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Direct Detection of the Interaction between Recombinant Soluble Extracellular Regions in the Heterodimeric Metabotropic γ-Aminobutyric Acid Receptor

2007; Elsevier BV; Volume: 283; Issue: 8 Linguagem: Inglês

10.1074/jbc.m705202200

1083-351X

Rei Nomura, Yoshikazu Suzuki, Akira Kakizuka, Hisato Jingami,

Receptor Mechanisms and Signaling

The γ-aminobutyric acid, type B (GABAB) receptor is a heterodimeric receptor consisting of two complementary subunits, GABAB1 receptor (GBR1) and GABAB2 receptor (GBR2). GBR1 is responsible for GABA binding, whereas GBR2 is considered to perform a critical role in signal transduction toward downstream targets. Therefore, precise communication between GBR1 and GBR2 is thought to be essential for the proper signal transduction process. However, biochemical data describing the interaction of the two subunits, especially for the extracellular regions, are not sufficient. Thus we began by developing a protein expression system of the soluble extracellular regions. One of the soluble recombinant GBR1 proteins exhibited a ligand binding ability, which is similar to that of the full-length GBR1, and thus the ligand-binding domain was determined. Direct interaction between GBR1 and GBR2 extracellular soluble fragments was confirmed by co-expression followed by affinity column chromatography and a sucrose density gradient sedimentation. In addition, we also found homo-oligomeric states of these soluble extracellular regions. The interaction between the two soluble extracellular regions caused the enhancement of the agonist affinity for GBR1 as previously reported in a cell-based assay. These results not only open the way to future structural studies but also highlight the role of the interaction between the extracellular regions, which controls agonist affinity to the heterodimeric receptor. The γ-aminobutyric acid, type B (GABAB) receptor is a heterodimeric receptor consisting of two complementary subunits, GABAB1 receptor (GBR1) and GABAB2 receptor (GBR2). GBR1 is responsible for GABA binding, whereas GBR2 is considered to perform a critical role in signal transduction toward downstream targets. Therefore, precise communication between GBR1 and GBR2 is thought to be essential for the proper signal transduction process. However, biochemical data describing the interaction of the two subunits, especially for the extracellular regions, are not sufficient. Thus we began by developing a protein expression system of the soluble extracellular regions. One of the soluble recombinant GBR1 proteins exhibited a ligand binding ability, which is similar to that of the full-length GBR1, and thus the ligand-binding domain was determined. Direct interaction between GBR1 and GBR2 extracellular soluble fragments was confirmed by co-expression followed by affinity column chromatography and a sucrose density gradient sedimentation. In addition, we also found homo-oligomeric states of these soluble extracellular regions. The interaction between the two soluble extracellular regions caused the enhancement of the agonist affinity for GBR1 as previously reported in a cell-based assay. These results not only open the way to future structural studies but also highlight the role of the interaction between the extracellular regions, which controls agonist affinity to the heterodimeric receptor. γ-Aminobutyric acid (GABA) 3The abbreviations used are: GABAγ-aminobutyric acidGBR1GABAB1 receptorGBR2GABAB2 receptorGPCRG-protein-coupled receptormGluRmetabotropic glutamate receptorLBDligand-binding domainECRextracellular regionPEGpolyethylene glycol. is a major inhibitory neurotransmitter of the central nervous system that activates two types of receptor, the ionotropic GABAA/C receptors, to produce fast synaptic inhibition, and the metabotropic GABAB receptor, to elicit a slow and prolonged inhibitory response. The GABAB receptor, a G-protein-coupled receptor (GPCR), modulates neurotransmitter release from pre-synaptic neurons or hyperpolarization of post-synaptic membranes (1Bowery N.G. Hill D.R. Hudson A.L. Doble A. Middlemiss D.N. Shaw J. Turnbull M. Nature. 