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Phosphorylation of Glutamate Receptor Interacting Protein 1 Regulates Surface Expression of Glutamate Receptors

2006; Elsevier BV; Volume: 282; Issue: 4 Linguagem: Inglês

10.1074/jbc.m606471200

ISSN

1083-351X

Autores

Karina Kulangara, Michel Kropf, Liliane Glauser, Sarah Magnin, Stefano Alberi, Alexandre Yersin, Harald Hirling,

Tópico(s)

Lipid Membrane Structure and Behavior

Resumo

The number of synaptic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-d-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas full-length GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane. The number of synaptic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-d-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas full-length GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane. AMPA-type glutamate receptors mediate most of the fast excitatory synaptic transmission in the brain. Various forms of synaptic plasticity depend on changes in AMPA receptor (AMPAR) 2The abbreviations used are: AMPAR, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor; ABP, AMPAR-binding protein; AFM, atomic force microscopy; GRIP, glutamate receptor interacting protein; NEEP21, neuron enriched endosomal protein of 21 kDa; NMDA, N-methyl-d-aspartate; OA, okadaic acid; PDZ, postsynaptic density 95/disc large/zonula occludens-1; PICK1, protein interacting with C kinase 1; aa, amino acids; GST, glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate; wt, wild type; GFP, green fluorescent protein; PKC, protein kinase C; W, Western blotting; IF, immunofluorescence; ct, carboxyl terminus. transmission. Trafficking of AMPAR and their lateral diffusion to and from synapses are key mechanisms that govern synaptic strength (1Malinow R. Malenka R.C. Annu. Rev. 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NMDA receptor-dependent internalization of AMPAR activates protein phosphatases and leads to a rapid reinsertion of AMPAR into the plasma membrane (24Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). GRIP1 plays a crucial role in AMPAR sorting as well as in AMPAR stabilization at synapses and intracellular compartments. It interacts with the carboxyl-terminal PDZ-binding motifs of GluR2, GluR3, and GluR4c (25Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar). GRIP belongs to a protein family comprising the splice variants GRIP1a (25Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar) and GRIP1b (26Yamazaki M. Fukaya M. Abe M. Ikeno K. Kakizaki T. Watanabe M. Sakimura K. Neurosci. Lett. 2001; 304: 81-84Crossref PubMed Scopus (56) Google Scholar), both having seven PDZ domains (PDZ 1–7), and GRIP1c 4–7, with only PDZ 4–7 (27Charych E.I. Yu W. Li R. Serwanski D.R. Miralles C.P. Li X. Yang B.Y. Pinal N. Walikonis R. De Blas A.L. J. Biol. Chem. 2004; 279: 38978-38990Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). ABP-L, also called GRIP2 (25Dong H. Zhang P. Song I. Petralia R.S. Liao D. Huganir R.L. J. Neurosci. 1999; 19: 6930-6941Crossref PubMed Google Scholar, 28Bruckner 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 (308) Google Scholar), and the short form ABP (29Srivastava 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 (325) Google Scholar) are highly homologous to GRIP1 but encoded by a different gene. GRIP1b and pABP-L are palmitoylated (26Yamazaki M. Fukaya M. Abe M. Ikeno K. Kakizaki T. Watanabe M. Sakimura K. Neurosci. Lett. 2001; 304: 81-84Crossref PubMed Scopus (56) Google Scholar, 30DeSouza S. Fu J. States B.A. Ziff E.B. J. Neurosci. 