A Four PDZ Domain-containing Splice Variant Form of GRIP1 Is Localized in GABAergic and Glutamatergic Synapses in the Brain
2004; Elsevier BV; Volume: 279; Issue: 37 Linguagem: Inglês
10.1074/jbc.m405786200
ISSN1083-351X
AutoresErik I. Charych, Wendou Yu, Rong‐wen Li, David R. Serwanski, Celia P. Miralles, Xuejing Li, Bih Y. Yang, Noelia Pinal, Randall S. Walikonis, Angel L. De Blas,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoWe have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is γ-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with γ-aminobutyric acid, type A, receptor (GABAAR) clusters than with α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABAAR clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses. We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is γ-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with γ-aminobutyric acid, type A, receptor (GABAAR) clusters than with α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABAAR clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses. GRIP1 (glutamate receptor interacting protein 1) is a 7-PDZ domain-containing protein (1Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (760) Google Scholar, 2Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar) belonging to a family of highly homologous proteins that includes GRIP2 (2Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar, 3Wyszynski M. Kim E. Yang F.C. Sheng M. Neuropharmacology. 1998; 37: 1335-1344Crossref PubMed Scopus (65) Google Scholar, 4Bruckner 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 (310) Google Scholar) and the AMPA 1The abbreviations used are: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid; PSD, postsynaptic density; GABAA, γ-aminobutyric acid, type A; GABAAR, GABAA receptor; HA, hemagglutinin; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; UTR, untranslated region; 5′-RACE, 5′-rapid amplification of cDNA ends; RT, room temperature; PBS, phosphate-buffered saline; PSDs, postsynaptic densities; Y2H, yeast two-hybrid; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; E, embryonic day; P, postnatal day; mAb, monoclonal antibody; VGAT, vesicular GABA transporter; AMCA, 7-amino-4-methylcoumarin-3-acetic acid; LTD, long term depression; GAD, glutamic acid decarboxylase. receptor-binding protein ABP (5Srivastava S. Osten P. Vilim F.S. Khatri L. Inman G. States B. Daly C. DeSouza S. Abagyan R. Valtschanoff J.G. Weinberg R.J. Ziff E.B. Neuron. 1998; 21: 581-591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 6Srivastava S. Ziff E.B. Ann. N. Y. Acad. Sci. 1999; 868: 561-564Crossref PubMed Scopus (14) Google Scholar). GRIP2 is also a 7-PDZ domain-containing protein, whereas ABP is a shorter splice variant of GRIP2, lacking the seventh PDZ domain of GRIP2. Both GRIP2 and ABP are derived from the same gene, whereas GRIP1 is encoded by a separate gene. GRIP1, GRIP2, and ABP interact with the C-terminal tail of AMPA receptor subunits GluR2/3/4c through PDZ domains 3-6 (1Dong H. O'Brien R.J. Fung E.T. Lanahan A.A. Worley P.F. Huganir R.L. Nature. 1997; 386: 279-284Crossref PubMed Scopus (760) Google Scholar, 3Wyszynski M. Kim E. Yang F.C. Sheng M. Neuropharmacology. 1998; 37: 1335-1344Crossref PubMed Scopus (65) Google Scholar, 5Srivastava S. Osten P. Vilim F.S. Khatri L. Inman G. States B. Daly C. DeSouza S. Abagyan R. Valtschanoff J.G. Weinberg R.J. Ziff E.B. Neuron. 1998; 21: 581-591Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). GRIP1 may play a role in the postsynaptic localization of AMPA receptors (7Li P. Kerchner G.A. Sala C. Wei F. Huettner J.E. Sheng M. Zhuo M. Nat. Neurosci. 1999; 2: 972-977Crossref PubMed Scopus (174) Google Scholar, 8Song I. Huganir R.L. Trends Neurosci. 