Mass Spectrometric Analysis of Glycine Receptor-associated Gephyrin Splice Variants
2006; Elsevier BV; Volume: 281; Issue: 46 Linguagem: Inglês
10.1074/jbc.m607764200
ISSN1083-351X
AutoresIngo Paarmann, Bertram Schmitt, Bjoern Meyer, Michael Karas, Heinrich Betz,
Tópico(s)Cellular transport and secretion
ResumoGephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRβ subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5–40 amino acids, are subject to alternative splicing (C1–C7, C4′–C6′). Since one of the cassettes, C5′, has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyRβ loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6′, already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5′ was coisolated mainly from liver. Notably C5′-containing gephyrin bound to the GlyRβ loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms. Gephyrin is an ubiquitously expressed protein that, in the nervous system, is essential for synaptic anchoring of glycine receptors (GlyRs) and major GABAA receptor subtypes. The binding of gephyrin to the GlyR depends on an amphipathic motif within the large intracellular loop of the GlyRβ subunit. The mouse gephyrin gene consists of 30 exons. Ten of these exons, encoding cassettes of 5–40 amino acids, are subject to alternative splicing (C1–C7, C4′–C6′). Since one of the cassettes, C5′, has recently been reported to exclude GlyRs from GABAergic synapses, we investigated which cassettes are found in gephyrin associated with the GlyR. Gephyrin variants were purified from rat spinal cord, brain, and liver by binding to the glutathione S-transferase-tagged GlyRβ loop or copurified with native GlyR from spinal cord by affinity chromatography and analyzed by mass spectrometry. In addition to C2 and C6′, already known to be prominent, C4 was found to be abundant in gephyrin from all tissues examined. The nonneuronal cassette C3 was easily detected in liver but not in GlyR-associated gephyrin from spinal cord. C5 was present in brain and spinal cord polypeptides, whereas C5′ was coisolated mainly from liver. Notably C5′-containing gephyrin bound to the GlyRβ loop, inconsistent with its proposed selectivity for GABAA receptors. Our data show that GlyR-associated gephyrin, lacking C3, but enriched in C4 without C5, differs from other neuronal and nonneuronal gephyrin isoforms. Glycine and γ-aminobutyrate (GABA) 2The abbreviations used are: GABA, γ-aminobutyrate; GlyR, glycine receptor; GST, glutathione S-transferase; MS, mass spectrometry. are the major inhibitory neurotransmitters in the mammalian central nervous system. Both amino acids activate postsynaptic ligand-gated chloride channel proteins, the glycine receptor (GlyR), and GABAA receptor proteins. These receptors are clustered in the postsynaptic membrane by the receptor-anchoring protein gephyrin (1Prior 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 (281) Google Scholar), which links both GlyRs and selected GABAA receptors to the cytoskeleton (2Kirsch J. Betz H. J. Neurosci. 1995; 15: 4148-4156Crossref PubMed Google Scholar, 3Kirsch J. Meyer G. Betz H. Mol. Cell Neurosci. 1996; 8: 93-98Crossref Scopus (58) Google Scholar, 4Kneussel M. Brandstatter J.H. Laube B. Stahl S. Muller U. Betz H. J. Neurosci. 1999; 19: 9289-9297Crossref PubMed Google Scholar). Apart from its role in clustering GlyRs and GABAA receptors, gephyrin is involved in the synthesis of the molybdenum cofactor (5Stallmeyer B. Schwarz G. Schulze J. Nerlich A. Reiss J. Kirsch J. Mendel R.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1333-1338Crossref PubMed Scopus (137) Google Scholar). This explains the ubiquitous expression of gephyrin also in nonneuronal tissues (1Prior 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 (281) Google Scholar, 6Ramming M. Kins S. Werner N. Hermann A. Betz H. Kirsch J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10266-10271Crossref PubMed Scopus (71) Google Scholar, 7Hermann A. Kneussel M. Betz H. Biochem. Biophys. Res. Commun. 