2002; 22: 3493-3503Crossref PubMed Google Scholar) and associated with the plasma membrane, whereas the non-palmitoylated forms are associated with internal vesicular structures. GRIP1a has numerous binding partners and can form large macromolecular complexes. PDZ 4–5 bind the prolactin-releasing peptide receptor as well as GluR2 and GluR3, and these receptors might compete for this same binding site (31Lin S.H. Arai A.C. Wang Z. Nothacker H.P. Civelli O. Mol. Pharmacol. 2001; 60: 916-923Crossref PubMed Scopus (39) Google Scholar). PDZ 6 interacts with liprin-α (32Wyszynski 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 (214) Google Scholar) and ephrinB, EphB2, and EphA7 (33Torres 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 (400) Google Scholar, 28Bruckner 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 (308) Google Scholar, 34Lin D. Gish G.D. Songyang Z. Pawson T. J. Biol. Chem. 1999; 274: 3726-3733Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). The linker region between PDZ 6 and 7 binds to the motor protein kinesin (35Setou M. Seog D.H. Tanaka Y. Kanai Y. Takei Y. Kawagishi M. Hirokawa N. Nature. 2002; 417: 83-87Crossref PubMed Scopus (412) Google Scholar, 36Hoogenraad C.C. Milstein A.D. Ethell I.M. Henkemeyer M. Sheng M. Nat. Neurosci. 2005; 8: 906-915Crossref PubMed Scopus (179) Google Scholar). PDZ 7 interacts with GRASP-1, a neuronal RasGEF (37Ye B. Liao D. Zhang X. Zhang P. Dong H. Huganir R.L. Neuron. 2000; 26: 603-617Abstract Full Text Full Text PDF PubMed Google Scholar). We have previously shown that NEEP21 interacts with a carboxyl-terminal site of GRIP1 and forms a regulated complex with GRIP1, GluR2, and the endosomal trafficking SNARE protein syntaxin 13 (38Steiner P. Alberi S. Kulangara K. Yersin A. Sarria J.C. Regulier E. Kasas S. Dietler G. Muller D. Catsicas S. Hirling H. EMBO J. 2005; 24: 2873-2884Crossref PubMed Scopus (91) Google Scholar). NEEP21 is localized to endosomes in the soma and dendritic shafts and is essential for correct receptor recycling and synaptic transmission (39Steiner P. Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar, 40Debaigt C. Hirling H. Steiner P. Vincent J.P. Mazella J. J. Biol. Chem. 2004; 279: 35687-35691Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 41Alberi S. Boda B. Steiner P. Nikonenko I. Hirling H. Muller D. Mol. Cell. Neurosci. 2005; 29: 313-319Crossref PubMed Scopus (49) Google Scholar). Previous studies proposed a possible phosphorylation of GRIP1 (28Bruckner 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 (308) Google Scholar, 31Lin S.H. Arai A.C. Wang Z. Nothacker H.P. Civelli O. Mol. Pharmacol. 2001; 60: 916-923Crossref PubMed Scopus (39) Google Scholar). Here we show that GRIP1a is indeed phosphorylated on serine 917 in the linker region between amino acids (aa) 881 and 933. This phosphorylation modulates the binding of GRIP1 to NEEP21 and alters surface expression of GluR2. Antibodies—Antibodies against the following proteins were applied for Western blotting (W), immunofluorescence (IF), or immunoprecipitation: monoclonal antibodies GRIP (W, 1:10000; IF, 1:300; BD Biosciences), GluR2 (W, 1:100; IF, 1:10, BD Biosciences), actin (W 1:2000, Chemicon, Temecula, CA); polyclonal antibodies GluR1 (IF, 1:5, Calbiochem), GluR2/3 (W 1:1000, Chemicon), NEEP21 (W 1:6000; IF, 1:300 (39Steiner P. Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar)). We used the following secondary antibodies: W, horseradish peroxidase-coupled goat anti-rabbit or anti-mouse IgG (W, 1:2000, Calbiochem) or Alexa Fluor®-coupled goat anti-rabbit or anti-mouse IgG (W, 1:2000, Molecular Probes, Inc., Eugene, OR); IF, Cy3-coupled (IF, 1:200; Jackson ImmunoResearch, West Grove, PA), Oregon Green-coupled (IF, 1:200; Molecular Probes), Alexa Fluor® 594-coupled (IF, 1:200; Molecular Probes) anti-mouse or anti-rabbit IgG. DNA Constructs and Glutathione S-Transferase (GST) Fusion Protein Expression—For bacterial expression of recombinant fusion proteins between GST and the indicated GRIP1a domains, DNA fragments were amplified by PCR on the cDNA encoding full-length GRIP1a (kindly provided by Dr. R. Huganir, Baltimore, MD, herein called GRIP) and subcloned between SmaI and XbaI of pGex-KG. The plasmid pGex-4T1-GST-GluR2-short, encoding a fusion protein between GST and the cytosolic domain of GluR2 (GST-GluR2ct), was kindly provided by Dr. H. Hirbec, Montpellier, France. The fusion protein GST-NEEP21-(104–185) has been described (38Steiner P. Alberi S. Kulangara K. Yersin A. Sarria J.C. Regulier E. Kasas S. Dietler G. Muller D. Catsicas S. Hirling H. EMBO J. 2005; 24: 2873-2884Crossref PubMed Scopus (91) Google Scholar). Fusion proteins were expressed in Escherichia coli BL21 and bound to glutathione-agarose beads (Sigma-Aldrich). For GST-pull-down assays beads were equilibrated in buffer B (20 mm HEPES/KOH, pH 7.4, 2 mm EDTA, 2 mm EGTA, 0.1 mm DTT, 0.1 m KCl, 1% Triton X-100) and for in vitro kinase assay in phosphorylation buffer (20 mm HEPES/KOH, pH 7.4, 10 mm MgCl2, 50 μm ATP). Cell Culture and Transfection—Hippocampal neurons were prepared from P0 Sprague-Dawley rats as previously described (38Steiner P. Alberi S. Kulangara K. Yersin A. Sarria J.C. Regulier E. Kasas S. Dietler G. Muller D. Catsicas S. Hirling H. EMBO J. 2005; 24: 2873-2884Crossref PubMed Scopus (91) Google Scholar). Neurons were plated either on plastic dishes coated with poly-d-lysine (5 μg/cm2; BD Biosciences) and laminin (0.7 μg/cm2; Invitrogen) at 23,000 cells/cm2 for biochemical experiments or on poly-d-lysine/laminin-coated borosilicate glass coverslips (Marienfeld GmbH, Lauda-Königshofen, Germany) at 12,500 cells/cm2 for immunocytochemistry. Neurons were transfected at days in vitro 10 with pcDNA3-GFP-GRIP1-(810–1112) (38Steiner P. Alberi S. Kulangara K. Yersin A. Sarria J.C. Regulier E. Kasas S. Dietler G. Muller D. Catsicas S. Hirling H. EMBO J. 2005; 24: 2873-2884Crossref PubMed Scopus (91) Google Scholar) using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol. HEK293T cells were cultured as previously described (38Steiner P. Alberi S. Kulangara K. Yersin A. Sarria J.C. Regulier E. Kasas S. Dietler G. Muller D. Catsicas S. Hirling H. EMBO J. 2005; 24: 2873-2884Crossref PubMed Scopus (91) Google Scholar) and transfected using the calcium phosphate technique. Immunoprecipitation, Surface Biotinylation, and Cell Fractionation—Preparation of brain membrane extracts from postnatal day 10 rats and covalent cross-linking of antibodies to protein A or G beads were done as previously described (38Steiner P. Alberi S. Kulangara K. Yersin A. Sarria J.C. Regulier E. Kasas S. Dietler G. Muller D. Catsicas S. Hirling H. EMBO J. 2005; 24: 2873-2884Crossref PubMed Scopus (91) Google Scholar). Antibody beads (10 μl) were incubated with membrane extract (2 mg) for 4 h and washed 3 times with buffer B (20 mm HEPES/KOH, pH 7.4, 2 mm EDTA, 2 mm EGTA, 0.1 mm dithiothreitol, 0.1 m KCl, 1% Triton X-100). Bound proteins were either glycine-eluted and neutralized by Tris-buffer for in vitro kinase assays, or sample buffer was added for Western blotting. For GST pulldown experiments 4 dishes of neurons were treated with the indicated drugs at 37 °C, washed in phosphate-buffered saline, and lysed in buffer B. The extract was incubated for 2 h at 4 °C with GST fusion protein beads. After washing bound proteins were analyzed by Western blots. Surface biotinylation was done as previously described (47Chung H.J. Xia J. Scannevin R.H. Zhang X. Huganir R.L. J. Neurosci. 