Molecular Basis of Gephyrin Clustering at Inhibitory Synapses
2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês
10.1074/jbc.m610290200
ISSN1083-351X
AutoresTaslimarif Saiyed, Ingo Paarmann, Bertram Schmitt, Svenja Haeger, Marı́a Solà, Guönther Schmalzing, Winfríed Weissenhorn, Heinrich Betz,
Tópico(s)Porphyrin and Phthalocyanine Chemistry
ResumoGephyrin is a bifunctional modular protein that, in neurons, clusters glycine receptors and γ-aminobutyric acid, type A receptors in the postsynaptic membrane of inhibitory synapses. By x-ray crystallography and cross-linking, the N-terminal G-domain of gephyrin has been shown to form trimers and the C-terminal E-domain dimers, respectively. Gephyrin therefore has been proposed to form a hexagonal submembranous lattice onto which inhibitory receptors are anchored. Here, crystal structure-based substitutions at oligomerization interfaces revealed that both G-domain trimerization and E-domain dimerization are essential for the formation of higher order gephyrin oligomers and postsynaptic gephyrin clusters. Insertion of the alternatively spliced C5′ cassette into the G-domain inhibited clustering by interfering with trimerization, and mutation of the glycine receptor β-subunit binding region prevented the localization of the clusters at synaptic sites. Together our findings show that domain interactions mediate gephyrin scaffold formation. Gephyrin is a bifunctional modular protein that, in neurons, clusters glycine receptors and γ-aminobutyric acid, type A receptors in the postsynaptic membrane of inhibitory synapses. By x-ray crystallography and cross-linking, the N-terminal G-domain of gephyrin has been shown to form trimers and the C-terminal E-domain dimers, respectively. Gephyrin therefore has been proposed to form a hexagonal submembranous lattice onto which inhibitory receptors are anchored. Here, crystal structure-based substitutions at oligomerization interfaces revealed that both G-domain trimerization and E-domain dimerization are essential for the formation of higher order gephyrin oligomers and postsynaptic gephyrin clusters. Insertion of the alternatively spliced C5′ cassette into the G-domain inhibited clustering by interfering with trimerization, and mutation of the glycine receptor β-subunit binding region prevented the localization of the clusters at synaptic sites. Together our findings show that domain interactions mediate gephyrin scaffold formation. The precise localization and a high density of neurotransmitter receptors at postsynaptic sites is a prerequisite for proper synaptic transmission. During the development of inhibitory synapses, the peripheral membrane protein gephyrin accumulates beneath the postsynaptic plasma membrane and plays a key role in recruiting inhibitory receptors under the contacting nerve terminals (1Kneussel M. Betz H. Trends Neurosci. 2000; 23: 429-435Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 2Moss S.J. Smart T.G. Nat. Rev. Neurosci. 2001; 2: 240-250Crossref PubMed Scopus (396) Google Scholar). Both attenuation of gephyrin expression by antisense oligonucleotides and targeted disruption of the gephyrin gene prevent the synaptic clustering of glycine receptors (GlyRs) 4The abbreviations used are: GlyR, glycine receptor; GlyRβ, glycine receptor β-subunit; BN-PAGE, blue-native PAGE; DIV, days in vitro; GFP, green fluorescent protein; GABAA, γ-aminobutyrate acid, type A; GABAAR, GABAA receptor; HEK, human embryonic kidney; VIAAT, vesicular inhibitory amino acid transporter. 4The abbreviations used are: GlyR, glycine receptor; GlyRβ, glycine receptor β-subunit; BN-PAGE, blue-native PAGE; DIV, days in vitro; GFP, green fluorescent protein; GABAA, γ-aminobutyrate acid, type A; GABAAR, GABAA receptor; HEK, human embryonic kidney; VIAAT, vesicular inhibitory amino acid transporter. (3Kirsch J. Wolters I. Triller A. Betz H. Nature. 