Serine phosphorylation of ephrinb2 regulates trafficking of synaptic

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1 Serine phosphorylation of ephrinb2 regulates trafficking of synaptic AMPA receptors Clara L. Essmann, Elsa Martinez, Julia C. Geiger, Manuel Zimmer, Matthias H. Traut, Valentin Stein, Rüdiger Klein and Amparo Acker-Palmer SUPPLEMENTARY INFORMATION Supplementary Figure 1. EphrinB2 reverse signaling inhibits GluR1 endocytosis. (a) Endocytosis of AMPA receptors under various stimulation conditions were visualized and quantified by the antibody feeding assay. GluR1 that remains at the surface is labeled in green and internalized in red. Scale bar, 5 µm. (b) Quantification of GluR1 internalization based on fluorescence intensities, pictured as the percentage of internalized GluR1 (red) versus total GluR1 (red + green) (SEM, *** P < ).

2 2 Supplementary Figure 2. Characterization of eb2ko. (a, b) EphrinB2 protein is absent in NestinCre+; eb2lox/lox (eb2ko) mice. Clustering of ephrinb2 assessed by the binding of EphB4-Fc to surface ephrinb2 showed no staining in hippocampal neurons isolated from eb2ko (a). EphB4-Fc pulldowns from mouse cortex E17 show absence of ephrinb2 protein in eb2ko (b). (c) Lack of ephrinb2 does not affect the number of mature synapses in cultured hippocampal neurons (15DIV). Quantification of PSD95 and synaptophysin clusters per 100 μm dendrite stretches in hippocampal neurons from eb2ko and control littermates.

3 3 Supplementary Figure 3. EphrinB2 and PICK1 do not co-localize at the surface in hippocampal neurons. Neurons were stimulated with EphB4-Fc and ephrinb2 clusters at the surface detected with anti-human Fc (green) before permeabilization. Co-localization with PICK1 or GRIP1 (red) was assessed after permeabilization of the cells. EphrinB2 clusters significantly co-localized with GRIP1 but not with PICK1. Scale bar, 5 µm.

4 4 Supplementary Figure 4. Overexpression of GRIP1-PDZ7 does not interfere with the ephrinb2-mediated stabilization of AMPA receptors. (a, b) Levels of AMPA receptor internalization in hippocampal neurons expressing GRIP1-PDZ7 and in control cells under various stimulation conditions were visualized and quantified using the antibody feeding assay. GluR2 that remains on the cell surface is indicated in green, internalized GluR2 in red (a). Quantification of AMPA receptor internalization in PDZ7-CFP expressing versus control transfected hippocampal neurons shown representatively in a. Quantification was done as in Fig. 1b (SEM, *** P < ) (b) Scale bars, 20 µm (whole neurons), 5 µm (enlargements).

5 5 Supplementary Figure 5. Efficient clustering of mutant ephrinb2 forms. CFP-ephrinB2 WT, CFP-ephrinB2 S9>A and CFP-ephrinB2 S9>E are expressed properly at the membrane and are able to form functional clusters upon binding to the EphB receptors. Cluster formation assay is monitored by the binding of EphB4-Fc to the surface ephrinb2. Scale bar, 10 µm.

6 6 Supplementary Figure 6. Serine phosphorylation of EphB receptors regulates GRIP binding. (a) Schematic C-terminal sequence of EphB2 receptor. In boxes are highlighted the PDZ target site as well as the adjacent serine residue. (b) Binding of GRIP to EphB2 is regulated by ligand engagement. HeLa cells transfected with Flag-EphB2 together with myc-grip were stimulated with pre-clustered ephrinb1-fc for 10 and 60 min or with pre-clustered Fc control for 10 min. Cell lysates were immunoprecipitated with anti-flag antibodies and analysed by Western Blot using anti-flag and anti-myc antibodies. (c) Binding of GRIP to EphB2 is regulated by serine phosphorylation. HeLa cells transfected with Flag-EphB2 or Flag-EphB2 S-4>A mutant together with myc-grip were stimulated as in b. Cell lysates were immunoprecipitated with anti-ephb2 antibodies and analysed by Western Blot using anti-ephb2 and anti-myc antibodies.