2001; 282: 67-70Crossref PubMed Scopus (8) Google Scholar). The mouse gephyrin gene consists of ∼30 exons (6Ramming M. Kins S. Werner N. Hermann A. Betz H. Kirsch J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10266-10271Crossref PubMed Scopus (71) Google Scholar). Alternative splicing has been reported so far for 10 of these exons, which encode cassettes of 5–40 amino acids, thereby allowing for the generation of a large number of possible gephyrin splice variants (1Prior 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 (281) Google Scholar, 6Ramming M. Kins S. Werner N. Hermann A. Betz H. Kirsch J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10266-10271Crossref PubMed Scopus (71) Google Scholar, 8Heck S. Enz R. Richter-Landsberg C. Blohm D.H. Brain Res. Dev. Brain Res. 1997; 98: 211-220Crossref PubMed Scopus (24) Google Scholar, 9Meier J. De Chaldee M. Triller A. Vannier C. Mol. Cell. Neurosci. 2000; 16: 566-577Crossref PubMed Scopus (63) Google Scholar). The gephyrin protein is composed of a N-terminal G-domain that is homologous to the E. coli MogA protein, a central linker region, and a C-terminal E-domain with homology to the E. coli MoeA protein. Gephyrin shares with these bacterial proteins common enzymatic functions in the biosynthesis of the molybdenum cofactor (5Stallmeyer B. Schwarz G. Schulze J. Nerlich A. Reiss J. Kirsch J. Mendel R.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1333-1338Crossref PubMed Scopus (137) Google Scholar), an essential coenzyme of oxidoreductases (10Johnson J.L. Wadman S.K. Molybdenum cofactor deficiency. In: Stanbury, J. B., and Wyngaarden, J. B. (eds)..Inherited Basis of Metabolic Disease. McGraw-Hill, New York1989Google Scholar). In all three domains, alternative splicing can generate diversity. The G-domain of gephyrin forms a trimer, whereas the E-domain can form dimers (11Sola M. Kneussel M. Heck I.S. Betz H. Weissenhorn W. J. Biol. Chem. 2001; 276: 25294-25301Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Sola 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 Journal. 2004; 23: 2510-2519Crossref PubMed Scopus (124) Google Scholar). Both N-terminal trimerization and C-terminal dimerization have been proposed to be essential for postsynaptic receptor clustering (12Sola 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 Journal. 2004; 23: 2510-2519Crossref PubMed Scopus (124) Google Scholar, 13Kneussel M. Betz H. Trends Neurosci. 2000; 23: 429-435Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The interaction between gephyrin and the GlyRβ subunit is well characterized and stable enough to allow copurification of gephyrin with the GlyR from rat spinal cord (14Pfeiffer F. Graham D. Betz H. J. Biol. Chem. 1982; 257: 9389-9393Abstract Full Text PDF PubMed Google Scholar, 15Schmitt B. Knaus P. Becker C.M. Betz H. Biochemistry. 1987; 26: 805-811Crossref PubMed Scopus (154) Google Scholar). A peptide containing 49 amino acids of the cytoplasmic loop between the third and fourth transmembrane domain of GlyRβ is sufficient to obtain robust gephyrin binding (16Meyer G. Kirsch J. Betz H. Langosch D. Neuron. 1995; 15: 563-572Abstract Full Text PDF PubMed Scopus (349) Google Scholar) and can be used for affinity purification of gephyrin splice variants from different organs (7Hermann A. Kneussel M. Betz H. Biochem. Biophys. Res. Commun. 2001; 282: 67-70Crossref PubMed Scopus (8) Google Scholar). Within gephyrin, the GlyRβ binding site is located within the E-domain (12Sola 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 Journal. 2004; 23: 2510-2519Crossref PubMed Scopus (124) Google Scholar, 17Schrader 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 (63) Google Scholar). Cassette 5′, which is located in the G-domain, has been reported to interfere with binding to the GlyR and proposed to prevent GlyR accumulation at gephyrin scaffolds of GABAergic synapses (9Meier J. De Chaldee M. Triller A. Vannier C. Mol. Cell. Neurosci. 