FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019341013> ?p ?o ?g. }

• W2019341013 endingPage "3598" @default.
• W2019341013 startingPage "3590" @default.
• W2019341013 abstract "The proteoglycan NG2 is expressed by immature glial cells in the developing and adult central nervous system. Using the COOH-terminal region of NG2 as bait in a yeast two-hybrid screen, we identified the glutamate receptor interaction protein GRIP1, a multi-PDZ domain protein, as an interacting partner. NG2 exhibits a PDZ binding motif at the extreme COOH terminus which binds to the seventh PDZ domain of GRIP1. In addition to the published expression in neurons, GRIP1 is expressed by immature glial cells. GRIP1 is known to bind to the GluRB subunit of the AMPA glutamate receptor expressed by subpopulations of neurons and immature glial cells. In cultures of primary oligodendrocytes, cells coexpress GluRB and NG2. A complex of NG2, GRIP1, and GluRB can be precipitated from transfected mammalian cells and from cultures of primary oligodendrocytes. Furthermore, NG2 and GRIP can be coprecipitated from developing brain tissue. These data suggest that GRIP1 acts as a scaffolding molecule clustering NG2 and AMPA receptors in immature glia. In view of the presence of synaptic contacts between neurons and NG2-positive glial cells in the hippocampus and the close association of NG2-expressing glial cells with axons, we suggest a role for the NG2·AMPA receptor complex in glial-neuronal recognition and signaling. The proteoglycan NG2 is expressed by immature glial cells in the developing and adult central nervous system. Using the COOH-terminal region of NG2 as bait in a yeast two-hybrid screen, we identified the glutamate receptor interaction protein GRIP1, a multi-PDZ domain protein, as an interacting partner. NG2 exhibits a PDZ binding motif at the extreme COOH terminus which binds to the seventh PDZ domain of GRIP1. In addition to the published expression in neurons, GRIP1 is expressed by immature glial cells. GRIP1 is known to bind to the GluRB subunit of the AMPA glutamate receptor expressed by subpopulations of neurons and immature glial cells. In cultures of primary oligodendrocytes, cells coexpress GluRB and NG2. A complex of NG2, GRIP1, and GluRB can be precipitated from transfected mammalian cells and from cultures of primary oligodendrocytes. Furthermore, NG2 and GRIP can be coprecipitated from developing brain tissue. These data suggest that GRIP1 acts as a scaffolding molecule clustering NG2 and AMPA receptors in immature glia. In view of the presence of synaptic contacts between neurons and NG2-positive glial cells in the hippocampus and the close association of NG2-expressing glial cells with axons, we suggest a role for the NG2·AMPA receptor complex in glial-neuronal recognition and signaling. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor interaction protein glutathioneS-transferase monoclonal myelin oligodendrocyte glycoprotein neural cell adhesion molecule phosphate-buffered saline polyclonal 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside postsynaptic density 95/discs large/zona occludens 1 laminin/neurexin/sex hormone binding globulin The NG2 proteoglycan is as a large transmembrane glycoprotein expressed by oligodendrocyte progenitor cells but down-regulated upon differentiation into mature oligodendrocytes (1Levine J.M. Stincone F. Lee Y.S. Glia. 1993; 7: 307-321Crossref PubMed Scopus (248) Google Scholar,2Dawson M.R. Levine J.M. Reynolds R. J. Neurosci. Res. 2000; 61: 471-479Crossref PubMed Scopus (344) Google Scholar). The AN2 proteoglycan (3Niehaus A. Stegmüller J. Diers-Fenger M. Trotter J. J. Neurosci. 1999; 19: 4948-4961Crossref PubMed Google Scholar) is the mouse homolog of NG2 (4Schneider S. Bosse F. D'Urso D. Muller H. Sereda M.W. Nave K. Niehaus A. Kempf T. Schnolzer M. Trotter J. J. Neurosci. 2001; 21: 920-933Crossref PubMed Google Scholar). In the developing and adult nervous system many NG2-positive cells abound in both white and gray matter whose classification as oligodendrocyte or astrocyte lineage cells remains unclear but whose electrophysiological properties are typical of immature glial cells (5Diers-Fenger M. Kirchhoff F. Kettenmann H. Levine J.M. Trotter J. Glia. 2001; 34: 213-228Crossref PubMed Scopus (113) Google Scholar). Recently, Bergles and colleagues (6Bergles D.E. Roberts J.D. Somogyi P. Jahr C.E. Nature. 