1993; 366: 745-748Crossref PubMed Scopus (371) Google Scholar, 4Feng G. Tintrup H. Kirsch J. Nichol M.C. Kuhse J. Betz H. Sanes J.R. Science. 1998; 282: 1321-1324Crossref PubMed Scopus (341) Google Scholar) and γ2-subunit-containing GABAAR subtypes (5Essrich C. Lorez M. Benson J.A. Fritschy J.M. Luscher B. Nat. Neurosci. 1998; 1: 563-571Crossref PubMed Scopus (717) Google Scholar, 6Kneussel M. Brandstatter J.H. Laube B. Stahl S. Muller U. Betz H. J. Neurosci. 1999; 19: 9289-9297Crossref PubMed Google Scholar, 7Kneussel M. Brandstatter J.H. Gasnier B. Feng G. Sanes J.R. Betz H. Mol. Cell. Neurosci. 2001; 17: 973-982Crossref PubMed Scopus (128) Google Scholar). Although a direct interaction with GABAARs has not yet been demonstrated, gephyrin binding to the large intracellular loop of GlyRβ has been shown to be of high affinity (8Meyer G. Kirsch J. Betz H. Langosch D. Neuron. 1995; 15: 563-572Abstract Full Text PDF PubMed Scopus (348) Google Scholar, 9Schrader N. Kim E.Y. Winking J. Paulukat J. Schindelin H. Schwarz G J. Biol. Chem. 2004; 279: 18733-18741Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Additional interaction partners of gephyrin include proteins implicated in the regulation of the cytoskeleton, intracellular trafficking, and protein synthesis (1Kneussel M. Betz H. Trends Neurosci. 2000; 23: 429-435Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 10Paarmann I. Saiyed T. Schmitt B. Betz H. Biochem. Soc. Trans. 2006; 34: 45-47Crossref PubMed Scopus (8) Google Scholar).Gephyrin is a modular protein consisting of an N-terminal G-domain, a C-terminal E-domain, and a connecting linker region (1Kneussel M. Betz H. Trends Neurosci. 2000; 23: 429-435Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 11Prior P. Schmitt B. Grenningloh G. Pribilla I. Multhaup G. Beyreuther K. Maulet Y. Werner P. Langosch D. Kirsch J. Betz H. Neuron. 1992; 8: 1161-1170Abstract Full Text PDF PubMed Scopus (277) Google Scholar). The G- and E-domains of gephyrin show significant homology to Escherichia coli, Drosophila, and plant proteins and are involved in the synthesis of a coenzyme of oxidoreductases, the molybdenum cofactor (4Feng G. Tintrup H. Kirsch J. Nichol M.C. Kuhse J. Betz H. Sanes J.R. Science. 1998; 282: 1321-1324Crossref PubMed Scopus (341) Google Scholar, 11Prior P. Schmitt B. Grenningloh G. Pribilla I. Multhaup G. Beyreuther K. Maulet Y. Werner P. Langosch D. Kirsch J. Betz H. Neuron. 1992; 8: 1161-1170Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 12Stallmeyer B. Schwarz G. Schulze J. Nerlich A. Reiss J. Kirsch J. Mendel R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1333-1338Crossref PubMed Scopus (134) Google Scholar). This enzymatic activity explains the widespread expression of the gephyrin gene also in non-neuronal tissues (11Prior P. Schmitt B. Grenningloh G. Pribilla I. Multhaup G. Beyreuther K. Maulet Y. Werner P. Langosch D. Kirsch J. Betz H. Neuron. 1992; 8: 1161-1170Abstract Full Text PDF PubMed Scopus (277) Google Scholar). Crystallographic analysis of the isolated G- and E-domains indicates that they have trimeric and dimeric structures, respectively (13Sola M. Kneussel M. Heck I.S. Betz H. Weissenhorn W. J. Biol. Chem. 2001; 276: 25294-25301Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Schwarz G. Schrader N. Mendel R.R. Hecht H.J. Schindelin H. J. Mol. Biol. 2001; 312: 405-418Crossref PubMed Scopus (84) Google Scholar, 15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar, 16Kim E.Y. Schrader N. Smolinsky B. Bedet C. Vannier C. Schwarz G. Schindelin H. EMBO J. 2006; 25: 1385-1395Crossref PubMed Scopus (96) Google Scholar). Bacterially expressed full-length gephyrin forms trimers that can assemble into higher order structures (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar). This oligomerization behavior of gephyrin and its subdomains is thought to provide the basis for the formation of submembranous hexagonal gephyrin scaffolds that cluster inhibitory neurotransmitter receptors at postsynaptic membrane specializations (1Kneussel M. Betz H. Trends Neurosci. 