2002; 25: 578-588Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar) and in targeting AMPA receptors to the synapse (2Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar, 4Bruckner 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 (310) Google Scholar, 9Osten P. Khatri L. Perez J.L. Kohr G. Giese G. Daly C. Schulz T.W. Wensky A. Lee L.M. Ziff E.B. Neuron. 2000; 27: 313-325Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). GRIP1 has been implicated in activity-dependent synaptic reorganization of AMPA receptors (10Lu X. Wyszynski M. Sheng M. Baudry M. J. Neurochem. 2001; 77: 1553-1560Crossref PubMed Scopus (56) Google Scholar) during LTD (8Song I. Huganir R.L. Trends Neurosci. 2002; 25: 578-588Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 11Bredt D.S. Nicoll R.A. Neuron. 2003; 40: 361-379Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar). GRIP1 also binds to the microtubule-based motor protein kinesin 5 through the region located between PDZ domains 6 and 7, thus being involved in vesicular trafficking of AMPA receptors along dendritic microtubules (12Setou M. Seog D.H. Tanaka Y. Kanai Y. Takei Y. Kawagishi M. Hirokawa N. Nature. 2002; 417: 83-87Crossref PubMed Scopus (415) Google Scholar), targeting AMPA receptors to synapses (2Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar, 4Bruckner 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 (310) Google Scholar). Furthermore, PDZ6 of both GRIP1 and GRIP2 interacts with the C termini of EphB2/EphA7 receptors and EphrinB1 ligands, recruiting cytoplasmic GRIPs to membrane lipid rafts (4Bruckner 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 (310) Google Scholar, 13Torres R. Firestein B.L. Dong H. Staudinger J. Olson E.N. Huganir R.L. Bredt D.S. Gale N.W. Yancopoulos G.D. Neuron. 1998; 21: 1453-1463Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). PDZ6 of both GRIP1 and GRIP2 also interacts with members of the liprin-α family, and disruption of this interaction prevents AMPA receptor surface expression and clustering (14Wyszynski M. Kim E. Dunah A.W. Passafaro M. Valtschanoff J.G. Serra-Pages C. Streuli M. Weinberg R.J. Sheng M. Neuron. 2002; 34: 39-52Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). AMPA receptors and GRIP1 as well as other components of glutamatergic synapses are present in lipid rafts (15Hering H. Lin C.C. Sheng M. J. Neurosci. 2003; 23: 3262-3271Crossref PubMed Google Scholar). GRIP-/- mouse mutants have shown that GRIP1 is important for embryonic development (16Bladt F. Tafuri A. Gelkop S. Langille L. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6816-6821Crossref PubMed Scopus (57) Google Scholar, 17Takamiya K. Kostourou V. Adams S. Jadeja S. Chalepakis G. Scambler P.J. Huganir R.L. Adams R.H. Nat. Genet. 2004; 36: 172-177Crossref PubMed Scopus (122) Google Scholar). Two splice forms of GRIP1 (GRIP1a and GRIP1b) differ in a short N-terminal peptide sequence such that GRIP1b can be palmitoylated, whereas GRIP1a cannot (18Yamazaki M. Fukaya M. Abe M. Ikeno K. Kakizaki T. Watanabe M. Sakimura K. Neurosci. Lett. 2001; 304: 81-84Crossref PubMed Scopus (56) Google Scholar). Similar palmitoylated and nonpalmitoylated forms have been described for ABP/GRIP2 (19DeSouza S. Fu J. States B.A. Ziff E.B. J. Neurosci. 2002; 22: 3493-3503Crossref PubMed Google Scholar). Palmitoylation of GRIP1/2 allows the anchoring of GRIP1/2 to the membrane and accumulation of AMPA receptors to the synaptic membrane (19DeSouza S. Fu J. States B.A. Ziff E.B. J. Neurosci. 