) Google Scholar, 10Okamoto T. Sekiyama N. Otsu M. Shimada Y. Sato A. Nakanishi S. Jingami H. J. Biol. Chem. 1998; 273: 13089-13096Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Recently, it has been shown that both the mGluR1 (11Robbins M.J. Ciruela F. Rhodes A. McLlhinney J. Neurochem. 1999; 72: 2539-2547Crossref PubMed Scopus (70) Google Scholar) and the Ca2+ receptor (12Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar,13Ray K. Hauschild B.C. Steinbach P.J. Goldsmith P.K. Hauache O. Spiegel A.M. J. Biol. Chem. 1999; 274: 27642-27650Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) are expressed at the cell surface as intermolecular disulfide-linked dimers. For mGluR1 (10Okamoto T. Sekiyama N. Otsu M. Shimada Y. Sato A. Nakanishi S. Jingami H. J. Biol. Chem. 1998; 273: 13089-13096Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), mGluR4 (14Han G. Hampson D.R. J. Biol. Chem. 1999; 274: 10008-10013Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and Ca2+ receptor (15Goldsmith P.K. Fan G.F. Ray K. Shiloach J. McPhie P. Rogers K.V. Spiegel A.M. J. Biol. Chem. 1999; 274: 11303-11309Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), the ECD of each receptor, purified as a secreted protein, exists as a disulfide-linked dimer, suggesting that one or more cysteines in the ECD is involved in receptor dimer formation. The rat mGluR subtype 1α (described as mGluR1 from now on) ECD contains a total of 19 cysteines (16Masu M. Tanabe Y. Tsuchida K. Shigemoto R. Nakanishi S. Nature. 1991; 349: 760-765Crossref PubMed Scopus (989) Google Scholar) of which several are highly conserved in other mGluRs, Ca2+ receptor, V2Rs, and T1Rs. Proteolysis of the mGluR5 receptor localized cysteine(s) critical for dimer formation to the first 17 kDa of the ECD (17Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar). This region contains three cysteines conserved in all mGluRs, and we mutated these cysteines of the mGluR1 and investigated the role of these cysteine mutants in receptor dimerization and function. This study led to the identification of a conserved Cys-140 as critical for disulfide-linked dimerization of mGluR1, and functional study indicated that, like the wild type receptor, C140S mutant receptor is also capable of intracellular signaling via Gq-phospholipase C pathway. Using Turbo Pfu DNA polymerase (Stratagene Inc.), a polymerase chain reaction (PCR) was performed to add 20 amino acid residues (MNGTEGPNFYVPFSNKTGVV) corresponding to the amino terminus of bovine rhodopsin to the mGluR1 before amino acid residue 584. The 1.8-kilobase PCR product was subcloned to the pCR3.1 expression vector (Invitrogen) as a HindIII-XhoI fragment. The entire nucleotide sequence of the PCR product was confirmed by using a dRhodamine terminator cycle sequencing reaction kit and ABI prizm-377 DNA sequencer (Applied Biosystems). The constructed truncation mutant, designated as RhodC-R1, was devoid of 1–583 amino-terminal ECD of the mGluR1 but included 20 amino acids of the amino terminus rhodopsin tag and the amino acid residues 584–1199 of mGluR1. mGluR1 cDNA was cloned in pCR3.1 expression vector (Invitrogen) as aBamHI-NotI fragment. Site-directed mutagenesis was performed using a commercial kit (QuikChange site-directed mutagenesis kit, Stratagene) as described by Ray et al.(18Ray K. Clapp P. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1998; 273: 34558-34567Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Three cysteine point mutants, C67S, C107S, and C140S were created by changing cysteine at a given site to serine. Seven Cys-scanning mutants, S119C, S126C, S129C, K134C, D143C, T146C, and T152C were created in a second round of mutagenesis by using C140S mutant plasmid DNA as a template. The mutations were confirmed by automated DNA sequencing using a dRhodamine terminator cycle sequencing reaction kit and the ABI prizm-377 DNA sequencer (Applied Biosystems). Receptor plasmid DNAs were prepared by a maxi-plasmid preparation kit (Qiagen) and were transiently transfection in HEK-293 cells using LipofectAMINE (Life Technologies) as described previously (19Ray K. Fan G.F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Protein extraction for immunoblotting or biotinylation-immunoprecipitation experiments was performed 48 h after transfection. Crude membrane extracts were prepared as described previously by Ray et al. (19Ray K. Fan G.F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Briefly, confluent cells in 75-cm2 flasks or 6-well plates were rinsed with ice-cold phosphate-buffered saline and scraped on ice in buffer A (5 mm Tris (pH 7.2), 2 mm EDTA), containing 10 mm iodoacetamide with freshly added Complete protease inhibitors mixture (Roche Molecular Biochemicals). The cells were forced through a 22-gauge needle five to eight times, and the lysate was spun in a TLA-45 centrifuge at 45,000 rpm for 30 min at 4 °C to collect a crude membrane pellet. The pellet was resuspended in buffer B containing 20 mm Tris-HCl (pH 6.8), 150 mmNaCl, 10 mm EDTA, 1 mm EGTA, 1% Triton X-100 with 10 mm iodoacetamide and freshly added protease inhibitors mixture. Whole cell extracts were prepared by solubilizing cells directly in buffer B containing 1% Triton-100 as described earlier (13Ray K. Hauschild B.C. Steinbach P.J. Goldsmith P.K. Hauache O. Spiegel A.M. J. Biol. Chem. 1999; 274: 27642-27650Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The protein content of each sample was determined by the modified Bradford method (Bio-Rad) and 20–30 μg of protein per lane was separated on 5% gel by SDS-PAGE. Electrotransferred proteins to nitrocellulose membranes were incubated with monoclonal anti-mGluR1 antibody (Transduction laboratories, catalog # M72620) at a dilution of 1: 10,000 or anti-mGluR1 polyclonal antibody at a dilution of 1:500 (Chemicon, catalog # AB1553). Subsequently, the nitrocellulose membrane was incubated with a secondary goat anti-mouse or anti-rabbit antibody conjugated to horseradish peroxidase (Kierkegaard and Perry Laboratories) at a dilution of 1: 5000, respectively. The mGluR1 bands were detected with an Enhanced Chemiluminescence system (ECL) (Amersham Corp). Biotinylated protein bands were detected using peroxidase-conjugated streptavidin-POD followed by visualization of the biotinylated bands using a BM chemiluminescence kit (Roche Molecular Biochemicals). 48 h after transfection, cell surface proteins of the intact HEK-293 cells were labeled with membrane-impermeant Biotin-7-NHS using the cellular labeling kit (Roche Molecular Biochemicals) as described earlier (19Ray K. Fan G.F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Briefly, intact cells were labeled with 50 μg/ml Biotin-7-NHS in biotinylation buffer (50 mm sodium borate, 150 mm NaCl) to biotinylate cell surface proteins. The reaction was stopped by adding 50 mm NH4Cl for 15 min on ice. The cells were washed with phosphate-buffered saline and solubilized with lysis buffer B. 300 μl (approximately 600 μg of total protein) of the whole cell lysate of biotin-labeled cells was further diluted with 300 μl of buffer B and incubated with 5 μl of mouse monoclonal mGluR1-specific (made against the carboxyl-terminal peptide corresponding to amino acids 1042–1160; 0.1 mg/ml stock; Transduction laboratories, Lexington, KY) for 1–2 h at 4 °C. Subsequently, 25 μl of Protein A/G-agarose (Santa Cruz Biotechnologies) was added, and the incubation was continued for an additional 2 h. The Protein A/G-agarose was washed three times with buffer B containing 0.5% SDS, and the immunoreactive proteins were eluted in sample buffer containing either no β-mercaptoethanol or 300 mm β-mercaptoethanol. Samples were analyzed by SDS-PAGE, and immunoblotting was performed as described before. For cleavage with PNGase-F or Endo-H (Roche Molecular Biochemicals), whole cell extracts (30 μl) were diluted in 20 μl of 50 mmsodium acetate (pH 4.8). Samples were incubated with 0.5 milliunits of Endo-H or 1.0 unit of PNGase-F for 2 h at 37 °C. Phosphoinositide (PhI) hydrolysis assay has been described previously (18Ray K. Clapp P. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1998; 273: 34558-34567Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Briefly, 24 h after transfection, transfected cells from a confluent 75-cm2 flask was replated in 24-well plates in medium containing 3.0 μCi/ml of [3H]myoinositol (PerkinElmer Life Sciences) in complete Dulbecco's modified Eagle's medium containing no glutamine for another 24 h, followed by 1-h preincubation with 1× PhI buffer (120 mm NaCl, 0.5 mm CaCl2, 5 mm KCl, 5.6 mm glucose, 0.4 mmMgCl2, 20 mm LiCl in 25 mm PIPES buffer, pH 7.2). After removal of PhI buffer, cells were incubated for an additional 30 min with different concentrations ofl-glutamate in PhI buffer. The reactions were terminated by addition of 1 ml of acid-methanol (1:1000, v/v) per well. Total inositol phosphates were purified by chromatography on Dowex 1-X8 columns. To determine the domain(s) involved in mGluR1 dimerization, we sought to construct a truncated form of the mGluR1 lacking the ECD. The amino terminus of this truncated mutant (RhodC-R1) consisted of the first 20 amino acid residues of rhodopsin followed by the remainder of the mGluR1 beginning with residue 584. The rhodopsin N terminus tag was added, because it has been shown to enhance proper processing and cell surface expression of several GPCRs (20Krautwurst D. Yau K.W. Reed R.R. Cell. 1998; 97: 917-926Abstract Full Text Full Text PDF Scopus (495) Google Scholar, 21Chandrashekar J. Mueller K.L. Hoon M.A. Adler E. Feng L. Guo W. Zuker C.S. Ryba N.J. Cell. 2000; 100: 703-711Abstract Full Text Full Text PDF PubMed Scopus (1067) Google Scholar). Next, the wild type mGluR1 and RhodC-R1 cDNAs were transiently transfected in HEK-293 cells, and after 48 h, whole cell extracts were prepared. To prevent non-specific disulfide bond formation during protein extraction, 10 mm iodoacetamide was included in the lysis buffer. On an immunoblot, as shown in Fig.1 A (left), under non-reducing conditions, a mGluR1-specific monoclonal antibody made against a carboxyl-terminal epitope detected two major bands of the wild type mGluR1 around ∼260- to 270-kDa molecular mass range and two fainter 130- and 135-kDa bands. The same four immunoreactive bands were also recognized by a mGluR1-specific polyclonal antibody (Fig. 1 B) and not detected in vector-transfected cells (data not shown). The intensity of the lower two 130- and 135-kDa bands varied from no immunoreactivity to faint immunoreactivity between different immunoblotting experiments. After reduction with β-mercaptoethanol, a majority of the wild type mGluR1 ∼260- to 270-kDa bands were reduced to a broad monomeric ∼133-kDa band, and a small portion remained as SDS-resistant dimeric aggregates. The RhodC-R1 mutant expression pattern revealed a major ∼90-kDa immunoreactive band, and the protein was expressed as a monomer, because the presence or absence of β-mercaptoethanol did not shift this band (Fig. 1 A, right).Figure 1Determination of cell surface expression of wild type mGluR1 and RhodC-R1 mutant receptors. Whole cell extracts were prepared from HEK-293 cells transiently transfected with either wild type (WT-R1) or with RhodC-R1 cDNAs. 