2000; 23: 429-435Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar) by reducing their lateral mobility (17Dahan M. Levi S. Luccardini C. Rostaing P. Riveau B. Triller A. Science. 2003; 302: 442-445Crossref PubMed Scopus (1335) Google Scholar, 18Jacob T.C. Bogdanov Y.D. Magnus C. Saliba R.S. Kittler J.T. Haydon P.J. Moss S.J. J. Neurosci. 2005; 25: 10469-10478Crossref PubMed Scopus (206) Google Scholar).In this study, we investigated whether G-domain trimerization and E-domain dimerization are essential for gephyrin scaffold formation. Using structure-deduced mutations that disrupt oligomerization interfaces, we found that both G- and E-domain interactions are required for gephyrin scaffolding. In addition, we report that the postsynaptic localization of the gephyrin scaffold depends on the GlyRβ binding region of the E-domain. Intact E- and G-domains are also a prerequisite for the formation of gephyrin hexamers, which we propose to represent novel intermediates of the scaffold assembly reaction. Together, our data indicate that oligomerization via the G- and E-domains is essential for gephyrin scaffold formation and, hence, the clustering of inhibitory receptors at developing synapses.EXPERIMENTAL PROCEDURESGeneration of Gephyrin Constructs—The region encoding the G-domain of gephyrin (amino acids 1-181) was amplified by PCR using Geph-pRSET (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar) as a template and subcloned into pBluescript II SK (+) (pBSK) (Stratagene) using XmaI/XhoI to generate G-pBSK. The full-length coding region of wild-type P1-gephyrin (gephyrin containing the cassettes 2 and 6′) was excised from Geph-pBSK (11Prior P. Schmitt B. Grenningloh G. Pribilla I. Multhaup G. Beyreuther K. Maulet Y. Werner P. Langosch D. Kirsch J. Betz H. Neuron. 1992; 8: 1161-1170Abstract Full Text PDF PubMed Scopus (277) Google Scholar) using XmaI/NsiI restriction sites and cloned between the XmaI and blunted ApaI sites of the pNKS 2 vector (19Gloor S. Pongs O. Schmalzing G. Gene. 1995; 160: 213-217Crossref PubMed Scopus (63) Google Scholar) to generate Geph-pNKS 2. Codons for an AHHHHHH sequence tag were inserted directly behind the initiator ATG by using the QuikChange mutagenesis kit (Stratagene) to yield His-Geph-pNKS 2. The additional alanine codon serves to maintain the Kozak initiation sequence of Geph-pNKS 2. Using PCR-based mutagenesis, the mutations F90R, L113R, L128R, and L168R were introduced into G-pBSK at the corresponding positions of P1-gephyrin to yield G4xR-pBSK. Wild-type and mutant G-domains were further subcloned into pQE-30 (Qiagen) using the XmaI/SalI sites to generate G-pQE-30 and G4xR-pQE-30, respectively. The mutant G-domain coding region of G4xR-pBSK was introduced into Geph-pBSK (11Prior P. Schmitt B. Grenningloh G. Pribilla I. Multhaup G. Beyreuther K. Maulet Y. Werner P. Langosch D. Kirsch J. Betz H. Neuron. 1992; 8: 1161-1170Abstract Full Text PDF PubMed Scopus (277) Google Scholar), using a PCR-based strategy and NotI/PstI sites, to generate the full-length construct Geph4xR-pBSK. Excision of the Geph4xR cDNA fragment allowed cloning into pEGFP-C2 (Clontech) via SacI/KpnI sites to generate Geph4xR-pEGFP-C2, into pQE-30 using XmaI/SalI sites to generate Geph4xR-pQE-30, and into His-Geph-pNKS 2 using BglII/NdeI sites to generate His-Geph4xR-pNKS 2.The point mutations G483R, R523E, and A532R were introduced into the E-domain (amino acids 316-736) by PCR-based mutagenesis using a PstI fragment of P1-gephyrin (bp 984-2789) cloned in pBSK. The mutated domain (ERER) was subcloned further into E-pRSET and Geph-pRSET (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar) using EcoRI/NcoI sites and into Geph-pEGFP-C2 (20Fuhrmann J.C. Kins S. Rostaing P. El Far O. Kirsch J. Sheng M. Triller A. Betz H. Kneussel M. J. Neurosci. 