2000; 16: 566-577Crossref PubMed Scopus (63) Google Scholar, 18Meier J. Grantyn R. J. Neurosci. 2004; 24: 1398-1405Crossref PubMed Scopus (63) Google Scholar). This indicates that only specific cassettes are present in GlyR-associated gephyrin. Therefore, we examined which gephyrin splice variants are bound to the GlyR. In the present study, we copurified gephyrin with the native GlyR from rat spinal cord and, for comparison, from rat spinal cord, brain, and liver via GST pull-down using the GlyRβ loop sequence. The presence of the different cassettes was investigated by mass spectrometry. So far, proteomic approaches have been seldom used to identify splice variants in higher eukaryotes, due to limitations in sequence coverage and sensitivity (19Godovac-Zimmermann J. Kleiner O. Brown L.R. Drukier A.K. Proteomics. 2005; 5: 699-709Crossref PubMed Scopus (49) Google Scholar, 20Roth M.J. Forbes A.J. Boyne M.T. Kim 2nd, Y.B. Robinson D.E. Kelleher N.L. Mol. Cell. Proteomics. 2005; 4: 1002-1008Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21Thiede B. Hohenwarter W. Krah A. Mattow J. Schmid M. Schmidt F. Jungblut P.R. Methods. 2005; 35: 237-247Crossref PubMed Scopus (189) Google Scholar). Here, affinity purification combined with mass spectrometry was found to reliably disclose differences in cassette composition of tissue-specific and receptor-bound gephyrin isoforms. Cassettes C2 and C6′ were found in all preparations examined. In addition, GlyR-associated gephyrin was revealed to significantly differ from the GlyRβ loop-isolated proteins in showing a strict absence of the C3 cassette and a conserved presence of the C4 cassette. Protein Expression and Purification—pGEX-4T-1 and pGEX-5X-1 were obtained from Amersham Biosciences, and GlyRβ-(378–426)-pGEX-5X-1 (16Meyer G. Kirsch J. Betz H. Langosch D. Neuron. 1995; 15: 563-572Abstract Full Text PDF PubMed Scopus (349) Google Scholar) and gephyrin-P1-pRSET (12Sola 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 Journal. 2004; 23: 2510-2519Crossref PubMed Scopus (124) Google Scholar) have been described previously. E-domain-pGEX-4T-1 was generated by cloning the E-domain of GE45-pRSET (12Sola 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 Journal. 2004; 23: 2510-2519Crossref PubMed Scopus (124) Google Scholar) into pGEX-4T-1. The proteins GST, GlyRβ-(378–426)-GST, E-domain-GST, and His-gephyrin-P1 were produced in E. coli BL21 (DE3) (Merck). Expression was induced by the addition of 0.2 mm isopropyl-1-thio-β-d-galactopyranoside at 25 °C overnight. Clear lysates were prepared according to the manufacturer's protocol (Amersham Biosciences). His-tagged gephyrin-P1 was purified according to standard protocols (Qiagen, Hilden, Germany). The GST pull-down procedure was performed at 4 °C as follows: 30-μl bed volume of glutathione-Sepharose beads (Amersham Biosciences) was incubated with 0.2–0.5 ml of clear bacterial lysate for 2 h. After washing with phosphate-buffered saline, the beads were incubated with 7.5 ml of a Triton X-100 extract prepared from rat brain, liver, or spinal cord for 2 h and washed four times with phosphate-buffered saline and 0.5% (w/v) Triton X-100. The beads were eluted three times with 30 μl each of 10 mm reduced glutathione in 50 mm Tris-Cl, pH 8.0, for 10 min at 25 °C under vigorous mixing. The eluates were combined. Preparation of Tissue Extracts—Triton X-100 extracts were prepared by adding 5 volumes of cold phosphate-buffered saline containing protease inhibitor mixture (complete; Roche Applied Science) and 100 μm phenylmethylsulfonyl fluoride to the tissue followed by homogenization for 3 min at 1000 rpm on ice using a Potter homogenizer (Braun Melsungen AG, Melsungen, Germany). The homogenate then was centrifuged at 2000 × g for 10 min at 4 °C. To the supernatant, Triton X-100 was added to a final concentration of 1% (w/v), and dithiothreitol was added to 1 mm. After 75 min under mild mixing at 4 °C, samples were centrifuged at 27,000 × g for 30 min at 4 °C. The supernatant was used directly in the GST pull-down. GlyR-associated gephyrin was copurified with the glycine receptor from spinal cord using an aminostrychnine column as described (14Pfeiffer F. Graham D. Betz H. J. Biol. Chem. 1982; 257: 9389-9393Abstract Full Text PDF PubMed Google Scholar, 15Schmitt B. Knaus P. Becker C.M. Betz H. Biochemistry. 