2000; 20: 7258-7267Crossref PubMed Google Scholar, 48Snyder E.M. Philpot B.D. Huber K.M. Dong X. Fallon J.R. Bear M.F. Nat. Neurosci. 2001; 4: 1079-1085Crossref PubMed Scopus (459) Google Scholar). Briefly, cells were biotinylated with 1 mg/ml sulfo-NHS-biotin (4 °C, 30 min). Cells were then lysed, and biotinylated proteins were precipitated with streptavidin-linked beads. For fractionation of hippocampal neurons, post-nuclear supernatants were prepared by cell lysis in buffer B without detergent by 10 strokes through a G25 needle followed by a 1000 × g centrifugation. It was further separated into a cytosolic fraction by centrifugation for 60 min at 100,000 × g. To recover the membrane fraction, the pellet was rehomogenized, solubilized by 1% Triton X-100, and submitted to another 100,000 × g centrifugation. In Vitro Kinase Assay—Fusion proteins were immobilized on glutathione-agarose beads and incubated for 8 min at 30 °C in the presence of [γ-32P]ATP (10 μCi, Amersham Biosciences) in phosphorylation buffer with either 10 μg of neuron extract or eluted proteins from IgG or anti-NEEP21-immunoprecipitations. When neuron extracts were used cells were either untreated or incubated with 100 nm okadaic acid (OA) or 1 μm calphostin C or 1 μm PMA for 1 h. Stimulation with 100 μm AMPA or 50 μm NMDA was for 3 min followed by further incubation for 7 or 17 min. When immune pellets were used, PMA (1 μm) was added to the phosphorylation reaction where indicated. Beads were then washed once with phosphorylation buffer, and bound proteins were separated on SDS-PAGE and stained with Coomassie Blue (Sigma-Aldrich), and the dried gel was exposed to Kodak X-Omat film (Amersham Biosciences). Interaction Force Measurement by Atomic Force Microscopy (AFM)—AFM tips and substrates were prepared as previously described (42Yersin A. Hirling H. Steiner P. Magnin S. Regazzi R. Huni B. Huguenot P. De Los Rios P. Dietler G. Catsicas S. Kasas S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8736-8741Crossref PubMed Scopus (74) Google Scholar). Briefly, GST fusion proteins (200–300 ng/μl) were covalently cross-linked by glutaraldehyde (0.5%) to the AFM tip (Digital Instruments, Santa Barbara; nominal spring constant 0.06 newton/m, calibrated by thermal noise analysis) and to a freshly cleaved mica sheet that had been functionalized by aminopropyltriethoxysilane. This method had previously been verified to keep the functionality of proteins intact (43Allen S. Chen X. Davies J. Davies M.C. Dawkes A.C. Edwards J.C. Roberts C.J. Sefton J. Tendler S.J. Williams P.M. Biochemistry. 1997; 36: 7457-7463Crossref PubMed Scopus (316) Google Scholar). Experiments were performed at room temperature on a Nanoscope III® (Digital Instruments) with force volume mode operating in Tris-buffered saline or injected neuron cell extract in phosphorylation buffer containing 300 μm ATP at a constant retraction speed of 355 nm/s. Force curves were analyzed off-line by a fuzzy logic algorithm developed in our laboratory (44Kasas S. Riederer B.M. Catsicas S. Cappella B. Dietler G. Rev. Sci. Instrum. 