2002; 22: 3493-3503Crossref PubMed Google Scholar). In this paper, we report the identification and characterization of GRIP1c 4-7, a novel splice form of GRIP1 that lacks PDZ domains 1-3 of GRIP1a/b but contains PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 is not just a short form of GRIP1a/b. GRIP1c 4-7 also contains specific 35-amino acid N-terminal and 12-amino acid C-terminal peptide sequences that differ from GRIP1a/b. GRIP1c 4-7 readily distributes to the plasma membrane and concentrates not only in glutamatergic synapses but also in GABAergic synapses both in cultured neurons and in the intact brain. Thus, some forms of GRIP1 might play a more significant role in GABAergic synapses than previously recognized. All the animal protocols have been approved by the Institutional Animal Care and Use Committee and follow the National Institutes of Health guidelines. Antibodies—All the anti-GABAAR, anti-GRIP1c 4-7 antibodies were raised in our laboratory. Rabbit and guinea pig antibodies specific for GRIP1c 4-7 were raised to amino acids 12-26 (KPHNFHHASHPPLRK) of rat GRIP1c 4-7. This sequence is not present in GRIP1a/b (Fig. 1). For anti-GRIP1c 4-7 antibody production, this peptide was covalently linked, via a C-terminal cysteine, to diphtheria toxoid (Mimotopes, San Diego, CA). A guinea pig antibody specific for GRIP1a/b was raised to the C-terminal amino acids 1100-1112 of rat GRIP1a/b (GGNLETREPTNTL). This amino acid sequence is not present in GRIP1c 4-7 (Fig. 1). For anti-GRIP1a/b antibody production, this peptide sequence was coupled via an N-terminal cysteine to keyhole limpet hemocyanin. A guinea pig and/or New Zealand rabbit were injected subcutaneously with a 1:1 emulsion of either diphtheria toxoid- or keyhole limpet hemocyanin-coupled peptide in complete Freund's adjuvant (for the first immunization) and with incomplete Freund's adjuvant (for all subsequent immunizations) once per month. The antibody titer in the sera was monitored by enzyme-linked immunosorbent assay. Sera were collected after 4 months of immunizations and were affinity-purified on immobilized peptide. The anti-GRIP1c 4-7 antibodies showed specificity for GRIP1c 4-7 but not for GRIP1a/b as shown by immunofluorescence of transfected HEK293 cells and immunoblots of brain membranes (see "Results"). Likewise, the anti-GRIP1a/b antibodies showed specificity for GRIP1a/b but not for GRIP1c 4-7 as shown by immunofluorescence of HEK293 cells (not shown) and immunoblots of brain membranes (see "Results"). The guinea pig anti-rat α1 subunit antibody was raised to amino acids 1-15, and the rabbit anti-rat γ2 subunit antibody was raised to amino acids 1-15 (20Miralles C.P. Li M. Mehta A.K. Khan Z.U. De Blas A.L. J. Comp. Neurol. 1999; 413: 535-548Crossref PubMed Scopus (55) Google Scholar, 21Christie S.B. Miralles C.P. De Blas A.L. J. Neurosci. 2002; 22: 684-697Crossref PubMed Google Scholar). The monoclonal mouse anti-β2/3 (62Brandon N.J. Jovanovic J.N. Smart T.G. Moss S.J. J. Neurosci. 2002; 22: 6353-6361Crossref PubMed Google Scholar-3G1) was raised in our laboratory to affinity-purified bovine GABAARs (22De Blas A.L. Vitorica J. Friedrich P. J. Neurosci. 1988; 8: 602-614Crossref PubMed Google Scholar, 23Vitorica J. Park D. Chin G. De Blas A.L. J. Neurosci. 1988; 8: 615-622Crossref PubMed Google Scholar). This antibody recognizes an extracellular epitope that is common to β2 and β3 subunits (β2/3) of the rat GABAARs but is not present in β1 (24Ewert M. De Blas A.L. Mohler H. Seeburg P.H. Brain Res. 1992; 569: 57-62Crossref PubMed Scopus (80) Google Scholar). All the rabbit and guinea pig antibodies used in light microscopy and EM immunocytochemistry were affinity-purified on immobilized peptide. The generation, affinity purification, specificity, and characterization of these GABAAR antibodies have been described elsewhere (20Miralles C.P. Li M. Mehta A.K. Khan Z.U. De Blas A.L. J. Comp. Neurol. 1999; 413: 535-548Crossref PubMed Scopus (55) Google Scholar, 21Christie S.B. Miralles C.P. De Blas A.L. J. Neurosci. 