20 μg of protein were loaded per lane with sample buffer containing no β-mercaptoethanol (−) or 300 mm β-mercaptoethanol (+) and fractionated on a 5% gel by SDS-PAGE (A). For the biotin-streptavidin labeling experiment (B), cell surface proteins were labeled with Biotin-7-NHS as described under "Materials and Methods." The cell lysate was immunoprecipitated with an anti-mGluR1 monoclonal antibody (Transduction Laboratories), and proteins eluted with sample buffer containing no β-mercaptoethanol (−) were separated by SDS-PAGE. WT-R1 and RhodC-R1 mutant receptor bands were detected with anti-mGluR1 polyclonal antibody purchased from Chemicon (blot labeled Anti-mGluR1). These same bands of the WT-R1 and RhodC-R1 mutant receptors were detected with peroxidase-conjugated streptavidin (labeled Strep-POD) in a separate blot of the same samples. C, enzymatic deglycosylation studies with PNGase-F (second lane fromleft for WT-R1 or RhodC-R1 blot) and Endo-H (third lane from left for WT-R1 or RhodC-R1 blot) to identify asparagine-linked glycosylation states of WT-R1 and RhodC-R1 mutant receptors. 30 μl of whole cell extract of each sample was incubated without (−) or with PNGase-F (+) or with Endo-H (+) for 2 h at 37 °C. The reaction was stopped by adding sample buffer, and digested samples were analyzed under non-reducing condition by immunoblotting with anti-mGluR1 monoclonal antibody. The positions of molecular weight standards are indicated on the left for blots shown in A, B, and C. Similar results were seen in additional experiments with independent transfections and immunoblots.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further determine whether the dimeric mGluR1 and monomeric RhodC-R1 forms were expressed at the cell surface, cell surface proteins were labeled with membrane impermeant Biotin-7-NHS prior to lysing the cells. The wild type mGluR1 and the RhodC-R1 were then immunoprecipitated with anti-mGluR1 monoclonal antibody and eluted with gel loading sample buffer containing no β-mercaptoethanol. Immunoprecipitates were analyzed by SDS-PAGE, and immunoblots were stained either with streptavidin-POD to detect biotinylated cell surface proteins or with a mGluR1 polyclonal antibody to detect all the immunoreactive bands. As shown in Fig. 1 B, under non-reducing conditions, streptavidin-POD detected the upper ∼260- to 270-kDa dimeric bands but no visible 130- or 135-kDa monomeric bands of the mGluR1. Similarly, streptavidin-POD detected the 90-kDa band of the RhodC-R1 mutant. A duplicate blot of these samples with anti-mGluR1 polyclonal antibody detected all the immunoreactive bands of the wild type mGluR1 and RhodC-R1. mGluR1 ECD contains four potential asparagine-linked glycosylation sites (Asn-Xaa-Ser/Thr) and is shown to undergo glycosylation (11Robbins M.J. Ciruela F. Rhodes A. McLlhinney J. Neurochem. 1999; 72: 2539-2547Crossref PubMed Scopus (70) Google Scholar), and the RhodC-R1 mutant contains two potential asparagine-linked glycosylation sites within the rhodopsin tag sequence. Therefore, to determine further the biochemical identity of the ∼260- to 270-kDa dimeric bands of the wild type and the 90-kDa RhodC-R1 mutant receptors, we conducted deglycosylation experiments with two glycosidase enzymes, PNGase-F and Endo-H. The PNGase-F enzyme cleaves all asparagine-linked carbohydrates (both intermediate high mannose forms and fully processed complex carbohydrate forms) from glycoproteins (22Barsomian G.D. Johnson T.L. Borowski M. Denman J. Ollington J.F. Hirani S. McNeilly D.S. Rasmussen J.R. J. Biol. Chem. 1990; 265: 6967-6972Abstract Full Text PDF PubMed Google Scholar). Sensitivity to Endo-H digestion distinguishes between the fully processed mGluR1 forms that are modified with complex carbohydrates (Endo-H-resistant) and intermediate high mannose modified forms (Endo-H-sensitive), which have not trafficked from the endoplasmic reticulum to the Golgi (13Ray K. Hauschild B.C. Steinbach P.J. Goldsmith P.K. Hauache O. Spiegel A.M. J. Biol. Chem. 1999; 274: 27642-27650Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 18Ray K. Clapp P. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1998; 273: 34558-34567Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 19Ray K. Fan G.F. Goldsmith P.K. Spiegel A.M. J. Biol. Chem. 1997; 272: 31355-31361Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). As shown in Fig.1 C, under non-reducing conditions, whole cell extracts prepared from mGluR1- and RhodC-R1-transfected cells digested with PNGase-F showed a decrease in the size of both the ∼260- to 270-kDa bands of the wild mGluR1 and the 90-kDa band of the RhodC-R1 mutant receptors. However, these bands remained mostly resistant to Endo-H digestion. These data suggest that the dimeric wild type mGluR1 and monomeric RhodC-R1 receptors are expressed at the cell surface as asparagine-linked glycosylated and mostly fully processed receptor forms. We generated single C → S mutants of three cysteines at positions 67, 109, and 140 in the mGluR1 ECD. The C → S mutants were further analyzed by determining their dimerization patterns on immunoblots ran under non-reducing conditions. Transiently transfected cells expressing these mutant receptors were treated with iodoacetamide to prevent aggregates forming secondary to non-specific disulfide bond formation prior to crude membrane preparation. The membrane extracts were run on immunoblots, and immunoreactive bands were detected with an anti-mGluR1 monoclonal antibody. As seen in Fig.2 A, the wild type mGluR1 receptor expressed as two dimeric bands with little or no monomeric forms visible on immunoblot. C67S and C109S mutant receptors expressed predominantly as two 130- and 135-kDa monomeric forms, and one or two fainter upper dimeric band corresponding to the upper ∼260- to 270-kDa dimeric bands of the wild type mGluR1 were sometimes visible. These fainter dimeric bands were more visible when samples ran on gels by SDS-PAGE were enriched as crude membrane extracts or as immunoprecipitated samples but not routinely seen in whole cell extracts prepared from C67S- and C109S-transfected cells. Interestingly, C140S mutant receptor expressed as a broad ∼140-kDa monomeric band that ran at slightly higher molecular mass than the 130- and 135-kDa monomeric bands of C67S and C109S mutants and also generated a ∼300-kDa dimeric band. This dimeric band of the C140S mutant ran higher than the dimeric bands of the wild type mGluR1 or C67S or C109S mutant receptors. The intensities of the immunoreactive dimeric bands relative to the monomeric bands for each mutant receptor and wild type receptor were then measured by densitometric scanning (Table I). The data showed that C67S, C109S, and C140S mutant receptors expressed mostly as monomeric forms and generated a small amount of dimeric forms. In contrast, the wild type receptor expressed predominantly as dimers and generated a small amount of monomers. After reduction (Fig. 2 B), a majority of the dimeric ∼260- to 270-kDa bands of the wild type receptor were reduced to a broad monomeric ∼133-kDa band and a small portion remained as SDS-resistant dimeric aggregates, whereas the dimeric bands generated by C67S, C109S, and C140S mutant receptors remained mostly as SDS-resistant dimeric aggregates. Also, the 140-kDa monomeric band of the C140S mutant showed a small shift of molecular mass to a 133-kDa band like the wild type receptor. Taken together, the results suggest that substituting serine for cysteines 67, 109, and 140 directly or indirectly blocks dimerization.Table IDensitometric measurement of dimeric versus monomeric band intensities of the wild type, C67S, C109S, and C140S mutant receptors expressed transiently in HEK-293 cellsTransfectionImmunodetectable relative amountDimeric bandsMonomeric bandsWild type mGluR11.85 ± 0.040.15 ± 0.06C67S0.25 ± 0.051.36 ± 0.04C109S0.20 ± 0.061.42 ± 0.04C140S0.36 ± 0.041.30 ± 0.03Immunoblotting experiments were performed under non-reducing