2000; 405: 187-191Crossref PubMed Scopus (737) Google Scholar) identified a novel type of synapse in the developing and adult rat hippocampus where pyramidal neurons of the CA3 area form morphologically identifiable synaptic boutons on NG2-positive cells. Furthermore, stimulation of the neurons resulted in a Ca2+signal in the NG2-positive glial cells which was dependent on the activity of the AMPA1 class of glutamate receptors. AMPA receptors are known to be expressed by subclasses of glial cells including oligodendrocyte precursor cells (7Patneau D.K. Wright P.W. Winters C. Mayer M.L. Gallo V. Neuron. 1994; 12: 357-371Abstract Full Text PDF PubMed Scopus (282) Google Scholar) where their function is thought to include an inhibitory influence on proliferation and lineage progression of oligodendrocyte progenitors (8Yuan X. Eisen A.M. McBain C.J. Gallo V. Development. 1998; 125: 2901-2914PubMed Google Scholar). NG2 is a single pass transmembrane proteoglycan with a large extracellular domain and two LAM G (LNS) domains near the NH2 terminus, suggesting a role as an adhesion molecule. NG2 has a short intracellular tail of 76 amino acids. The PDZ protein MUPP1 was identified recently by yeast two-hybrid screening of a library from E9.5/10.5 mouse embryo as an intracellular binding partner of rat NG2 (9Barritt D.S. Pearn M.T. Zisch A.H. Lee S.S. Javier R.T. Pasquale E.B. Stallcup W.B. J. Cell. Biochem. 2000; 79: 213-224Crossref PubMed Scopus (82) Google Scholar), although the biological significance of this finding remains unclear. Because the COOH terminus of NG2 exhibits a putative PDZ binding motif and PDZ domain proteins are adaptor proteins that target and cluster protein complexes including neurotransmitter receptors to the cell surface (10Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1041) Google Scholar), we sought to identify intracellular partners of NG2 in glial progenitor cells. The cytoplasmic domain of murine NG2 was used as bait in yeast two-hybrid analysis to screen a library from early postnatal mouse brain. This screen revealed the glutamate receptor interaction protein (GRIP) 1 as a binding partner. We show that GRIP1 acts as a direct molecular link between AMPA receptors and the glycoprotein NG2 in immature glial cells. This protein complex, together with as yet to be identified additional members, may contribute to a postsynaptic microdomain in immature glial cells and contribute to glial-neuronal signaling. NMRI mice were obtained from the central animal facilities of the University of Heidelberg. The following primary antibodies were used: monoclonal (mc) GRIP1 (Becton Dickinson), polyclonal (pc) antibodies against GRIP1 (kind gift from Dr. R. Klein), and pc GluRB 2The AMPA receptor subunits GluRA, B, C, and D are sometimes referred to as GluR1, 2, 3, and 4, respectively. (Chemicon, Hofheim, Germany). The antibodies recognize specifically the short GluRB form (COOH-terminal peptide: SVKI*), which binds to GRIP1. We also used pc AN2 antibodies (3Niehaus A. Stegmüller J. Diers-Fenger M. Trotter J. J. Neurosci. 1999; 19: 4948-4961Crossref PubMed Google Scholar), anti-GST880S16 (pc AN2 against a GST fusion protein consisting of amino acids 1255–1545, mc AN2 antibodies (3Niehaus A. Stegmüller J. Diers-Fenger M. Trotter J. J. Neurosci. 1999; 19: 4948-4961Crossref PubMed Google Scholar), pc antibodies against NCAM (11Trotter J. Bitter-Suermann D. Schachner M. J. Neurosci. Res. 1989; 22: 369-383Crossref PubMed Scopus (140) Google Scholar), mc anti-Myc (Sigma), and mc 8-18-C5 against MOG (a kind gift from Dr. C. Linington). The COOH-terminal region of mouse NG2 (NH2-RKRNKT … NGQYWV-COOH, GenBank accession number AF352400) was fused to the GAL4 binding domain by cloning it into the pGBT9 vector (Clontech) with EcoRI/BamHI. The resulting bait construct was designated pGBT9cyto. Using the lithium acetate method, the yeast strain CG1945 was transformed sequentially with pGBT9cyto and a postnatal mouse brain MATCHMAKER cDNA library in pACT2 (Clontech). 