2002; 22: 5393-5402Crossref PubMed Google Scholar) using PstI restriction sites to generate the constructs ERER-pRSET, GephRER-pRSET, and GephRER-pEGFP-C2, respectively. Transfer of the mutant E-domain from GephRER-pRSET into Geph4xR-pBSK via EcoRI/HindIII sites generated the double domain mutant Geph4xR,RER-pBSK. From this construct, the mutant E-domain was introduced into His-Geph-pNKS 2 using NdeI/XhoI sites to yield His-GephRER-pNKS 2, whereas introduction of both mutant G- and E-domain sequences at the BglII/XhoI sites yielded His-Geph4xR,RER-pNKS 2.The mutants Gephmut and Emut deficient in GlyRβ binding have been described previously (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar). In these mutants, residues 713-721 in the E-domain of gephyrin were replaced by the homologous loop of the E. coli MoeA protein. This E-domain mutation abolishes GlyRβ binding but does not affect C-terminal dimerization (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar). The same mutation was further introduced into His-Geph-pNKS 2 from Geph-MoeA-pBSK (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar) using NdeI/XhoI sites to generate His-Gephmut-pNKS 2. GephC5′-pEGFP-C2, where the cassette C5′ encoding 13 amino acids was introduced after the 98th gephyrin codon, was obtained from G. A. O’Sullivan (Max Planck Institute for Brain Research). The gephyrin insert containing the cassette C5′ was introduced into the pQE-31 vector (Qiagen) using SacI/XmaI sites to yield GephC5′-pQE-31. The GC5′ domain was excised from this construct and introduced into G-pQE-30 via XbaI/BglII sites and into His-Geph-pNKS 2 via BglII/NdeI sites to generate GC5′-pQE-30 and His-GephC5′-pNKS 2, respectively. The construct Gephmut-pEGFP-C2 (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar) has been described previously. All constructs were verified by DNA sequencing.Expression and Purification of Recombinant Proteins—N-terminal His6-tagged wild-type and mutant domain proteins were expressed using the pQE-30/31 (Qiagen) expression system in E. coli BL21 DE3 (Novagen), whereas G4xR was expressed in E. coli C41 DE3 (21Miroux B. Walker J.E. J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1558) Google Scholar). Recombinant proteins were purified as described (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar) and directly used for gel filtration chromatography.Size Exclusion Chromatography—The recombinant wild-type and mutant G- and E-domain proteins were used. The purified proteins were subjected to chromatography on a Superdex 200 column (2.4 ml) in His6 elution buffer (50 mm sodium phosphate, pH 8.0, 300 mm NaCl, 250 mm imidazole, 20 mm β-mercaptoethanol) using a SMART separation unit (Amersham Biosciences). All samples including standard marker proteins (Bio-Rad) were analyzed under identical conditions (6 °C, flow rate 40 μl/min, 50-μl fractions).BN-PAGE of [35S]Methionine-labeled Full-length Gephyrin Purified from Xenopus laevis Oocytes—Collagenase-defolliculated oocytes were injected with capped cRNAs and metabolically labeled by overnight incubation at 19 °C in frog Ringer’s solution (90 mm NaCl, 1 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 10 mm HEPES, pH 7.4) supplemented with ∼40 MBq/ml l-[35S]methionine (>40 TBq/mmol, Amersham Biosciences, ∼0.1 MBq/oocyte). Oocytes were lysed in homogenization buffer consisting of 1% (w/v) digitonin (Merck Biosciences) in 0.1 m phosphate buffer, pH 8.0, 10 mm iodoacetamide, and protease inhibitors (10 μm antipain, 5 μm pepstatin A, 50 μm leupeptin, 100 μm Pefabloc SC). Full-length wild-type and mutant gephyrin proteins were purified as His-tagged proteins under non-denaturing conditions from the centrifugation-cleared digitonin extracts using nickel-nitrilotriacetic acid-agarose (Qiagen) essentially as described previously (22Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (481) Google Scholar). Pilot experiments revealed that the migration of gephyrin in the BN-PAGE gel was not affected by the inclusion of digitonin in the washing and elution buffers. Accordingly, digitonin was only used for the initial homogenization of the oocytes and excluded from all further purification steps. Proteins were eluted from the beads by two subsequent incubations with 250 mm imidazole/HCl, pH 7.4, each for 15 min at ambient temperature. Within 1-2 h of purification, proteins were separated on BN-PAGE gels (4-16% acrylamide) (23Schaögger H. Cramer W.A. von Jagow G. Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1032) Google Scholar) as described (22Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (481) Google Scholar). Gels were fixed, dried, and exposed to a PhosphorImager screen, which was scanned with a Storm 820 PhosphorImager (Amersham Biosciences) and analyzed using the ImageQuant software.Transfection of HEK 293T Cells and Hippocampal Neurons—HEK 293T cells were cultured on glass coverslips and transfected with cDNAs encoding gephyrin constructs using the calcium phosphate co-precipitation method as detailed previously (24Kirsch J. Kuhse J. Betz H. Mol. Cell. Neurosci. 1995; 6: 450-461Crossref PubMed Scopus (118) Google Scholar). After 24 h of transfection, cells were fixed and processed for immunocytochemistry. Primary hippocampal neurons were prepared from 18 day-old rat embryos and newborn gephyrin knock-out mice and cultured as described (20Fuhrmann J.C. Kins S. Rostaing P. El Far O. Kirsch J. Sheng M. Triller A. Betz H. Kneussel M. J. Neurosci. 2002; 22: 5393-5402Crossref PubMed Google Scholar). Neurons were transfected at days in vitro (DIV) 12 or 13 using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol and fixed at DIV 18.Immunofluorescence Staining—HEK 293T cells and hippocampal neurons were fixed with 4% (w/v) paraformaldehyde for 10-12 min. Fixation and immunostaining were performed essentially as described (20Fuhrmann J.C. Kins S. Rostaing P. El Far O. Kirsch J. Sheng M. Triller A. Betz H. Kneussel M. J. Neurosci. 2002; 22: 5393-5402Crossref PubMed Google Scholar). Cells were blocked with 1% (w/v) bovine serum albumin in phosphate-buffered saline for 1 h and incubated with primary antibody for 90 min. GFP was visualized by autofluorescence. For the detection of VIAAT, a primary rabbit antibody (1:1000) from Synaptic Systems (Goöttingen, Germany), and the secondary antibody Alexa Fluor 546 (1:1000) from Molecular Probes were used. Immunostainings were analyzed using a Leica TCS-SP confocal laser scanning microscope. All confocal images are displayed as flattened stacks obtained from sections in the z-axis.RESULTSDesign of Gephyrin Constructs with Impaired Oligomerization Properties—The crystal structure of the G-domain of gephyrin (13Sola M. Kneussel M. Heck I.S. Betz H. Weissenhorn W. J. Biol. Chem. 2001; 276: 25294-25301Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) shows that 4 hydrophobic amino acid residues (Phe-90, Leu-113, Leu-128, and Leu-168) are located at the trimer interface (Fig. 1B). We used site-directed mutagenesis to replace these G-domain residues by 4 arginines, which due to hydrophilicity and charge were anticipated to abolish the interactions required for trimerization (Fig. 1A). Similarly, based on the crystallographic data available for the E-domain dimer (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar), 3 amino acids (Gly-483, Arg-523, and Ala-532) predicted to be located at the dimer interface (Fig. 1C) were substituted with arginines or glutamic acid (Fig. 1A). The murine gephyrin gene comprises 30 exons. Of these, 10 exons or “cassettes”, named C1 to C7 and C4′ to C6′, have been found to be subject to alternative splicing, thus giving rise to a potentially large diversity of gephyrin isoforms (11Prior P. Schmitt B. Grenningloh G. Pribilla I. Multhaup G. Beyreuther K. Maulet Y. Werner P. Langosch D. Kirsch J. Betz H. Neuron. 