2002; 22: 684-697Crossref PubMed Google Scholar, 22De Blas A.L. Vitorica J. Friedrich P. J. Neurosci. 1988; 8: 602-614Crossref PubMed Google Scholar, 23Vitorica J. Park D. Chin G. De Blas A.L. J. Neurosci. 1988; 8: 615-622Crossref PubMed Google Scholar, 25Moreno J.I. Piva M.A. Miralles C.P. De Blas A.L. J. Comp. Neurol. 1994; 350: 260-271Crossref PubMed Scopus (39) Google Scholar, 26Christie S.B. Li R.W. Miralles C.P. Riquelme R. Yang B.Y. Charych E. Yu W. Daniels S.B. Cantino M.E. De Blas A.L. Prog. Brain Res. 2002; 136: 157-180Crossref PubMed Scopus (43) Google Scholar, 27Christie S.B. De Blas A.L. J. Comp. Neurol. 2003; 456: 361-374Crossref PubMed Scopus (42) Google Scholar). The mouse monoclonal anti-PSD-95, the rabbit anti-GluR1 antibody, and the rabbit anti-HA antibody were from Upstate Biotechnology, Inc. (Lake Placid, NY), and the anti-HA mouse monoclonal antibody was from Covance (Princeton, NJ). Rabbit anti-GluR2/3 was a gift from Dr. Robert Wenthold (National Institutes of Health, Bethesda, MD); mouse monoclonal anti-SV2 was a gift from Dr. Kathleen M. Buckley (Harvard Medical School); sheep anti-GAD was a gift from Dr. I. Kopin (National Institutes of Health); and guinea pig anti-GABA was from Chemicon (Temecula, CA). Rabbit and guinea pig anti-vesicular glutamate transporter-1 (vGlut-1) were from Synaptic Systems (Goettingen, Germany) and Chemicon (Temecula, CA), respectively. Mouse monoclonal anti-GRIP1 antibody, to a region of GRIP1 that includes part of the linker region between PDZ domains 6 and 7 and all of PDZ domain 7, was from Transduction Laboratories. Fluorophore-labeled secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Colloidal gold-labeled (10 nm) goat anti-mouse secondary antibody was from ICN (Irvine, CA). All other colloidal gold-labeled secondary antibodies were from Jackson ImmunoResearch. Yeast Two-hybrid—All vectors and yeast strains for bait analysis and yeast two-hybrid (Y2H) screening were from Dr. Roger Brent (University of California, San Francisco) or Origene Technologies (Rockville, MD). Sense and antisense oligonucleotide primers were designed to amplify the C-terminal 50 amino acids of the AMPA receptor subunit GluR3 including its natural stop codon, and the PCR product was directionally cloned into the polylinker of pEG202. We confirmed that the bait fusion protein could be expressed in yeast by immunoblotting the cell lysate of yeast transformants with mouse anti-LexA monoclonal antibody (Origene Technologies, Rockville, MD). We also tested that this fusion protein did not activate the lacZ reporter by itself. For this purpose, Saccharomyces cerevisiae EGY48 was transformed with pSH18-34 and pEG202 (containing the bait insert), and the lacZ reporter activity was tested by replica-plating the transformants on the appropriate X-gal-containing media. For the positive control, the yeast was transformed with pSH18-34 and pSH17-4, the latter of which contains the LexA DNA binding domain fused to the B42 transcriptional activator domain. For a negative control, the yeast was transformed with pSH18-34 and pRHFM1 or with pSH18-34 and pEG202, the empty bait vector. We also confirmed that the bait did not activate the genomic LEU2 reporter gene, because the transformants containing the bait did not grow in the absence of leucine. For library screening, the yeast strain EGY48, previously transformed with pEG202 (containing the bait) and pSH18-34, was transformed with pJG4-5 containing the oligo(dT)-primed rat brain cDNA library (Origene Technologies). An aliquot of the pooled transformants was then diluted 1:10 in liquid YNB medium containing galactose (to activate the GAL1 promoter) and allowed to incubate for 4 h at 30 °C to induce cDNA library expression. Only clones with an activated leucine reporter grew on the medium