1980; 283: 92-94Crossref PubMed Scopus (895) Google Scholar, 2Bettler B. Kaupmann K. Mosbacher J. Gassmann M. Physiol. Rev. 2004; 84: 835-867Crossref PubMed Scopus (711) Google Scholar). The GABAB receptor belongs to the class C GPCR, together with metabotropic glutamate receptors (mGluR1–8), a Ca2+-sensing receptor and some pheromone and taste receptors. Each class C GPCR member consists of three functional regions: a large N-terminal extracellular region (ECR) that binds ligands, a seven-spanning transmembrane region, and a cytoplasmic region. Another feature of these receptors is that they function as dimers. mGluRs and Ca2+-sensing receptor form functional homodimers, and the crystal structures of the homodimeric ligand-binding domains (LBDs) of mGluR1, mGluR3, and mGluR7 have been determined (3Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1124) Google Scholar, 4Muto T. Tsuchiya D. Morikawa K. Jingami H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3759-3764Crossref PubMed Scopus (316) Google Scholar). On the other hand, the GABAB receptor forms a heterodimer consisting of two subunits, GABAB1 receptor (GBR1) and GABAB2 receptor (GBR2). γ-aminobutyric acid GABAB1 receptor GABAB2 receptor G-protein-coupled receptor metabotropic glutamate receptor ligand-binding domain extracellular region polyethylene glycol. The two GBR subunits share ∼35% amino acid sequence similarity. The first GBR subunits, isolated by expression cloning using a specific synthetic radiolabeled ligand (5Kaupmann 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 (883) Google Scholar, 6Froestl W. Bettler B. Bittiger H. Heid J. Kaupmann K. Mickel S.J. Strub D. Neuropharmacology. 1999; 38: 1641-1646Crossref PubMed Scopus (18) Google Scholar), are two isoforms, named GBR1a and GBR1b. They differ in the N terminus, where GBR1a possesses a repeat of consensus sequences for the complement protein (also called Sushi domain), and it is missing in the GBR1b. Because no significant differences have been revealed about the two main GBR1 isoforms in a number of pharmacological studies, we focused on GBR1a to simplify the biochemical study (GBR1a is termed GBR1 hereafter). When a recombinant GBR1 is expressed by itself in COS cells, it remains intracellular, and the transfected cells lack ligand binding ability (7Couve A. Filippov A.K. Connolly C.N. Bettler B. Brown D.A. Moss S.J. J. Biol. Chem. 1998; 273: 26361-26367Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). In further studies, the GBR2 subunit was discovered by several groups (8Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.-J. Johnson M. Gunwaldsen C. Huang L.-Y. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (932) Google Scholar, 9White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1022) Google Scholar, 10Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1022) Google Scholar, 11Kuner R. Köhr G. Grünewald S. Eisenhardt G. Bach A. Kornau H.-C. Science. 1999; 283: 74-77Crossref PubMed Scopus (503) Google Scholar), and its co-localization with GBR1 in the brain was reported (8Jones K.A. Borowsky B. Tamm J.A. Craig D.A. Durkin M.M. Dai M. Yao W.-J. Johnson M. Gunwaldsen C. Huang L.-Y. Tang C. Shen Q. Salon J.A. Morse K. Laz T. Smith K.E. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Nature. 1998; 396: 674-679Crossref PubMed Scopus (932) Google Scholar, 10Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1022) Google Scholar, 11Kuner R. Köhr G. Grünewald S. Eisenhardt G. Bach A. Kornau H.-C. Science. 1999; 283: 74-77Crossref PubMed Scopus (503) Google Scholar). Subsequently, when GBR1cDNA was co-transfected with GBR2cDNA simultaneously, both subunits were expressed on the cell surface, and the cells clearly showed ligand binding activity. Interestingly, GBR2 increased the affinities of agonists to GBR1, but not that of an antagonist (9White J.H. Wise A. Main M.J. Green A. Fraser N.J. Disney G.H. Barnes A.A. Emson P. Foord S.M. Marshall F.H. Nature. 1998; 396: 679-682Crossref PubMed Scopus (1022) Google Scholar, 10Kaupmann K. Malitschek B. Schuler V. Heid J. Froestl W. Beck P. Mosbacher J. Bischoff S. Kulik A. Shigemoto R. Karschin A. Bettler B. Nature. 