33 × 106 transformants were screened. Transformants were grown on SD medium-Leu-Trp-His plates; 5 mm3-amino-1,2,4-triazole was added to the medium to suppress leaky HIS3 reporter gene expression. Positive clones were tested for β-galactosidase gene activity: yeast colonies were grown on SD-Leu-Trp-His, transferred onto reinforced nitrocellulose membrane, submerged in liquid nitrogen, and placed on a Z-buffer/X-gal-soaked Whatman (Z-buffer: 16.1 g/liter Na2HPO4*7H2O, 5.5 g/liter NaH2PO4*H2O, 0.75 g/liter KCl, 0.246 g/liter MgSO4*7H2O, pH 7; Z-buffer/X-gal solution: 100 ml of Z-buffer, 0.27 ml of β-mercaptoethanol, 1.67 ml of 20 mg/ml X-gal stock solution). Blue color was allowed to develop for 30 min-3 h. DNA-sequencing results revealed 48 isolates and 10 independent clones of GRIP1. All clones included the seventh PDZ domain and the COOH terminus of GRIP1. The specificity of the NG2-GRIP1 interaction was confirmed by a β-galactosidase test and growth selection of cotransformed yeast cells with pGBT9cyto and isolated library plasmids. Unspecific NG2 interactions were excluded by contransformation of yeast cells with pGBT9cyto and GRIP1 constructs in pGADGH lacking the seventh PDZ domain (kind gift from Dr. R. Klein). Unspecific interactions of the seventh PDZ domain of GRIP1 were excluded by cotransformation of yeast with a pGBT9 construct encoding the cytoplasmic COOH terminus of an unrelated transmembrane protein M6A. 3H. Werner and K. Nave, unpublished data. The yeast two-hybrid system was used to map the interaction site of GRIP1 with NG2. Results from the sequencing of GRIP1 revealed the seventh PDZ domain as putative interaction site. Mouse GRIP1 deletion constructs in pACT2 were used as resulting from the screen: mouse GRIP1 4p-7 (LEIEFD … EPTNTL), mouse GRIP1 5–7 (KHSVEL … EPTNTL), mouse GRIP1, 7 (ATIMSG … EPTNTL). Further control constructs of rat GRIP1 were kind gifts from Dr. R. Klein: rat GRIP1 4–6 (TSPRGT … KLSDVY), rat GRIP1, 6 (EDNSDE … KQTDAQ), rat GRIP1 6–7 (GAIIYT … EPTNTL). The NH2- and COOH-terminal sequences above correspond to sequences from mouse GRIP1 library plasmids or to the published rat GRIP1 sequence (12Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (754) Google Scholar). All constructs were verified by DNA sequencing. Cotransformed yeast cells were grown on double dropout medium and assayed for β-galactosidase gene activity and additionally selected for growth on triple dropout medium. The yeast two-hybrid system was used to test the interaction of NG2 and the seventh PDZ domain of GRIP2. The GRIP2 cDNA sequence was generated by reverse transcription PCR, which introduced EcoRI/BamHI sites and then cloned into the pACT2 vector: rat GRIP2, 7 (RSREVGT … SSPQMI); the sequence corresponds to the published rat sequence (13Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar) and was verified by sequencing. The yeast two-hybrid system was used to map the PDZ binding motif at the extreme COOH terminus of NG2. Individual mutations of the 0, −1, −2, and −3 positions of the COOH-terminal peptide QYWV* were introduced by PCR, cloned into pGBT9, and designated as NG2 0G (Val mutated to Gly), NG2 −1G (Trp mutated to Gly), NG2 −2G (Tyr mutated to Gly), NG2 −2F (Tyr mutated to Phe), NG2 −3G (Gln mutated to Gly). Mutant NG2 constructs were contransformed with mouse GRIP1 PDZ7 and rat GRIP2 PDZ7. Yeast cells were grown on double dropout medium and assayed for β-galactosidase gene activity and additionally selected for growth on triple dropout medium. COS7 cells were transfected by electroporation (0.25 kV, 250 microfarads). Plasmids were used at 15 μg/300 μl of cell suspension (4 × 106 cells/ml, Dulbecco's modified Eagle's medium and 10% fetal calf serum). The NG2 deletion mutant was generated by trimolecular ligation of two PCR-amplified regions of NG2 with artificially introduced restriction sites in the pRK5 vector (14Schall T.J. Lewis M. Koller K.J. Lee A. Rice G.C. Wong G.H. Gatanaga T. Granger G.A. Lentz R. Raab H. Cell. 1990; 61: 361-370Abstract Full Text PDF PubMed Scopus (846) Google Scholar). The deletion mutant consists of the signal sequence, one-fourth of the very NH2-terminal extracellular portion (including both LNS domains), the transmembrane domain, and the complete intracellular region. Mouse GRIP1/PDZ7 and mouse GRIP1/PDZ5–7 expression constructs were generated by cloning murine sequences into pRK5 vector withEcoRI/BamHI andEcoRI/HindIII sites introduced by PCR, respectively. The constructs contain a translation initiation sequence (15Kozak M. Gene (Amst.). 