lacking leucine. After allowing 4-6 days of growth, the fastest growing colonies were replica-plated onto solid YNB galactose growth medium containing X-gal. Plasmids from yeast clones showing lacZ reporter activity (presumably expressing bait interactors) were rescued by mechanical disruption and detergent lysis. The DNA was extracted with phenol/chloroform and was used to transform the trp-E. coli KC8 strain. Growth medium lacking tryptophan was used to select KC8 cells containing pJG4-5. Plasmid preparations from KC8 transformants were subjected to restriction analysis with EcoRI and XhoI, the enzymes used for directional cloning of the cDNA library into pJG4-5. The coding and noncoding strands of the cDNA clones were independently sequenced by using the BigDye Terminator DNA sequencing kit (Applied Biosystems, Foster City, CA) and read using the ABI377 Prism DNA sequencer, model 377XL (Applied Biosystems). Isolation and Sequencing of the 5′-UTR of GRIP1c 4-7 cDNA—By using the adaptor-ligated Marathon-ready rat brain cDNA library (Clontech) as template in a PCR, we completed the full cDNA sequence of the GRIP1c 4-7 clone that was isolated by yeast two-hybrid screening. For this purpose, we used an antisense, gene-specific primer that corresponds to part of the GRIP1c 4-7 5′-UTR (5′-TTCTCTAGAGGCAAGGGGTGGTGACT-3′) and a sense primer corresponding to the 5′ end adaptor of the cDNA library. For PCR amplification, the Advantage 2 Polymerase Mix (Clontech) was used under the following thermocycling conditions: 94 °C for 30 s followed by 20 cycles, each consisting of 94 °C for 5 s and 68 °C for 2 min. After obtaining a PCR product of about 500 bp, the specificity of the reaction was confirmed by using nested primers to the 5′ adaptor sequence and the GRIP1c 4-7 5′-UTR (5′-TATAAGACCCTCACGGAGGACCGACGAT-3′). The 500-bp PCR product obtained from the initial 5′-rapid amplification of cDNA ends (5′-RACE) was used as the template. The nested 5′-RACE product was then cloned, by the T/A cloning method, into pT-Adv plasmid (Clontech) for DNA sequencing. We have submitted the rat GRIP1c 4-7 cDNA full-length sequence to the GenBank™ (accession number AY437398). Preparation of Tissue Fractions—For crude synaptosomal fraction, forebrains (cerebral cortex and hippocampus) from two 6-8-week-old Sprague-Dawley rats were homogenized with a glass/Teflon homogenizer in 10 ml of solution A (0.32 m sucrose, 1 mm NaHCO3, 1 mm MgCl2, 0.5 mm CaCl2, 1 mg/liter leupeptin, 0.1 mm phenylmethylsulfonyl fluoride) at 4 °C. The homogenate was diluted to 20 ml with solution A and centrifuged for 10 min at 1400 × g at 4 °C, and the supernatant was saved. The pellet was suspended in 20 ml of solution A and centrifuged as above. The two supernatants were pooled and centrifuged at 13,800 × g at 4 °C for 10 min, and the pellet was suspended in 16 ml of solution B (0.32 m sucrose and 1 mm NaHCO3) and homogenized with a glass Dounce homogenizer at 4 °C. This crude synaptosomal fraction, containing membranes and cytosol, was stored in aliquots at -70 °C. Synaptosomes were prepared from homogenates by the method of Carlin et al. (28Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-845Crossref PubMed Scopus (624) Google Scholar). A fraction enriched in postsynaptic densities (PSDs) was prepared by treating synaptosomes with 0.5% Triton X-100 for 15 min at 4 °C followed by centrifugation at 100,000 × g for 1 h at 4 °C. The pellet containing PSDs was resuspended in 50 mm Tris-HCl, pH 7.4 (29Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1020) Google Scholar). Samples were subjected to SDS-PAGE and immunoblotted with specific antibodies as described elsewhere (30De Blas A.L. Cherwinski H.M. Anal. Biochem. 