1998; 396: 683-687Crossref PubMed Scopus (1022) Google Scholar). In addition, yeast two-hybrid experiments indicated that both subunits interact at their coiled-coil intracellular regions. Truncation and amino acid mutation experiments revealed that this association is involved in shielding an ER retention signal close to the coiled-coil region within the GBR1 intracellular region and enables the combined GBR subunits to transport to the cell surface (12Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Neuron. 2000; 27: 97-106Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 13Pagano A. Rovelli G. Mosbacher J. Lohmann T. Duthey B. Stauffer D. Ristig D. Schuler V. Meigel I. Lampert C. Stein T. Prézeau L. Blahos J. Pin J.-P. Froestl W. Kuhn R. Heid J. Kaupmann K. Bettler B. J. Neurosci. 2001; 21: 1189-1202Crossref PubMed Google Scholar). However, this C-terminal interaction between both subunits is not essential for dimerizing the receptor complex per se, because removal of the GBR1 C terminus allows the assembly of a functional heterodimer (13Pagano A. Rovelli G. Mosbacher J. Lohmann T. Duthey B. Stauffer D. Ristig D. Schuler V. Meigel I. Lampert C. Stein T. Prézeau L. Blahos J. Pin J.-P. Froestl W. Kuhn R. Heid J. Kaupmann K. Bettler B. J. Neurosci. 2001; 21: 1189-1202Crossref PubMed Google Scholar, 14Calver A.R. Robbins M.J. Cosio C. Rice S.Q.J. Babbs A.J. Hirst W.D. Boyfield I. Wood M.D. Russell R.B. Price G.W. Couve A. Moss S.J. Pangalos M.N. J. Neurosci. 2001; 21: 1203-1210Crossref PubMed Google Scholar). Fluorescence resonance energy transfer experiments also showed that both subunits interact at the ECR on the cell surface (15Liu J. Maurel D. Etzol S. Brabet I. Ansanay H. Pin J.-P. Rondard P. J. Biol. Chem. 2004; 279: 15824-15830Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Therefore, these previous data indicated that GBR1 and GBR2 interact in the extracellular region and/or transmembrane region; however, the interaction manner at the molecular level has not been completely deciphered. The amino acids responsible for ligand binding in the GBR1 subunit have been postulated by homology modeling to the crystal structures of the mGluR1-LBD and amino acid mutagenesis experiments (16Kniazeff J. Galvez T. Labesse G. Pin J.-P. J. Neurosci. 2002; 22: 7352-7361Crossref PubMed Google Scholar). The amino acids that possibly participate in the ligand binding are well conserved among several species. However, the corresponding residues in the GBR2 subunit vary among species, and amino acid substitutions of these residues did not prevent GABA activation of the receptor (16Kniazeff J. Galvez T. Labesse G. Pin J.-P. J. Neurosci. 2002; 22: 7352-7361Crossref PubMed Google Scholar). Therefore, GBR2 itself is considered not to bind ligands, whereas G-proteins interact with specific intracellular sites in GBR2 (17Galvez T. Duthey B. Kniazeff J. Blahos J. Rovelli G. Bettler B. Prézeau L. Pin J.-P. EMBO J. 2001; 20: 2152-2159Crossref PubMed Scopus (317) Google Scholar, 18Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14649-14654Crossref PubMed Scopus (156) Google Scholar, 19Robbins M.J. Calver A.R. Filippov A.K. Hirst W.D. Russell R.B. Wood M.D. Nasir S. Couve A. Brown D.A. Moss S.J. Pangalos M.N. J. Neurosci. 2001; 21: 8043-8052Crossref PubMed Google Scholar, 20Duthey B. Caudron S. Perroy J. Bettler B. Fagni L. Pin J.-P. Prézeau L. J. Biol. Chem. 2002; 277: 3236-3241Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The extracellular signals received by GBR1 are transmitted to the cytoplasmic region of GBR2 through an allosteric interaction between the two subunits. Thus, signal transmission in the GABAB receptor has been proposed to occur via initial ligand binding to the binding pocket within the GBR1 subunit, followed by allosteric conformational changes in the transmembrane and cytoplasmic regions of the GBR2 subunit, and, finally, G-protein activation. This scenario is consistent with experimental data to date; however, the mechanistic details remain elusive. As a first step toward understanding how the ligand-binding signal is propagated within a receptor protein, it is crucial to determine the LBD of the GABAB receptor and to obtain a sufficient amount of the purified protein for biophysical studies. Previously, Malitschek et al. (21Malitschek B. Schweizer C. Keir M. Heid J. Froestl W. Mosbacher J. Kuhn R. Henley J. Joly C. Pin J.