1999; 234: 187-208Crossref PubMed Scopus (1128) Google Scholar) and encode an NH2-terminal Myc tag. GluRB full-length, flop, short form was a kind gift from Dr. H. Monyer. Constructs were verified by sequencing. Transfected COS7 cells were washed with phosphate-buffered saline (PBS) 24 h after transfection, starved for 1 h in methionine/cysteine-free medium, then metabolically labeled with 100 μCi/ml [35S]Met/Cys for 4 h. Cells were washed twice with PBS and lysed (1% Triton X-100, 50 mm Tris, pH 7.5, 150 mm NaCl, protease inhibitors), and the lysates were chilled for 30 min and centrifuged at 3,000 rpm for 5 min to remove nuclei. For immunoprecipitation the following antibodies were used: mouse mc Myc (Sigma), rabbit pc AN2 (3Niehaus A. Stegmüller J. Diers-Fenger M. Trotter J. J. Neurosci. 1999; 19: 4948-4961Crossref PubMed Google Scholar), pc GluRB (Chemicon). Lysates were preabsorbed with protein A-Sepharose (Amersham Biosciences) for 1 h at 4 °C, then subjected to immunoprecipitation overnight at 4 °C. Precipitation was performed with protein A-Sepharose. Precipitates were washed three times with radioimmune precipitation assay buffer (0.1% SDS, 1% Nonidet P-40, 1% sodium deoxycholate, 150 mm NaCl, 50 mm Tris, pH 7) and once with PBS before adding sample buffer and resolving the proteins by SDS-PAGE. Gels were dried, exposed to screens, and evaluated with a PhosphorImager. Total mouse brains (P7) were homogenized in buffer (50 mmTris, pH 7.8, 3 mm MgCl2, 320 mmsucrose, protease inhibitors) using an Ultra Turrax. The homogenate was centrifuged for 10 min at 1,000 rpm, 4 °C, the resulting supernatant was centrifuged for 1 h at 100,000 × g, 4 °C. The pellet was extracted in buffer (50 mm Tris, pH 7.8, 150 mm NaCl, 1 mm EDTA, 0.5% SDS, 0.05% sodium deoxycholate, 1% Triton X-100, protease inhibitors) for 1 h at 4 °C and centrifuged afterward for 1 h, 100,000 ×g, 4 °C. The supernatant was preabsorbed with protein A-Sepharose and subjected to immunoprecipitation as described above. Precipitates were washed three times with 1% Triton X-100 buffer and once with PBS and analyzed by SDS-PAGE and Western blotting. Primary oligodendrocytes were lysed in 1% Triton X-100, 50 mm Tris, pH 7.5, 150 mm NaCl, protease inhibitors, and the lysates were chilled on ice, centrifuged, and subjected to immunoprecipitation. Precipitates were washed three times with lysis buffer and once with PBS and analyzed by SDS-PAGE and Western blotting. SDS-PAGE was performed according to Laemmli (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206999) Google Scholar). Proteins were blotted onto polyvinylidene difluoride membrane (Hybond P, Amersham Biosciences), blocked with 4% milk powder in PBS and 0.1% Tween 20, and incubated with primary antibodies for 1 h. The blots were washed with PBST and incubated with appropriate secondary horseradish peroxidase-conjugated anti-mouse immunoglobulin (Dianova, Hamburg, Germany), horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Dianova). They were washed twice with PBST and once with PBS and subsequently developed by enhanced chemiluminescence (Amersham Biosciences). 12 × 106 cells (primary oligodendrocytes) were extracted in 750 μl of 1% Triton X-100 buffer, chilled for 30 min, and centrifuged (see “Coimmunoprecipitation”). 750 μl of extract was adjusted to 40% sucrose with 750 μl of 80% sucrose. The extract was overlaid with 1.75 ml of 30% sucrose and 1.5 ml of 5% sucrose in an SW60 tube (Beckman). After centrifugation (4 h at 218,000 × g, 4 °C) eight fractions were collected (17Klein C. Kramer E.M. Cardine A.M. Schraven B. Brandt R. Trotter J. J. Neurosci. 2002; 22: 698-707Crossref PubMed Google Scholar) and analyzed by 4–10% SDS-PAGE and Western blotting. Primary oligodendrocytes were cultured according to Trotter and colleagues (11Trotter J. Bitter-Suermann D. Schachner M. J. Neurosci. Res. 1989; 22: 369-383Crossref PubMed Scopus (140) Google Scholar,18Sontheimer H. Trotter J. Schachner M. Kettenmann H. Neuron. 