2000; 71: 2082-2086Crossref Scopus (42) Google Scholar). Immunocytochemistry—Okadaic acid (OA)-treated or transfected neurons were fixed in 4% paraformaldehyde, 4% sucrose for 5 min for surface staining or for 12 min for intracellular staining. For surface staining an extracellularly binding anti-GluR2 or anti-GluR1 antibody was added in the absence of detergent. For intracellular staining antibodies were added in presence of 0.3% Triton X-100 for 2 h. Neurons were washed in 66 mm NaCl, 150 mm Tris HCl, pH 7.4, and secondary antibodies were added for 1 h. After washing coverslips were mounted in 50% glycerol containing Mowiol (Fluka) and DABCO (1,4-diazabicyclo(2.2.2)octane; Sigma-Aldrich) to retard photobleaching. Quantitative Image Analysis—For quantification of surface GluR1 and GluR2 labeling, 10 confocal sections were acquired with identical parameters; an average projection was applied resulting in the average fluorescence intensity for each pixel. The fluorescence intensity was quantified on the cell body. A threshold of 80 was applied, and the red pixels summed using Metamorph software. The sum of the red pixels was divided by the size of the perimeter of the cells to normalize for different cell sizes. Colocalization was defined as the pixels that are positive for both GluR2 (see Fig. 1E, red) and NEEP21 (green). 100% in Fig. 1E corresponds to the sum of pixels that are red or green. The following numbers of cells n were analyzed in Fig. 1: for GluR1: control, 29; 1 h, 26; 2 h, 16; 3 h, 16; for GluR2: control, 71; 1 h, 47; 2 h, 87; 3 h, 45; for colocalization: control, 63; OA, 63. Site-directed Mutagenesis—In vitro site-directed mutagenesis was performed using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. Template DNA was either full-length rat GRIP1 or GFP-GRIP1-(810–1112). The antisense primers for the different mutated sites were as follows: S930A, 5′-CTGTCGCCCCAGCTGAGCGCGAGCCGTTGGAGC-3′; T927A, 5′-GCTGACTGCGAGCCGCTGGAGCCTCATGATTC-3′; S920A, 5′-GAGCCTCATGATTCAAAGCCATAGTGCTCCCCGAC-3′; T918A, 5′-CATGATTCAAACTCATAGCGCTCCCCGACATGATTG-3′; S917A, 5′-CATGATTCAAACTCATAGTGGCCCCCGACATGATTGTTGC-3′; S917D, 5′-CATGATTCAAACTCATAGTGTCCCCCGACATGATTGTTGC; S915A, 5′-CTCATAGTGCTCCCCGCCATGATTGTTGCCTCG-3′; T912A, 5′-CTCCCCGACATGATTGCTGCCTCGAGCTCTCTC-3′; S903A, 5′-CTCTCAGGATTCCCGCCTGGCCGCAGGTCTC-3′; T899A, 5′-CCCGACTGGCCGCAGGCCTCCAGGTCCTCCAATG-3′; S891A, 5′-GTCCTCCAATGCTTGAGCCCAGAAGTTTTCCTC-3′. The Phosphatase Inhibitor Okadaic Acid Decreases Surface GluR2 in Hippocampal Neurons—Endocytosis of AMPAR and subsequent sorting to either degradation or recycling back to the plasma membrane depends on differential stimuli and protein phosphatase cascades (18Lee S.H. Simonetta A. Sheng M. Neuron. 2004; 43: 221-236Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 24Ehlers M.D. Neuron. 2000; 28: 511-525Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar). To analyze the implication of phosphorylation in AMPAR surface expression, we treated hippocampal neurons at days in vitro 14 with the protein phosphatase 1 and 2A inhibitor OA. Then we performed surface labeling using extracellularly binding antibodies for the AMPAR subunits GluR1 or GluR2 (Fig. 1, A and B, respectively, upper row; corresponding DIC images are on the lower row). After quantitative image analysis, we found that treatment for 1 h with 100 nm OA resulted in a strong reduction of surface GluR2 (62.6%) compared with control untreated neurons (100%; p < 0.01). This lower GluR2 level persisted upon prolonged treatments of 2 or 3 h (Fig. 1C, black bars). Interestingly, the same OA treatments had no significant effect on the surface expression of GluR1 (Fig. 1C, gray bars). The total GluR2 content of the neurons was not altered, as verified by Western blotting of OA-treated neurons (Fig. 1D), excluding an effect on gene expression or degradation. Instead, this result indicated a change in GluR2 trafficking between the neuron surface and intracellular compartments. Costaining for the presynaptic marker synaptophysin indicated that GluR2 was