-P. Kaupmann K. Bettler B. Mol. Pharmacol. 1999; 56: 448-454Crossref PubMed Scopus (98) Google Scholar) reported that the GBR1 extracellular region can be produced as a soluble protein and that it retains the binding pharmacology of wild-type GBR1, as determined by photoaffinity labeling. Here we report a method to express and partially purify a soluble form of the ECRs of both GBR subunits in the insect cell/baculovirus system. Although the obtained GBR1-ECR showed imperfect binding activity possibly due to incorrect folding component (see "Discussion"), interaction between the soluble GBR1-ECR and GBR2-ECR and its effect on agonist affinity have been proved using several biochemical assays. These results directly demonstrated the function of the interaction between the extracellular regions of the two subunits, which has been previously suggested only in a cell-based assay. We also detected homo-oligomerization states of these soluble proteins. These results provide insights for the interaction manner of the extracellular domains of the heteromeric GABAB subunits. Our method reported here would facilitate structural studies to further understand the activation mechanism at an atomic resolution. Materials–Baclofen, CGP54626, and [3H]CGP54626 (53.31 Ci/mmol) were purchased from Tocris (UK). GABA was purchased from Wako (Osaka, Japan). Oligonucleotide primers were obtained from Proligo (Japan). Construction of Expression Vectors for GBR1-ECRs and GBR2-ECR–To make expression plasmids for the ECRs of GBR1 and GBR2, we performed PCR with the full-length human GBR1 and GBR2 cDNAs, which were kindly provided by Prof. Bernhard Bettler (University of Basel), as templates. In the case of GBR1-ECR, a forward primer was designed with a BamHI site just after the 3′-end of the coding sequence of an endogenous GBR1 signal peptide. The reverse primer was designed with a stop codon, followed by an XbaI site. The PCR product was cloned using the BamHI and XbaI sites of pBlueScript, which contains a hemagglutinin signal sequence followed by a FLAG epitope (DYKDDDDK) tag just upstream of the BamHI site. The cDNA fragment was excised using the NotI and XbaI sites, which were in multiple cloning sites of pBlueScript, and the fragment was inserted into the pFastBac DUAL vector, resulting in the transfer vector pGBR1-ECR1. For the longer GBR1-ECR2 and -ECR3 constructs, PCRs were performed with a forward primer with an MfeI site and reverse primers designed at distinct positions with a stop codon followed by a NheI site. The two PCR products were replaced with the MfeI-XbaI fragment in pGBR1-ECR1, resulting in pGBR1-ECR2 and -ECR3. For GBR2-ECR, the forward primer was designed with an Eco47III site just after the coding region of the endogenous signal sequence, and the reverse primer was designed with a sequence encoding His6, followed by an XbaI site, at the putative 3′-end of the GBR2-ECRcDNA. The PCR product was exchanged with the mGluR1cDNA previously cloned into the pFastBac DUAL vector using the Eco47III and XbaI sites. Consequently, an artificial GBR2cDNA, containing a DNA sequence encoding the mGluR1 signal sequence instead of the original signal sequence, was prepared. Production of Baculoviruses for Protein Expression–Baculoviruses for protein expression were obtained by the Bac-to-Bac baculovirus expression system (Invitrogen). Spodoptera frugiperda (Sf-9) cells were propagated in a monolayer at 27 °C in TNM-FH (Grace's powder medium, 0.4% yeastolate, 0.3% lactalbumin hydrolysate, 0.1% pluronic F-68, 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B). The vector DNA was transformed into DH10Bac Escherichia coli cells (Invitrogen). The recombinant bacmid DNA purified from the DH10Bac cells was then transfected into Sf-9 insect cells, using the Cellfectin reagent (Invitrogen). After incubation for 72 h at 27 °C, the viruses were amplified by re-infecting the Sf-9 cells to enhance the viral