1989; 2: 1135-1145Abstract Full Text PDF PubMed Scopus (221) Google Scholar), shaken-off oligodendrocytes were cultured on poly-l-lysine-coated glass coverslips for 2 days in SATO medium containing 1% horse serum, 10 ng/ml platelet-derived growth factor, and 5 ng/ml basic fibroblast growth factor. Cells were washed with PBS, fixed for 10 min with 4% paraformaldehyde, washed with PBS, permeabilized for 5 min with 0.05% Triton X-100, washed with PBS, and blocked with β-mercaptoethanol and 10% horse serum. Primary antibodies were diluted in blocking buffer and incubated for 30 min. Cells were washed three times with β-mercaptoethanol and 10% horse serum and incubated for 30 min with appropriate secondary Cy2- and Cy3-conjugated pc antibodies (Dianova), diluted in blocking buffer. The washed coverslips were mounted in Moviol and analyzed by confocal microscopy (Leica, Bensheim, Germany). The complete mouse NG2 cytoplasmic region consisting of 76 amino acids (RKRN … QYWV*, * = translation stop codon) was used as a bait in a yeast two-hybrid analysis to screen a postnatal mouse brain cDNA library. 33 × 106 transformants were screened. Ten independent library plasmids represented fragments of the same sequence, which was identical to murine GRIP1, a multi-PDZ domain protein (Fig. 1 a). GRIP1 was originally identified as an interacting protein of AMPA receptor subunits GluRB and GluRC (12Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (754) Google Scholar) The GRIP1 clones isolated in the screen were grouped according to their PDZ domain composition: group1 encodes a partial PDZ domain 4 through PDZ domain 7 (GRIP1/4p-7), group2 PDZ domain 5 through 7 (GRIP1/5–7), group3 a partial PDZ domain 6 through 7 (GRIP1/6p-7), group4 encodes PDZ domain 7 only (GRIP1/7). This analysis already identified the seventh PDZ domain as the putative interaction site. Yeast two-hybrid technology was used to verify the interaction of GRIP1 with the cytoplasmic region of NG2 and to confirm the interaction site by excluding binding of NG2 to GRIP1 PDZ domains other than PDZ7. Yeast was cotransformed with isolated GRIP1 plasmids and pGBT9cyto, grown on double dropout medium, and subsequently assayed for β-galactosidase activity. The yeast colonies were additionally selected for growth on triple dropout medium. As shown in Fig. 1 b, yeast two-hybrid analysis and subsequent β-galactosidase assay confirmed the binding of NG2 to GRIP1/PDZ4p-7, GRIP1/PDZ5–7, GRIP1/PDZp6–7, and GRIP1/PDZ7. When rat GRIP1 constructs consisting of PDZ domains 4–6 and the sixth domain only were tested for interaction, the yeast cells were negative for β-galactosidase activity, and failed to grow on triple dropout medium. Rat constructs containing the seventh PDZ domain such as GRIP1/PDZ6p-7 and GRIP1/PDZ6–7 restored the interaction of NG2 and GRIP1 and clearly identified the seventh PDZ domain of GRIP as the interaction site. GRIP2, a GRIP1 homolog, also consists of seven PDZ domains and is expressed in the central nervous system (13Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar, 19Bruckner K. Pablo Labrador J. Scheiffele P. Herb A. Seeburg P.H. Klein R. Neuron. 1999; 22: 511-524Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Because the sequences of the corresponding PDZ domains of GRIP1 and GRIP2 are very well conserved, we tested whether the interaction with NG2 was similarly conserved. The results in Fig. 1 c demonstrate that NG2 also interacts with the seventh PDZ domain of GRIP2, even though no clone for GRIP2 was isolated in the original screen. PDZ domains bind to COOH-terminal peptides of the interacting protein. The COOH-terminal tetrapeptide of NG2 (QYWV*) is conserved among rat, mouse, human, and Drosophila. It is similar to the PDZ binding motifs of ephrin B1 (YYKV*), which binds to PDZ6 of GRIP1/2 (19Bruckner K. Pablo Labrador J. Scheiffele P. Herb A. Seeburg P.H. Klein R. Neuron. 