conditions as described in the legend of Fig 2 A. The intensities of the total dimeric and total monomeric immunoreactive bands on x-ray film were measured by densitometric scanning. The results are presented as the mean S.E. of six immunoblotting experiments. Open table in a new tab Immunoblotting experiments were performed under non-reducing conditions as described in the legend of Fig 2 A. The intensities of the total dimeric and total monomeric immunoreactive bands on x-ray film were measured by densitometric scanning. The results are presented as the mean S.E. of six immunoblotting experiments. Because point mutations of three cysteine residues in mGluR1 generated monomeric receptors, we wanted to distinguish between properly processed cell surface versusintracellularly trapped monomeric forms. Cell surface proteins of the HEK-293 cells transiently transfected with wild type, C67S, C109S, and C140S mutant receptors were labeled with membrane impermeant Biotin-7-NHS. The whole cell extracts were immunoprecipitated with anti-mGluR1 monoclonal antibody, ran under non-reduced condition, and analyzed on immunoblots stained either with streptavidin-POD to detect biotinylated cell surface proteins or with anti-mGluR1 polyclonal antibody to detect total mGluR1 immunoreactive species. As seen in Fig.3 A, streptavidin-POD detected only the dimeric ∼260- to 270-kDa bands of the wild type mGluR1 and a fainter dimeric band of C67S and C109S mutants but no monomeric 130- to 135-kDa bands of these receptors. In contrast, streptavidin detected both the dimeric 300-kDa and monomeric 140-kDa bands of the C140S mutant receptor. A duplicate blot of the samples with anti-mGluR1 polyclonal antibody detected both the cell surface and the intracellular forms of the wild type and mutant receptors (Fig.3 A, anti-mGluR1 blot). To further determine the biochemical identity of the expressed bands of the C67S, C109S, and C140S mutant receptors, we tested for sensitivity to Endo-H digestion. Whole cell extracts prepared from HEK-293 cells transiently transfected with wild type, C67S, C109S, and C140S cDNAs were digested with Endo-H and analyzed by SDS-PAGE under non-reducing conditions. As seen in Fig.3 B, digestion with Endo-H caused no decrease in the sizes of the dimeric ∼260- to 270-kDa bands of the wild type mGluR1, which remained mostly resistant to Endo-H digestion. Whereas, both the lower 130- and 135-kDa bands of the wild type, C67S, and C109S mutant receptors showed sensitivity to Endo-H digestion with downward mobility shifts, indicating that these are intracellularly trapped, high mannose receptor forms. For the C140S mutant, however, both the 300- and 140-kDa bands were largely resistant to Endo-H digestion, and only a small fraction of both bands showed mobility shift after Endo-H digestion as shown by arrows in Fig. 3 B (right side). These results suggest that, of the different monomeric forms generated by C67S, C109S, or C140S mutants, only the 140-kDa monomeric form of C140S mutant receptor is expressed at the cell surface. The ability of the monomeric forms of the C140S mutant to express at the cell surface suggested that the Cys-140 residue is sufficient to form an intermolecular disulfide bond critical for dimerization. To further confirm this notion, we introduced ectopic cysteines at different positions on C140S mutant background to determine whether enforced apposition of these cysteine residues would rescue any dimeric receptor form from the C140S monomers. Band patterns of seven Cys-scanning mutants, namely S119C, S126C, S129C, K134C, D143C, T146K, and T152C generated on the C140S mutant background were analyzed under non-reducing conditions and compared with the wild type and C140S mutant receptors. As shown in Fig. 4, four mutants S129C, K134C, D143C, and T146C, expressed mostly as two dimeric ba