titer. Finally, the viral titer was checked by a plaque formation assay, using an immobilized monolayer culture of Sf-9 cells. Protein Expression and Purification–HighFive cells were cultured in a monolayer at 27 °C in Express Five serum-free medium (Invitrogen) supplemented with 18 mm l-glutamine. The GBR1-ECRs and GBR2-ECR proteins were expressed by inoculating the baculoviruses into the HighFive cells. The cell culture medium was collected 4 days after the inoculation. After the addition of protease inhibitors (10 μg/ml leupeptin, 2 μg/ml pepstatin, 0.1 mm phenylmethylsulfonyl fluoride), the cells were pelleted by centrifugation at 6000 × g for 20 min at 4 °C. Purification of the protein was facilitated by the FLAG and His tags. In the case of the GBR1-ECRs, the supernatant was applied directly to anti-FLAG M2-agarose (Sigma) packed in a disposable column (Bio-Rad). After the column was washed with low salt buffer (10 mm Tris-HCl (pH 7.5) and 20 mm NaCl), the protein bound to the column was eluted by high salt buffer (20 mm Tris-HCl (pH 7.5), 118 mm NaCl, 5.6 mm glucose, 1.2 mm KH2PO4, 1.2 mm MgSO4, 4.7 mm KCl, and 1.8 mm CaCl2) containing the FLAG peptide at a concentration of 150 μg/ml. The supernatant containing the GBR2-ECR protein was directly applied to nickel-Sepharose (Amersham Biosciences) packed in a disposable column (Bio-Rad). The column was washed with phosphate buffer (0.5 m NaCl and 20 mm sodium phosphate), and the protein bound to the column was eluted with the phosphate buffer containing 150 mm imidazole. Western Blotting–The culture medium was fractionated by SDS-PAGE, and the proteins were electroblotted onto a nitrocellulose membrane. The membrane was blocked for 1 h in TBST (10 mm Tris-HCl (pH 8.0), 150 mm NaCl, and 0.05% Tween 20) with 3% bovine serum albumin and then was incubated for 1 h with anti-FLAG and anti-His antibodies at room temperature. The membrane was washed and then incubated for 1 h with anti-mouse IgG conjugated with alkaline phosphatase at room temperature. Color development was done in ALP buffer (40 mm Tris-HCl (pH 9.0), 150 mm NaCl, 1 mm MgCl2) using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium Color Development Substrate (Promega). Ligand Binding Assay–The ligand binding assay was performed by the polyethylene glycol (PEG) precipitation method, as previously described (22Tsuji Y. Shimada Y. Takeshita T. Kajimura N. Nomura S. Sekiyama N. Otomo J. Usukura J. Nakanishi S. Jingami H. J. Biol. Chem. 2000; 275: 28144-28151Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). 3H-Labeled CGP54626, GABA, and the protein solution were mixed in 150 μl of the high salt buffer at 4 °C for 1 h. Then, 6-kDa PEG was added to the sample to a final concentration of 15%, along with 3 mg/ml γ-globulin. After vortexing and centrifugation, the precipitated material was washed twice with 1 ml of 40 mm HEPES (pH 7.4) and 2.5 mm CaCl2 containing 8% 6-kDa PEG, and then was dissolved in 1 ml of water. After the addition of 14 ml of ClearsolII (Nacalai Tesque), the radioactivity was measured using a scintillation counter. Density Gradient Centrifugation–The GBR1-ECR3 protein, partially purified by the anti-FLAG M2 column, the GBR2-ECR protein, partially purified by the nickel column, and a mixture of both proteins were sedimented through 15–35% sucrose gradients formed with the high salt buffer. The gradients were made using a Gradient Master (BioComp, Inc., Minneapolis, MN). Samples were applied to the top of the sucrose gradient and were centrifuged in an SW41 (Beckman) rotor for 22 h at 40,000 rpm. After the centrifugation, the samples were fractionated into 150-μl aliquots by a piston gradient fractionator (BioComp, Inc.). The fractionated samples were subjected to SDS-PAGE and Western blotting with anti-FLAG and anti-His antibodies. An anti-mouse secondary antibody conjugated with horseradish peroxidase was then applied. Proteins were visualized and quantified using an ECL detection kit (Amersham Biosciences). Expression and Purification of the Extracellular Regions of the GABAB Receptor–Fig. 1 shows diagrams of the recombinant proteins of the GABAB receptor ECRs used in the baculovirus