1999; 22: 511-524Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) and neurexins (EYYV*), which bind to CASK (20Hata Y. Butz S. Sudhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar), with a valine at position 0 and a tyrosine at the −2 position. To test whether QYWV* of NG2 is indeed a PDZ binding motif, positions 0, −1, −2, and −3 were mutated individually to glycine (Fig. 2). The mutations 0G, −1G, and −2G eliminated the interaction of NG2 and GRIP1/2, confirming that there is a PDZ binding motif at the extreme COOH terminus of NG2. Mutating the −3 position was without effect. The −2 position has been reported to be critical for the interaction. To characterize further the relevance of the amino acid at the −2 position, tyrosine was mutated to phenylalanine (Fig. 2), thus testing whether the hydroxyl group was essential for the interaction as is the case of PSD95 binding to the third PDZ domain of CRIPT (21Niethammer M. Valtschanoff J.G. Kapoor T.M. Allison D.W. Weinberg T.M. Craig A.M. Sheng M. Neuron. 1998; 20: 693-707Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), where a hydrogen bond forms with the residue of the binding groove. However, this mutation (−2F) did not interfere with the interaction of NG2 and GRIP, showing that it is not the OH group that is essential but rather the hydrophobic nature of the aromatic side chain. PDZ7 of GRIP1/2 thus belongs to class II PDZ domains, which bind to motifs with hydrophobic amino acids at positions 0 and −2: ΦXΦ* (where Φ is a hydrophobic amino acid, andX is any amino acid (10Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1041) Google Scholar, 22Songyang Z. Fanning A.S., Fu, C., Xu, J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1219) Google Scholar)). NG2 associates with GRIP1 or GRIP2 in mammalian cells. NG2-GRIP1 and NG2-GRIP2 associations were further confirmed by coimmunoprecipitation of NG2 together with GRIP1 or GRIP2 expressed in COS7 cells. First, cells were analyzed after transfection with an NG2 construct (NG2del, Fig. 3 a), comprising the signal sequence, approximately one-fourth of the extracellular portion, the transmembrane domain, and the complete intracellular region. The protein was incorporated into the membrane and recognized by pc AN2 antibodies (Fig. 3 b). Subsequently the cells were transfected with the NG2del construct together with Myc-tagged GRIP1/PDZ7 or GRIP2/PDZ7, respectively (Fig. 3 a). Anti-Myc antibodies precipitated GRIP1 and associated NG2del from cotransfected COS7 cells (Fig. 3 c, lane 2) and pc AN2 antibodies recognizing mouse NG2 precipitate NG2del and associated GRIP1 (Fig. 3 c, lane 3). Anti-Myc antibodies also precipitate GRIP2 and associated NG2del from transfected COS7 cells (Fig. 3 c, lane 4) expressing both these constructs. To confirm the NG2-GRIP1 interaction in vivo, extracts from P7 total mouse brain were subjected to immunoprecipitation using AN2 antibodies followed by Western blot analysis of coprecipitated GRIP1. The results (Fig. 3 d) demonstrate that GRIP1 associates with NG2 in vivo. Pc AN2 antibodies precipitate NG2 (lane 1) and associated GRIP1 as revealed by Western blotting with antibodies against GRIP1 (lane 2). Lanes 3 and4 are controls showing the presence of GRIP1 and NG2 in the brain extracts before precipitation. Biochemical studies of GRIP1 have largely focused on neuronal expression, where GRIP1 is enriched in the postsynaptic density and at synaptic plasma membranes (13Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar, 23Wyszynski M. Kim E. Yang F.C. Sheng M. Neuropharmacology. 1998; 37: 1335-1344Crossref PubMed Scopus (65) Google Scholar). Because NG2 is expressed by immature glial cells including oligodendrocyte progenitors (3Niehaus A. Stegmüller J. Diers-Fenger M. Trotter J. J. Neurosci. 1999; 19: 4948-4961Crossref PubMed Google Scholar), it was important to demonstrate that the interaction partner GRIP1 is also expressed in these cells. A Western blot analysis of lysates of the murine oligodendrocyte progenitor cell line Oli-neu (24Jung M. Kramer E. Grzenkowski M. Tang K. Blakemore W. Aguzzi A. Khazaie K. Chlichlia K. von Blankenfeld G. Kettenmann H. Trotter J. Eur. J. Neurosci. 1995; 7: 1245-1265Crossref PubMed Scopus (205) Google Scholar) and cultures of mouse primary oligodendrocytes consisting of a range of differentiation stages with antibodies against GRIP1 demonstrated a band of 130 kDa (Fig. 4 a,lanes 1 and 2). A total lysate from P9 mouse brain exhibited a band at the same molecular mass (lane 3) as in the control blot with rat cerebrum (lane 4). These results demonstrate that GRIP1 is indeed expressed in oligodendrocyte lineage cells. Because GRIP also binds to the GluRB subunit of AMPA receptors (12Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (754) Google Scholar) and AMPA receptors are expressed by glial cells (7Patneau D.K. Wright P.W. Winters C. Mayer M.L. Gallo V. Neuron. 