1992; 8: 1161-1170Abstract Full Text PDF PubMed Scopus (277) Google Scholar, 25Heck S. Enz R. Richter-Landsberg C. Blohm D.H. Brain Res. Dev. Brain Res. 1997; 98: 211-220Crossref PubMed Scopus (23) Google Scholar, 26Meier J. De Chaldee M. Triller A. Vannier C. Mol. Cell. Neurosci. 2000; 16: 566-577Crossref PubMed Scopus (61) Google Scholar, 27Ramming M. Kins S. Werner N. Hermann A. Betz H. Kirsch J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10266-10271Crossref PubMed Scopus (70) Google Scholar). One of these cassettes, C5′ (13 amino acids), encoded by exon 6, has been proposed to interfere with gephyrin binding to the GlyR and thereby to generate a GABAAR-specific postsynaptic gephyrin scaffold (26Meier J. De Chaldee M. Triller A. Vannier C. Mol. Cell. Neurosci. 2000; 16: 566-577Crossref PubMed Scopus (61) Google Scholar, 28Meier J. Grantyn R. J. Neurosci. 2004; 24: 1398-1405Crossref PubMed Scopus (63) Google Scholar). To examine the role of C5′ in gephyrin interaction, we also generated constructs containing this cassette for oligomerization studies (Fig. 1A).The different domain constructs were named G4xR (harboring substitutions F90R, L113R, L128R, and L168R), ERER (G483R, R523E, and A532R), and GC5′ (containing cassette C5′) and the corresponding full-length constructs Geph4xR, GephRER, and GephC5′, respectively. In addition, we used Gephmut containing an E-domain mutation (see “Experimental Procedures”), which abolishes GlyRβ binding but does not affect C-terminal dimerization (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar).Gel Filtration Chromatography of Gephyrin Domain Constructs—After bacterial expression and affinity purification, recombinant wild-type and mutant gephyrin domain proteins were subjected to gel filtration chromatography on a Superdex 200 column. The wild-type G-domain eluted at a position corresponding to a size of 58 ± 8 kDa (n = 5) (Fig. 1D). Because the calculated molecular mass of the recombinant G-domain is ∼22 kDa, this result is consistent with the previously reported trimer formation of the G-domain (13Sola M. Kneussel M. Heck I.S. Betz H. Weissenhorn W. J. Biol. Chem. 2001; 276: 25294-25301Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In contrast, recombinant G4xR eluted in a major peak at 21 ± 6 kDa, which corresponds to the molecular mass of the monomeric G-domain (Fig. 1D). Thus, the 4 arginine substitutions within the G-domain interface disrupted the trimerization of this N-terminal region of gephyrin. Recombinant GC5′ eluted in a major peak corresponding to 22 ± 4 kDa and a minor peak of 44 ± 5 kDa (Fig. 1D). Apparently, insertion of the C5′ cassette impairs G-domain trimerization.The isolated gephyrin E-domain has been shown to form dimers in solution (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar). In agreement with these earlier data, recombinant wild-type E-domain protein, with a calculated mass of 48 kDa, eluted from the column at a volume corresponding to 102 ± 13 kDa (Fig. 1E). In contrast, for the ERER mutant protein carrying 3 charged amino acid substitutions at its dimer interface a major peak was observed at a position corresponding to 59 ± 4 kDa, i.e. a molecular mass corresponding to the E-domain monomer (Fig. 1E). Additionally, a minor peak at the position of the dimer was detectable. Thus, the mutations introduced at the predicted dimer interface of the E-domain largely disrupted dimer formation. For Emut, an E-domain construct impaired in GlyRβ binding (Fig. 1A), a dimeric structure has been established previously (15Sola M. Bavro V.N. Timmins J. Franz T. Ricard-Blum S. Schoehn G. Ruigrok R.W.H. Paarmann I. Saiyed T. O'Sullivan G.A. Schmitt B. Betz H. Weissenhorn W. EMBO J. 2004; 23: 2510-2519Crossref PubMed Scopus (122) Google Scholar).Oligomerization Properties of Full-length Gephyrin Constructs—The gel filtration data shown above indicate that charge substitutions at G- and E-domain interfaces impair