infection experiments. To determine the C-terminal ends of the soluble forms of the ECRs by analogy, we first aligned the amino acid sequences of the GABAB receptor ECR and the mGluR1-LBD, and then constructed transfer vectors for the expression of the three GBR1-ECRs (GBR1-ECR1–3) and the GBR2-ECR. Because we expected that efficient secretion was a key step to obtain these soluble proteins, we replaced the original signal peptides by exogenous signal peptides, which had been working well in our laboratory. As shown in Fig. 1, the signal peptide from influenza virus hemagglutinin was added to the N termini of the GBR1-ECRs, whereas that of mGuR1 was exploited for GBR2-ECR. Consequently, both proteins were secreted well and accumulated in the culture medium. We then purified these soluble proteins by affinity chromatography. The expression and purification of these extracellular fragments were confirmed by SDS-PAGE followed by silver staining and Western blotting as shown in Fig. 2. The three GBR1 fragments (GBR1-ECR1–3) migrated to positions, slightly lower than 75 kDa. Their sizes correspond to the molecular mass of 64 kDa calculated from the amino acid compositions. The GBR2-ECR was detected at a position around 50 kDa. This size also corresponds to the molecular mass of GBR2-ECR. Thus, all three GBR1-ECRs and GBR2-ECR were confirmed to have been secreted into the culture medium. The purified GBR1-ECR3 and GBR2-ECR proteins were concentrated and analyzed by SDS-PAGE followed by Coomassie Brilliant Blue R250 staining, to assess the purity. Although a few extra bands were observed (Fig. 2, C and D), especially at the high molecular mass regions in the GBR1-ECR3 lane, each protein was pure enough to perform the following biochemical experiments.FIGURE 2Purification of the FLAG-tagged GBR1-ECRs and the His-tagged GBR2-ECR. GBR1-ECR1–3 were expressed and purified with anti-FLAG M2-agarose gel. Eluted samples were run on 9% SDS-polyacrylamide gels and analyzed by silver staining, Coomassie Brilliant Blue staining, and Western blotting using specific antibodies, as shown. The arrows indicate bands corresponding to GBR1-ECR1 (A), GBR1-ECR2 (B), and GBR1-ECR3 (C). GBR2-ECR was expressed and purified with a nickel-Sepharose column. The arrows indicate bands corresponding to GBR2-ECR (D). Samples of 1.0 μg (GBR1-ECR3) and 0.4 μg (GBR2-ECR) were fractionated and silver stained. Concentrated samples of 4.5 μg (GBR1-ECR3) and 7.3 μg (GBR2-ECR) were stained by Coomassie Brilliant Blue (CBB).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Ligand Binding Assay of the GBR1-ECRs–We investigated the ligand-binding abilities of GBR1-ECR1–3, using 3H-labeled CGP54626 ([3H]CGP54626) by the PEG-precipitation method, as described under "Experimental Procedures" (Fig. 3A). The final concentration of [3H]CGP54626 was 20 nm. Although [3H]CGP54626 did not bind to GBR1-ECR1 and -ECR2, its binding to GBR1-ECR3 was clear. Furthermore, in the presence of non-labeled GABA, the binding was displaced. This result was intriguing, because the difference between GBR1-ECR2 and GBR1-ECR3 was the five additional amino acid residues at the C terminus of GBR1-ECR3. As a result, these five amino acid residues were shown to be important for the proper protein folding required for ligand binding ability. However, the GBR2-ECR itself did not bind the ligand, consistent with previous reports (16Kniazeff J. Galvez T. Labesse G. Pin J.-P. J. Neurosci. 2002; 22: 7352-7361Crossref PubMed Google Scholar). We next investigated saturation binding. The amount of specific ligand binding to GBR1-ECR3 increased and plateaued at a concentration of ∼40 nm [3H]CGP54626 (Fig. 3B, squares), while significant binding to the GBR1-ECR2 was not observed (Fig. 3B, triangles). The dissociation constant (Kd) of this ligand for GBR1-ECR3 was 8.7 ± 2.3 nm. Bmax was 1.12 ± 0.154 nmol/mg of protein. This was 7% of the theoretical value, which was calculated on the assumption that all of the purified proteins had normal binding activity. Therefore only 7% of the obtained proteins were active. The other 93% were inactive probably because of incorrect protein folding (see "Discussion"). Although