1994; 12: 357-371Abstract Full Text PDF PubMed Scopus (282) Google Scholar), we examined whether NG2-positive cells also express GluRB. First, a Western blot analysis was performed using antibodies to GluRB (Fig. 4 b). A band of 108 kDa was detected in lysates of primary oligodendrocytes (lane 2) and in the control lysate (P9 total mouse brain, lane 3). No expression was detected in the Oli-neu cell line (lane 1). To define GluRB expression in individual cells, mixed cultures of primary oligodendrocytes were costained with AN2 antibodies and GluRB antibodies (Fig. 4 c). All oligodendrocyte lineage cells staining with antibodies to GluRB coexpressed NG2; however, not all NG2-positive cells expressed GluRB. GluRB staining was restricted to more immature cells with a few processes and revealed a punctate distribution of the protein along the major processes in addition to an intense intracellular staining. More mature oligodendrocytes characterized by a more complex morphology no longer expressed GluRB (see also Fig. 6 d, white star). NG2 expression shows a partial overlap with the expression of the O4 marker (3Niehaus A. Stegmüller J. Diers-Fenger M. Trotter J. J. Neurosci. 1999; 19: 4948-4961Crossref PubMed Google Scholar), which characterizes a late stage of oligodendrocyte progenitor cells (25Trotter J. Schachner M. Brain Res. Dev. Brain Res. 1989; 46: 115-122Crossref PubMed Scopus (122) Google Scholar) and is retained on more mature oligodendrocytes (26Sommer I. Schachner M. Dev. Biol. 1981; 83: 311-327Crossref PubMed Scopus (961) Google Scholar). In contrast, there is no overlap of NG2 and proteins expressed by more mature oligodendrocytes (3Niehaus A. Stegmüller J. Diers-Fenger M. Trotter J. J. Neurosci. 1999; 19: 4948-4961Crossref PubMed Google Scholar). To study the timing of GRIP1 and GluRB expression in oligodendrocyte lineage cells (Fig. 5), mixed cultures of primary oligodendrocytes were used as an in vitro model for oligodendrocyte maturation (27Pfeiffer S.E. Warrington A.E. Bansal R. Trends Cell Biol. 1993; 3: 191-197Abstract Full Text PDF PubMed Scopus (728) Google Scholar) and analyzed after 2, 8, and 14 days in culture by Western blotting. These cultures represent a dynamic population of different developmental stages that initially consists (div2) of predominantly immature cells. GRIP1 expression is high in immature cultures and decreases slowly as oligodendrocytes mature. In contrast, GluRB expression drops dramatically as oligodendrocytes mature. Maturation of oligodendrocytes is shown by increasing expression of MOG, a marker of mature oligodendrocytes. Staining of such cultures demonstrates that NG2 and MOG are never coexpressed and that GluRB-expressing cells are always MOG-negative. GRIP1 expression persists over a longer period of maturation than GluRB expression, implying that GRIP1 may have multiple binding partners during the course of oligodendroglial development. Transfected COS7 cells were subjected to radiolabeling and immunoprecipitation to investigate whether a complex consisting of NG2, GRIP1, and GluRB can be isolated. Cells were transfected with the NG2del, Myc-tagged GRIP1 and the AMPA subunit GluRB (flop, short form). NG2 as well as the GluRB subunit were incorporated into the plasma membrane. The GRIP1 construct encodes PDZ domains 5–7 (Fig. 6 a). GluRB was reported to bind to PDZ4–5 of GRIP (12Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (754) Google Scholar), and we have shown that NG2 binds to GRIP/PDZ7. Fig. 6 b shows immunoprecipitation of the complex: pc AN2" @default.
• W2019341013 created "2016-06-24" @default.
• W2019341013 creator A5038465445 @default.
• W2019341013 creator A5044498878 @default.
• W2019341013 creator A5066252977 @default.
• W2019341013 creator A5086750644 @default.
• W2019341013 date "2003-02-01" @default.
• W2019341013 modified "2023-10-17" @default.
• W2019341013 title "The Proteoglycan NG2 Is Complexed with α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors by the PDZ Glutamate Receptor Interaction Protein (GRIP) in Glial Progenitor Cells" @default.