1987; 26: 805-811Crossref PubMed Scopus (154) Google Scholar). Sample Preparation for Mass Spectrometry—To the samples, reducing SDS-PAGE loading buffer was added, followed by incubation for 5 min at 48 °C for the GlyR-associated gephyrin or at 95 °C for gephyrin purified via GST pull-down. The samples were resolved on one-dimensional 8–10% polyacrylamide gels. To visualize proteins, gels were silver-stained with a Silver Stain Plus kit (Bio-Rad). Gephyrin bands were identified by size (see Fig. 1), cut out, and stored at –80 °C. The protein bands were destained with 100 μl of 15 mm potassium ferricyanide III and 50 mm sodium thiosulfate, followed by two washes with 400 μl of H2O for 15 min and two washes with 400 μl of 50% (v/v) acetonitrile, 25 mm NH4HCO3, pH 8.0, for 15 min. Subsequent preparation of the samples for mass spectrometry was essentially performed as described (22Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7818) Google Scholar). After trypsin digestion, the samples were spun in a table-top microcentrifuge. The aqueous solution was collected, and the gel pieces were extracted once for 45 min with 20 mm NH4HCO3. After brief centrifugation, the supernatant was collected, followed by extraction of the gel pieces for 30 min with 70% (v/v) acetonitrile, 5% (v/v) formic acid. After centrifugation, the resulting supernatant was combined with the two previous supernatants from the same sample. The combined extracts were dried in a SpeedVac and stored at –20 °C until mass spectrometric analysis. Mass Spectrometry—All MS experiments were performed on the Voyager-DE™ STR (Applied Biosystems, MDS SCIEX). Additionally, MS/MS spectra were acquired with the 4800 MALDI-TOF/TOF™ analyzer (Applied Biosystems, MDS SCIEX) to verify the MS data. Prior to analysis, the pellets were dissolved in 5 μl of 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid under mixing for 3 h. The sample (0.4 μl) was mixed with 0.4 μl of matrix (5 mg/ml α-cyano-4-hydroxycinnamic acid; Bruker Daltonik, Bremen, Germany) in 50% (v/v) acetonitrile, 0.5% (v/v) trifluoroacetic acid directly on a stainless steel target (Applied Biosystems, MDS SCIEX) and dried in ambient air. The crystals were washed briefly with 1 μl of ice-cold 5% (v/v) formic acid. The acquisition range for the MS analysis was set from 600 to 5000 Da, and the low mass gate was set to 500 Da. The spectra were first externally calibrated using a peptide mass mixture (Peptide Calibration Standard II, Bruker Daltonik, Bremen, Germany). Trypsin autolysis peaks and prominent gephyrin peaks were then used for internal calibration. Between 400 and 1000 single scans were accumulated for each mass spectrum. Representative spectra are shown in Fig. 2 and supplemental Fig. 1. Selected peaks characterizing the gephyrin splice variants were fragmented to get sequence information (see supplemental Fig. 3 and supplemental Table 1). Analysis of Mass Spectrometric Data—All MS spectra were smoothed, noise-filtered, and deisotoped using the Data Explorer™ software (Applied Biosystems, MDS SCIEX). Deisotoped peaks were automatically labeled by the software, and the peaks were used for data base search. Gephyrin was identified using Mascot™ (23Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6773) Google Scholar) and MS-Fit (Expasy) via World Wide Web interface. Gephyrin splice variants not present in the NCBI and Swiss-Prot data bases were generated in silico. The presence and absence of the cassettes was determined using the World Wide Web-based FindMod (24Wilkins M.R. Gasteiger E. Gooley A.A. Herbert B.R. Molloy M.P. Binz P.A. Ou K. Sanchez J.C. Bairoch A. Williams K.L. Hochstrasser D.F. J. Mol. Biol. 1999; 289: 645-657Crossref PubMed Scopus (251) Google Scholar). For all cassettes, Aldente (Expasy) was used to identify false positive results due to keratin contamination. Additionally, the cassettes were checked for trypsin (25Harris W.A. Janecki D.J. Reilly J.P. Rapid Commun. Mass Spectrom. 