the incompleteness of protein purification can affect results of quantitative analyses, which need precise active protein concentration, it does not affect results of qualitative analyses such as a competition binding assay or detection of interaction with other proteins. Therefore we proceeded with the following qualitative analyses using this protein. The inhibition of [3H]CGP54626 binding by two agonists and an antagonist was examined. The dose-response curves are shown in Fig. 3C. The rank order of inhibition was CGP54626 ≫ GABA > baclofen (Table 1).TABLE 1Half-maximal inhibition (IC50) values of three ligands in [3H]CGP54626 binding to GBR1-ECR3 in the presence and absence of GBR2-ECR and mGluR1-LBDLigandIC50GABABaclofenCGP54626μmnmGBR1-ECR323.54 ± 0.344.44 ± 0.419.47 ± 0.8GBR1-ECR3 + GBR2-ECR6.71 ± 1.712.38 ± 0.227.45 ± 6.5GBR1-ECR3 + mGluR1-LBD19.43 ± 0.3NDaND, not determined.NDa ND, not determined. Open table in a new tab Interaction between GBR1-ECR3 and GBR2-ECR–Because both the GBR1-ECR3 and GBR2-ECR proteins were successfully expressed in soluble forms, we investigated whether GBR1-ECR3 and GBR2-ECR interact with each other in vitro. The viruses encoding the two proteins were co-infected into the insect cells simultaneously. The supernatant of the culture medium was concentrated and loaded on the anti-FLAG M2 column. After washing with low salt buffer, the bound material was eluted with a FLAG peptide and subjected to SDS-PAGE and Western blotting. The membranes were incubated with an anti-FLAG antibody and an anti-His antibody. The samples eluted from the column should contain GBR1-ECR3, because it was trapped by the FLAG peptide tag attached at the N terminus, but not the free GBR2 fragment, unless GBR2-ECR was associated with GBR1-ECR3. GBR1-ECR3 was detected with the anti-FLAG antibody, as shown in Fig. 4A, left, whereas a protein band of ∼50 kDa was detected with the anti-His antibody (Fig. 4A, right). This band is possibly GBR2-ECR, because the molecular mass of GBR2-ECR is 50 kDa and it contains the His tag at its C terminus. To confirm that this band was not due to nonspecific trapping by the anti-FLAG column, GBR2-ECR alone was expressed independently, and the culture medium was loaded onto the anti-FLAG column and eluted with the FLAG peptide. No protein band was detected in the elution by the anti-His antibody (Fig. 4B). Consequently, this indicates that the GBR2-ECR was bound to the anti-FLAG column through the interaction with the GBR1-ECR3, suggesting that the soluble GBR1 and GBR2 extracellular fragments interact with each other under these solution conditions. To confirm the direct interaction between GBR1-ECR3 and GBR2-ECR, we next performed a density gradient centrifugation analysis. We made a 15–35% sucrose gradient and applied each protein on the top of the gradients. After ultracentrifugation, the gradients were fractionated into 150-μl portions, and an aliquot of each fraction was subjected to SDS-PAGE under non-reducing conditions and Western blotting (Fig. 5). Monomeric GBR1-ECR3 was detected as a ∼70-kDa band in fractions 9–12 (Fig. 5A). Other oligomeric protein bands of >250 kDa were observed in fractions after fraction 20, and these oligomeric products shifted to the monomeric position under reducing conditions (data not shown), indicating that these oligomers were formed by intermolecular disulfide bonds. In contrast, GBR2-ECR was broadly found as a band around 50 kDa in fractions from 8 to 26 in Fig. 5D, suggesting that the GBR2 subunit exists in homo-oligomeric states. Then, 2 μg of GBR1-ECR3 and 10 μg of GBR2-ECR were mixed in a final volume of ∼10 μl and loaded onto the same gradient. We chose this protein amount on the basis of the ligand binding experiment of affinity change (described below, in the next paragraph). When the two subunits were mixed, a complex of GBR1-ECR3 and GBR2-ECR appeared in the delayed fractions 18–21, as detected by the anti-FLAG antibody (Fig. 5B). These emerged bands, which were reproducibly observed, were not detected when the GBR1-ECR3 was mixed and centrifuged with the control bovine serum albumin (Fig. 5C). When the mixed samples