• W2019341013 cites W1490096362 @default.
• W2019341013 cites W1498285527 @default.
• W2019341013 cites W1500368251 @default.
• W2019341013 cites W1535641516 @default.
• W2019341013 cites W1652174858 @default.
• W2019341013 cites W179352461 @default.
• W2019341013 cites W1822921207 @default.
• W2019341013 cites W1919311176 @default.
• W2019341013 cites W1953992154 @default.
• W2019341013 cites W1966232883 @default.
• W2019341013 cites W1969244755 @default.
• W2019341013 cites W1973106753 @default.
• W2019341013 cites W1973476654 @default.
• W2019341013 cites W1974776242 @default.
• W2019341013 cites W1975861627 @default.
• W2019341013 cites W1978050645 @default.
• W2019341013 cites W1987704449 @default.
• W2019341013 cites W1988594478 @default.
• W2019341013 cites W1993589817 @default.
• W2019341013 cites W1995691453 @default.
• W2019341013 cites W1996603253 @default.
• W2019341013 cites W1996691843 @default.
• W2019341013 cites W1997276312 @default.
• W2019341013 cites W2000626734 @default.
• W2019341013 cites W2001545716 @default.
• W2019341013 cites W2001888569 @default.
• W2019341013 cites W2002450368 @default.
• W2019341013 cites W2004747469 @default.
• W2019341013 cites W2005868265 @default.
• W2019341013 cites W2012160176 @default.
• W2019341013 cites W2016898056 @default.
• W2019341013 cites W2031611126 @default.
• W2019341013 cites W2033481426 @default.
• W2019341013 cites W2036567890 @default.
• W2019341013 cites W2049212652 @default.
• W2019341013 cites W2050989030 @default.
• W2019341013 cites W2051114185 @default.
• W2019341013 cites W2053626372 @default.
• W2019341013 cites W2054243450 @default.
• W2019341013 cites W2054587629 @default.
• W2019341013 cites W2058426472 @default.
• W2019341013 cites W2076540617 @default.
• W2019341013 cites W2078452348 @default.
• W2019341013 cites W2079832984 @default.
• W2019341013 cites W2081422930 @default.
• W2019341013 cites W2083156858 @default.
• W2019341013 cites W2094416985 @default.
• W2019341013 cites W2095549425 @default.
• W2019341013 cites W2098744846 @default.
• W2019341013 cites W2099564001 @default.
• W2019341013 cites W2100837269 @default.
• W2019341013 cites W2105006060 @default.
• W2019341013 cites W2108461170 @default.
• W2019341013 cites W2115810166 @default.
• W2019341013 cites W2118076209 @default.
• W2019341013 cites W2120558693 @default.
• W2019341013 cites W2121936687 @default.
• W2019341013 cites W2130101439 @default.
• W2019341013 cites W2133388060 @default.
• W2019341013 cites W2145492711 @default.
• W2019341013 cites W2146101256 @default.
• W2019341013 cites W2148981717 @default.
• W2019341013 cites W2150010887 @default.
• W2019341013 cites W2151444391 @default.
• W2019341013 cites W2164239948 @default.
• W2019341013 cites W2336952534 @default.
• W2019341013 doi "https://doi.org/10.1074/jbc.m210010200" @default.
• W2019341013 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12458226" @default.
• W2019341013 hasPublicationYear "2003" @default.
• W2019341013 type Work @default.
• W2019341013 sameAs 2019341013 @default.
• W2019341013 citedByCount "90" @default.
• W2019341013 countsByYear W20193410132012 @default.
• W2019341013 countsByYear W20193410132013 @default.
• W2019341013 countsByYear W20193410132014 @default.
• W2019341013 countsByYear W20193410132015 @default.
• W2019341013 countsByYear W20193410132016 @default.
• W2019341013 countsByYear W20193410132017 @default.
• W2019341013 countsByYear W20193410132018 @default.
• W2019341013 countsByYear W20193410132019 @default.
• W2019341013 countsByYear W20193410132020 @default.
• W2019341013 countsByYear W20193410132021 @default.
• W2019341013 countsByYear W20193410132022 @default.
• W2019341013 countsByYear W20193410132023 @default.
• W2019341013 crossrefType "journal-article" @default.
• W2019341013 hasAuthorship W2019341013A5038465445 @default.
• W2019341013 hasAuthorship W2019341013A5044498878 @default.
• W2019341013 hasAuthorship W2019341013A5066252977 @default.
• W2019341013 hasAuthorship W2019341013A5086750644 @default.

• next