1983; 133: 214-219Crossref PubMed Scopus (271) Google Scholar). Homogenates from various tissues (3-month-old male rats) or from forebrain from rats of different ages were prepared as above except that the non-neural tissue was first ground with a PowerGen 125 (Fisher) and then homogenized in a glass/Teflon homogenizer as with brain in buffer containing 0.32 m sucrose, 10 mm Tris-HCl, pH 7.4, and protease inhibitors 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 10 μg/ml aprotinin A, and 10 μg/ml leupeptin. The homogenates were used directly for SDS-PAGE and immunoblotting. Immunoprecipitations—Immunoprecipitation from sodium deoxycholate extracts of rat cortical/hippocampal membranes was done according to the method of Luo et al. (31Luo J. Wang Y. Yasuda R.P. Dunah A.W. Wolfe B.B. Mol. Pharmacol. 1997; 51: 79-86Crossref PubMed Scopus (354) Google Scholar). Briefly, a rat brain cortical/hippocampal crude and unlysed synaptosomal fraction, containing 5 mg/ml total protein in solution B (see above), was centrifuged at 13,800 × g at 4 °C for 10 min, and the pellet was suspended in a volume of TE buffer (10 mm Tris-HCl and 5 mm EDTA, pH 7.4) equal to that of the original suspension. One-tenth volume of ice-cold sodium deoxycholate buffer (10% sodium deoxycholate in 500 mm Tris-HCl, pH 9.0) was added, and the sample was incubated at 36 °C for 30 min, followed by the addition of one-tenth volume of Triton X-100 buffer (1% Triton X-100 and 500 mm Tris-HCl, pH 9.0). The sample was dialyzed against solution C (50 mm Tris-HCl, pH 7.4, 5 mm EDTA, and 0.1% Triton X-100) at 4 °C overnight. Detergent-insoluble material was pelleted by centrifugation at 37,000 × g for 40 min at 4 °C, and the supernatant was used for immunoprecipitation with an anti-GRIP1c 4-7 antibody. 40 μl of protein A-Sepharose beads, suspended in 450 μl of 50 mm Tris-HCl, pH 7.4, were incubated with 50 μl of guinea pig anti-GRIP1c 4-7 antiserum, or the preimmune serum, overnight at 4 °C with rotation. A volume of the extract, containing 200 μg of protein, was added to the antibody-coated beads and incubated overnight at 4 °C. Beads, washed with solution C, were incubated with SDS-PAGE sample buffer (0.01 m Tris-HCl, pH 6.8, 20% glycerol, 10% β-mercaptoethanol, 2.3% SDS, 0.005% bromphenol blue) for 10 min at 85 °C. The beads were pelleted by centrifugation, and the supernatant was subjected to SDS-PAGE and immunoblotted with a rabbit anti-GluR2/3 antibody. The immunoblotting procedure has been described elsewhere (30De Blas A.L. Cherwinski H.M. Anal. Biochem. 1983; 133: 214-219Crossref PubMed Scopus (271) Google Scholar). Low Density Hippocampal Cultures and Transfection—Hippocampal cultures were prepared by the method of Banker and Goslin (32Goslin K. Assmussen H. Banker G. Banker G. Goslin K. Culturing Nerve Cells. 2nd Ed. MIT Press, Cambridge, MA1998: 339-370Google Scholar) as described elsewhere (21Christie S.B. Miralles C.P. De Blas A.L. J. Neurosci. 2002; 22: 684-697Crossref PubMed Google Scholar). Briefly, dissociated neurons from embryonic day 18 Sprague-Dawley rat hippocampi were plated at a density of 3,000-8,000 cells per 18-mm diameter circular coverslip and maintained in glial cell conditioned culture medium for up to 21 days. Cultured hippocampal neurons were transfected with 3 μg of pcDNA3.1(+) containing GRIP1c 4-7 (with the hemagglutinin (HA) tag at its N terminus) at 9 days in culture using the Calphos transfection kit (BD Biosciences) according to the manufacturer's instructions. Transfected cells were cultured for 7 additional days and processed for immunofluorescence as described below. HEK293 Cell Culture and Transfection—HEK293 cells were maintained in high glucose Dulbecco's modified Eagle's medium (Invitrogen) with 5% fetal bovine serum (Invitrogen) in a 5% CO2 atmosphere. HEK293 cells were cultured