2002; 16: 1714-1722Crossref PubMed Scopus (53) Google Scholar) and other common contaminations (26Ding Q. Xiao L. Xiong S. Jia Y. Que H. Guo Y. Liu S. Proteomics. 2003; 3: 1313-1317Crossref PubMed Scopus (34) Google Scholar). FindMod was also used to check for GlyR contamination in GlyR-associated gephyrin and for GlyRβ-(378–426)-GST in case of the other gephyrin protein bands. MS/MS spectra were processed similarly and searched against the NCBI data base. Mass accuracy was set to 50 ppm for the precursor and 0.25 Da for the fragments. Unidentified spectra of splice variants were interpreted manually. Determination of Gephyrin Cassette Boundaries at the Amino Acid Level—The genomic rat and mouse sequences containing the gephyrin gene were obtained from the genome data bases (NCBI). Using the published gephyrin cDNA sequences and the intronexon boundaries described (6Ramming M. Kins S. Werner N. Hermann A. Betz H. Kirsch J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10266-10271Crossref PubMed Scopus (71) Google Scholar), the exact sequence of all cassettes, except for C1, could be verified. Since the genomic rat sequence is apparently incomplete, the mouse sequence was used when no published rat sequence was available, such as for C6 and C7. Statistical Analysis—For all bands examined, 10 independent experiments were performed. The statistical significance of differences between gephyrin protein bands for a given cassette was calculated using Fisher's exact test (27Sachs L. Angewandte Statistik. 6. Ed. Springer Verlag, Berlin/Heidelberg/New York/Tokyo1984Crossref Google Scholar). GlyR-associated gephyrin (SC GlyR) was copurified with the GlyR from spinal cord extracts on an aminostrychnine column as described (14Pfeiffer F. Graham D. Betz H. J. Biol. Chem. 1982; 257: 9389-9393Abstract Full Text PDF PubMed Google Scholar, 15Schmitt B. Knaus P. Becker C.M. Betz H. Biochemistry. 1987; 26: 805-811Crossref PubMed Scopus (154) Google Scholar). Additionally, gephyrin able to interact with the GlyR was isolated from detergent extracts of spinal cord, brain, and liver by a GST pull-down approach with the gephyrin binding motif of GlyRβ (16Meyer G. Kirsch J. Betz H. Langosch D. Neuron. 1995; 15: 563-572Abstract Full Text PDF PubMed Scopus (349) Google Scholar). This identified in both brain and spinal cord two major gephyrin bands of different apparent molecular weights in one-dimensional SDS-polyacrylamide gels, which we named brain 93 kDa and brain 90 kDa, and SC 93 kDa and SC 90 kDa, respectively (see Fig. 1). Such a separation of gephyrin bands was not seen in liver (see Fig. 1). To reveal whether these gephyrin polypeptides contained different alternatively spliced exons, or cassettes, the individual bands were cut out and subjected to mass spectrometry. For GlyR-associated gephyrin, the observed upper and lower bands were indistinguishable by mass spectrometry. A representative spectrum of a brain 90 kDa band is shown in Fig. 2 in its raw, deisotopized, and simplified form. Mass spectrometry data for the different gephyrin bands were obtained at comparable quality. For the calculation of average sequence coverage, a reference sequence with the following features was used: rat gephyrin containing cassettes C2 and C6′ (GenBank™ accession number CAA47009), methionine at position 1 replaced by an acetyl group, and alanine at position 242. C2 and C6′ were found in all samples analyzed. Gephyrin without N-terminal modification was found in only 3%, but gephyrin with N-terminal acetylation