oligomerization of the individual gephyrin subdomains. To assess the effect of these assembly mutations on full-length gephyrin, we used BN-PAGE, which permits gel electrophoresis under non-denaturing conditions and, thus, determination of the oligomeric structure of proteins (22Nicke A. Baumert H.G. Rettinger J. Eichele A. Lambrecht G. Mutschler E. Schmalzing G. EMBO J. 1998; 17: 3016-3028Crossref PubMed Scopus (481) Google Scholar, 29Schaögger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1885) Google Scholar). Recombinant full-length gephyrin purified by metal affinity chromatography from [35S]methionine-labeled X. laevis oocytes migrated upon BN-PAGE as a major band with an apparent mass of ∼640 kDa (Fig. 2, lane 1) as assessed by comparison with soluble mass markers. In addition, higher order complexes accumulated at the interface between stacking and separating gels. We then treated the natively purified gephyrin with urea and SDS to dissociate the protein oligomer into lower order intermediates by weakening non-covalent subunit interactions (30Gendreau S. Voswinkel S. Torres-Salazar D. Lang N. Heidtmann H. Detro-Dassen S. Schmalzing G. Hidalgo P. Fahlke C. J. Biol. Chem. 2004; 279: 39505-39512Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The 640-kDa gephyrin band and the high molecular mass complexes seen at the top of the separating gel proved to be very sensitive to SDS and were converted almost completely to monomeric gephyrin migrating at ∼110 kDa in SDS concentrations ≥0.01% (lane 7). By careful titration with low concentrations of SDS, intermediate oligomeric forms of gephyrin could be generated (lanes 3-6), which were judged to constitute dimers and trimers according to their apparent masses. Some trimers were also produced when full-length gephyrin was treated with 1 m urea (lane 2). By referring to the migration of monomers and trimers at ∼110 and ∼330 kDa, respectively, the ∼640-kDa band was concluded to represent a gephyrin hexamer.FIGURE 2BN-PAGE of full-length wild-type and mutant gephyrin proteins after affinity purification from Xenopus oocyte extracts. On the left, the putative oligomeric structures of respective protein bands are schematically indicated. Wild-type gephyrin (lane 1) runs as a hexamer that, upon treatment with urea, is partially dissociated into trimers (lane 2). Treatment with increasing concentrations of SDS (lanes 3-7) gives rise to lower order oligomeric states, down to the monomer (lane 7). Gephmut (lane 11) displays a predominantly hexameric structure, whereas Geph4xR (lane 8) and GephC5′ (lane 12) behave as dimeric proteins. GephRER (lane 9) migrates to a position consistent with a trimeric structure, whereas Geph4xR,RER (lane 10) behaves as a fully monomeric protein. Note that in addition to the hexamer, higher order complexes are seen at the interface between stacking and resolving gel in lanes 1-4 and 11. Positions of marker proteins are indicated by their corresponding mass in kDa (lane 13). * marks monomer (lane 5), dimer (lane 6), and trimer (lane 7).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The hexameric structure of full-length gephyrin can be readily reconciled with the existence of the two independent oligomerization interfaces that together define the overall assembly state. Accordingly, trimers formed through G-domain interactions dimerize through E-domain interactions into a hexameric complex. In support of this view, the G-domain mutants Geph4xR (lane 8) and GephC5′ (lane 12) migrated as dimers upon BN-PAGE. The somewhat slower mobility of Geph4xR and GephC5′ dimers as compared with that of the major dimers produced by partially denaturing SDS treatment of wild-type gephyrin (lane 6) may reflect conformational differences. Indeed, more slowly migrating dimers were also formed as a minor byproduct of SDS-induced dissociation of wild-type gephyrin (lanes 5-7).For the E-domain mutan
Referência(s)