on poly-l-lysine-coated 18-mm coverslips and transfected with 1 μg of plasmid DNA, all in pcDNA3.1(+), encoding GRIP1c 4-7, GRIP1a, or GRIP1a 4-7 or combinations of GRIP1c 4-7 and AMPA receptor subunits using the LipofectAMINE 2000 method following the manufacturer's instructions (Invitrogen). Cells were then processed for immunofluorescence as described below. Immunofluorescence of Hippocampal Cultures and HEK293 Cells—Double or triple label immunofluorescence detection of various antigens with specific antibodies raised in various species was done as described elsewhere (21Christie S.B. Miralles C.P. De Blas A.L. J. Neurosci. 2002; 22: 684-697Crossref PubMed Google Scholar, 26Christie S.B. Li R.W. Miralles C.P. Riquelme R. Yang B.Y. Charych E. Yu W. Daniels S.B. Cantino M.E. De Blas A.L. Prog. Brain Res. 2002; 136: 157-180Crossref PubMed Scopus (43) Google Scholar, 27Christie S.B. De Blas A.L. J. Comp. Neurol. 2003; 456: 361-374Crossref PubMed Scopus (42) Google Scholar). Hippocampal neurons or transfected HEK293 cells were fixed by immersion of coverslips in 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS) for 12 min at RT followed by permeabilization with 0.25% Triton X-100 in PBS for 5 min. The cultures were incubated with a mixture of the primary antibodies (defined in the legends for Figs. 3 and 5, 6, 7), diluted in 0.25% Triton X-100 PBS for 2 h at room temperature. Coverslips then were washed and incubated for 1 h at RT with a mixture of species-specific secondary antibodies all raised in donkey and conjugated to either Texas Red, FITC, and/or AMCA fluorophores (1:200 dilution in 0.25% Triton X-100 PBS, Jackson ImmunoResearch). Optimal primary antibody dilutions were determined by dilution series. In HEK293 cells, the cell nuclei were labeled with DAPI, and cell surface localization was determined with FITC-conjugated phalloidin. The coverslips were washed with PBS and mounted using Prolong anti-fade mounting solution (Molecular Probes; Eugene, OR). Specificity of the immunolabeling by the anti-GRIP1c 4-7 antibody was demonstrated by blocking the binding of the primary antibody with 20 μg/ml of the antigenic peptide. Moreover, no immunolabeling was obtained when the primary antibody was omitted. Images were collected using a ×60 pan-fluor objective on a Nikon Eclipse T300 microscope with a Sensys KAF 1401E CCD camera, driven by IPLab 3.0 (Scanalytics, Fairfax, VA) acquisition software. Image files were then processed and merged for color colocalization figures using Adobe Photoshop 4.01.Fig. 5GRIP1c 4-7 colocalizes with GABAARs and AMPA receptor clusters in mature cultures of hippocampal neurons. Triple label immunofluorescence using combinations of rabbit anti-GRIP1c 4-7 (A), guinea pig anti-GRIP1c 4-7 (D, G, J, and M), mouse mAb to β2/3 subunits of the GABAAR (B), sheep anti-GAD (C and F), rabbit anti-γ2 subunit of the GABAAR (E and K), mouse anti-PSD-95 (H), rabbit anti-GluR2/3 subunits of the AMPA receptors (N), rabbit anti-vGlut1 (I), and mouse mAb anti-SV2 (L and O) antibodies. A-F, GRIP1c 4-7, revealed with either the rabbit or the guinea pig antibody, colocalizes with GABAAR clusters both at GABAergic synapses (filled arrows) and outside GABAergic synapses (filled arrowheads) as determined by the presence or absence of GAD+ terminals, respectively. Some of the GRIP1c 4-7 clusters do not colocalize with GABAA receptor clusters or GAD+ terminals (open arrowheads). G-I, GRIP1c 4-7 colocalizes with PSD-95 clusters at glutamatergic synapses (vGlut1+, filled arrows). Some GRIP1c 4-7 clusters colocalize with PSD-95 but not with vGlut1 (filled arrowheads). J-L, GRIP1c 4-7 colocalizes with GABAAR clusters in the presence (filled arrows) or absence (filled arro
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