was found in 95% of the experiments. The N-terminal acetylation was confirmed by MS/MS (see supplemental Fig. 3F and supplemental Table 1). At position 242 of the published rat gephyrin sequence (1Prior 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 (281) Google Scholar), an arginine residue is present, whereas in all other mammals an alanine residue is conserved. However, in none of the experiments with the rat strain used (Wistar rats) was an arginine 242 found, but rather, an alanine was found in one-third of the experiments. Reexamination of our original clones (1Prior 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 (281) Google Scholar) revealed a sequencing error at position 242 in the published sequence. Deviations of peptide masses within one experiment were determined using Mascot™ (23Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6773) Google Scholar) and MS-Fit (Expasy). The average sequence coverage of the peptide mass fingerprints varied from 44 to 49% (see Fig. 3), a value characteristic of in-gel digests with trypsin. The average deviation was ∼20 ppm (see Fig. 3). This indicates that mass spectrometry results were obtained for all gephyrin protein bands with similar reliability. Under the same conditions, the presence and absence of all cassettes was correctly identified in recombinant gephyrin-P1 and the recombinant E-domain (see supplemental Fig. 2, A and B), whereas no false positive results were obtained from actin and myosin heavy chain 9 copurified as nonspecific proteins with gephyrin from liver (data not shown). The amino acid sequences of different peptides were confirmed by MS/MS (see supplemental Table 1 and supplemental Fig. 3). Only a limited number of theoretically possible peaks identified splice variants; presumably, this reflects difficulties with peptide extraction from the gel, unused cleavage sites, etc. Cassettes C4 and C5 were defined by the presence or absence of a single tryptic fragment. C3 could only be detected with a peak at 1056 Da. Cassettes C2 and C6′ were identified easily because multiple peaks were found in the MS spectra; their identities were verified in MS/MS experiments. Supplemental Fig. 3 shows a selection of the acquired MS/MS spectra. All MS/MS spectra resulted in significant data base identifications except for the peak at 1056 Da found for C3; this is consistent with fragmentations in the low mass region frequently creating only manually interpretable spectra (supplemental Table 1). The presence of the fragments at the hyphenation points of peptide 1056 Da (residues P and D) identified it unambiguously in this special chase (supplemental Fig. 3B). The borderline MS/MS data base identification of the tryptic fragment of cassette C4 (1786.8 Da) must be correct, because the MS/MS spectrum contains a satisfactory y-ion series (supplemental Fig. 3C). Additionally, N-terminal acetylation of gephyrin was proven in an MS/MS experiment (supplemental Fig. 3F). No significant differences were observed for the cassettes located in the E- and G-domain. In all experiments, the presence of the cassettes C2 and C6′ was observed (see Fig. 4), indicating that these cassettes are highly abundant. Additionally, they clearly do not interfere with GlyRβ binding, since they were also present in gephyrin obtained from affinity-purified GlyR preparations (SC GlyR). The GlyR binding site of gephyrin resides in the E-domain (12Sola 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 Journal. 