localized to synaptic as well as extra-synaptic sites, and this distribution did not change upon OA treatment (data not shown). To examine the site to which GluR2 redistributed after OA treatment, we performed double-immunolabeling for GluR2 and NEEP21. The latter protein of neuronal early endosomes associates with the endosomal SNARE protein syntaxin 13, the scaffolding protein GRIP, and GluR2, and it regulates GluR2 sorting (38Steiner P. Alberi S. Kulangara K. Yersin A. Sarria J.C. Regulier E. Kasas S. Dietler G. Muller D. Catsicas S. Hirling H. EMBO J. 2005; 24: 2873-2884Crossref PubMed Scopus (91) Google Scholar, 39Steiner P. Sarria J.C. Glauser L. Magnin S. Catsicas S. Hirling H. J. Cell Biol. 2002; 157: 1197-1209Crossref PubMed Scopus (94) Google Scholar). OA treatment significantly increased the colocalization between GluR2 and NEEP21 compared with control neurons (Fig. 1E; from 5.4 to 12.6%; p > 0.01). These results indicate that OA treatment causes a shift from surface GluR2 to intracellular GluR2. It also suggests that protein dephosphorylation is involved in trafficking of GluR2-containing AMPAR. The Interaction between GRIP and NEEP21 Is Altered by OA Treatment of Neurons—It has been suggested previously that phosphorylation of GRIP might regulate targeting of surface receptors (28Bruckner 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 (308) Google Scholar, 31Lin S.H. Arai A.C. Wang Z. Nothacker H.P. Civelli O. Mol. Pharmacol. 2001; 60: 916-923Crossref PubMed Scopus (39) Google Scholar). To assess whether OA treatment leads to a modification of GRIP correlating with the change in surface GluR2 localization, we analyzed the electrophoretic mobility of GRIP in total extract of OA-treated hippocampal neurons by Western blot. After 1 h of exposure, OA induced a dose-dependent retardation of GRIP migration consistent with a potential GRIP phosphorylation (Fig. 2A). We then analyzed by GST pulldown assays whether this OA-induced, slower-migrating form of GRIP has altered binding to immobilized fusion protein between GST and the cytosolic domain of NEEP21 spanning amino acids 104–185 (GST-NEEP21ct). GRIP was efficiently pulled down by GST-NEEP21ct beads from extracts of non-treated neurons and extracts of neurons treated with 10 nm OA (Fig. 2B). In contrast, treatment of neurons with 100 nm or 1 μm OA strongly decreased the binding of GRIP to NEEP21ct in this in vitro assay (Fig. 2B). No binding was observed to glutathione beads bound to a negative control of GST alone. These results suggest that the mechanism underlying the effect of OA treatment on GluR2 surface expression includes altered association of GRIP to NEEP21. The altered binding between NEEP21 and GRIP1 upon OA treatment of neurons was further analyzed by on-line AFM recordings. AFM allows a direct measurement of the number of binding events and the relative interaction forces between a protein on the tip of the AFM cantilever and another protein on the mica surface (42Yersin A. Hirling H. Steiner P. Magnin S. Regazzi R. Huni B. Huguenot P. De Los Rios P. Dietler G. Catsicas S. Kasas S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8736-8741Crossref PubMed Scopus (74) Google Scholar, 45Florin E.L. Moy V.T. Gaub H.E. Science. 1994; 264: 415-417Crossref PubMed Scopus (1735) Google Scholar). We have previously shown that NEEP21ct binds to a site located in the carboxyl-terminal aa 810–1112 of GRIP1, and our previous AFM measurements yielded an interaction force of around 111 piconewtons (38Steiner P. Alberi S. Kulangara K. Yersin A. Sarria J.C. Regulier E. Kasas S

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