2004; 23: 2510-2519Crossref PubMed Scopus (124) Google Scholar). In contrast to C2 and C6, cassettes C1, C6, and C7 were hardly or not at all detected (see Table 1). For C1, this might reflect the fact that this is a very rare cassette. In the case of C6, there exists an alternative explanation. Since it is located in the E-domain, it might interfere with GlyR binding. The most prominent feature of C7 is that its presence introduces a translational stop codon after amino acid position 463 in rat gephyrin containing C2 and C6′ (6Ramming M. Kins S. Werner N. Hermann A. Betz H. Kirsch J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10266-10271Crossref PubMed Scopus (71) Google Scholar). As a consequence, a major portion of the E-domain containing the GlyR binding site is deleted, and the calculated molecular mass of a gephyrin polypeptide containing C7 is ∼30 kDa smaller as compared with the gephyrin bands investigated in this study. Hence, it should not be contained in the 90–93 kDa bands analyzed here.TABLE 1Cassette detection and exclusion ratesCassetteLiverBrain 93 kDaBrain 90 kDaSC 93 kDaSC 90 kDaSC GlyR%%%%%%C11000000C450100408050100C60000100C7000000ΔC5′100100100100100100ΔC4′C4C5000101010ΔC6801001009060100ΔC7304020301020 Open table in a new tab The presence of C5′ was detected only occasionally. Although C5′ was observed more often in liver as compared with the other bands (see Fig. 4), this difference is not statistically significant (p < 0.25 for SC GlyR). Since C5′ has been reported to interfere with GlyRβ binding (9Meier J. De Chaldee M. Triller A. Vannier C. Mol. Cell. Neurosci. 2000; 16: 566-577Crossref PubMed Scopus (63) Google Scholar), its presence in affinity-purified GlyR preparations and GlyRβ-(378–426)-GST pull-downs was a surprising finding. However, binding of C5′-containing gephyrin to the GlyRβ loop used in this study has also been seen recently with the purified recombinant proteins. 3T. Saiyed, personal communication. For precise localization of the cassettes, see Fig. 6. Within the central linker region, differences for most of the cassettes were observed. In contrast to the cassettes C1, C2, C6, and C6′ located within the E- or G-domain, most of the cassettes found in the central linker region were differentially distributed between the bands defined above. C3 was abundantly represented in the liver, brain 93 kDa, and SC 93 kDa bands but not detected in brain 90 kDa, SC 90 kDa, and GlyR-associated gephyrin (see Fig. 5). This difference turned out to be highly significant (p < 0.005). Obviously, C3 does not directly interfere with GlyRβ binding. C3 is largely repressed in neurons by NOVA proteins, neuronal regulators of pre-mRNA splicing (28Ule J. Jensen K.B. Ruggiu M. Mele A. Ule A. Darnell R.B. Science. 2003; 302: 1212-1215Crossref PubMed Scopus (825) Google Scholar, 29Ule J. Ule A. Spencer J. Williams A. Hu J.S. Cline M. Wang H. Clark T. Fraser C. Ruggiu M. Zeeberg B.R. Kane D. Weinstein J.N. Blume J. Darnell R.B. Nat. Genet. 2005; 37: 844-852Crossref PubMed Scopus (396) Google Scholar), indicating that GlyR-associated gephyrin, brain 90 kDa, and SC 90 kDa are indeed derived from neurons, but brain 93 kDa and SC 93 kDa might originate from nonneuronal (e.g. glial) cells. C3 consists of 36 amino acids, corresponding to a molecular mass of 4 kDa. Therefore, the observed difference in the molecular mass of SC 93 kDa versus SC 90 kDa and of brain 93 kDa versus brain 90 kDa can be easily explained by the presence of the C3 cassette in the bands of higher molecular mass. C4 was readily disclosed in all bands, being most prominent in GlyR-associated gephyrin and brain 93 kDa but significantly less abundant in the SC 90 kDa, brain 90 kDa, and liver isolates (see Table 1). This was the most obvious difference observed between GlyR-associated gephyrin and the SC 90 kDa band (see Fig. 5), suggesting a special importance of the C4 cassette for the GlyR-associated variant (SC GlyR). C4 obviously seems not to impair binding to GlyRβ and might in addition provide some functionally relevant features, such as phosphorylation and protein binding sites. Among the splice variants containing C4, those without C5 were detected most frequently. C4 without C5 was found in significantly higher amounts in GlyR-associated gephyrin as compared with the SC 90 kDa and brain 90 kDa polypeptides (see Fig. 5). The results for
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