7 Supplementary methods Antibodies and expression constructs. The following primary antibodies were used: rabbit anti-glur2 (IP 5μg) (Chemicon), rabbit anti-phosphoserine 880 GluR2 (WB 1:5000) (Upstate), rabbit anti-glur1 (IF 1:50) (Calbiochem), rabbit anti-synaptophysin (IF 1:200) (Synaptic Systems), rabbit anti-grip1 (IF 1:200; WB 1:1000) (Upstate), rabbit anti-ephrinb (IP 5μl; WB 1:1000) (anti-lerk2a) 1 rabbit anti-ephrinb1 C-18 (IF 1:100) (Santa Cruz), rabbit anti-ephb2 (WB 1:1000) 2, rabbit anti-phospho-ephrinb (WB 1:500) 3, mouse anti-grip1 (IF 1:200; WB 1:1000) (Chemicon), mouse anti- Phosphotyrosine 4G10 (WB 1:1000) (Upstate), mouse anti-flag (WB 1:1000), mouse anti- N-Cadherin (WB 1:1000), mouse anti-psd95 (IF 1:200), mouse anti-myc (WB 1:1000) (Sigma), mouse anti-ha (WB 1:1000) (Roche), mouse anti-glur2 (IF 1:500; WB 1:1000), mouse anti-map2 (WB 1:1000) (Chemicon), mouse anti-β-tubulin (WB 1:2000) (Covance), mouse anti-src 2-17 (1:100) 4, goat anti-ephrinb1 (WB 1:1000), goat anti-ephrinb2 (WB 1:1000) (R&D), goat anti-human Fc (1:50) (Jackson Immuno Research). The secondary antibodies used include Alexa Fluor 488-conjugated goat antimouse and goat anti-rabbit (IF 1:100) (Molecular Probes), Cy2-, Cy3-, Cy5-, AMCA-, Texas Red- conjugated donkey anti-mouse and donkey anti-rabbit antibodies (IF 1:100) and Cy2-conjugated goat anti-human Fc antibody (IF 1:50) (Jackson ImmunoResearch). For Western blot analysis, the secondary antibodies used were HRP-conjugated goat antimouse, donkey anti-goat and goat anti-rabbit IgG (1:3000) (Jackson ImmunoResearch). Expression constructs of HA-ephrinB1, HA-ephrinB1-ΔC and HA-ephrinB1-6F were generated by subcloning from pjp104, pjp105 5 and pkb21, respectively, into pcdna3.1/hygro using EcoRI and XhoI restriction sites. HA-ephrinB1 S-9>A and HA-

8 8 ephrinb1 S-9>E were generated from HA-ephrinB1 by site directed mutagenesis (Stratagene). CFP-ephrinB2 WT was generated as described 3. CFP-ephrinB2 S-9>A and CFP-ephrinB2 S-9>E were generated from CFP-ephrinB2 WT by site directed mutagenesis. Myc-GRIP2 was generated as described 5. GRIP1-PDZ6-CFP was generated by subcloning from pjp127 5 into pecfp-n1 (Clontech) using EcoRI and XhoI restriction enzymes. GRIP1-PDZ7-CFP was generated by cloning the PCR product of GRIP1 sequence from pjp127 5 with flanking HindIII and KpnI restriction sites into pecfp-c1 (Clontech). Flag-EphB2 has been described 6. Flag-EphB2 S-4>A was generated from Flag-EphB2 by site directed mutagenesis. A bacterial expression construct encoding the cytoplasmic domain of ephrinb1 (cytob1) fused to GST was described 7. GST-cytoB1 S-9>A and GST-cytoB1 S-26>A were generated from GSTcytoB1 by site directed mutagenesis. Antibody Feeding Assay Live hippocampal neurons 15-21DIV were blocked at 37 C for 10 min in blocking solution, incubated with the primary antibody anti-glur2 (AA ; 1:500) (Chemicon) or GluR1( AA ; 1:50) (Calbiochem) for 18 min, washed with warm D-PBS + Ca 2+ Mg 2+ and stimulated with human Fc or EphB4-Fc in neurobasal medium at 37 C for 1 hr. 100 µm AMPA was added in the last 10 min prior fixation. For NMDA stimulation 50 µm NMDA was added to the cells for 2 min. Cells were incubated with Alexa Fluor 488- or Cy2- conjugated secondary antibodies for 2 hr at room temperature to detect pre-labeled surface receptors. After washing three times 5 min with PBS, neurons were permeabilized for 4 min with ice cold 0.1% Triton X-100 in PBS, blocked

9 9 again for 30 min and finally incubated with a Cy3-conjugated secondary antibody to visualize pre-labeled internalized receptors. Electrophysiology- Patch-clamp recordings Miniature excitatory postsynaptic currents (mepsc) were recorded from dissociated hippocampal neurons (15-19 DIV) at room temperature (20-22 C). The recording chamber was continuously perfused with carbogenated artificial cerebro-spinal fluid (ACSF) that contained (in mm): 119 NaCl; 2.5 KCl; 1.3 MgSO 4 ; 2.5 CaCl 2 ; 1 NaH 2 PO 4 ; 26.2 NaHCO 3 and 11.1 D-glucose supplemented with 100 µm picrotoxin (PTX), 100 µm D(-)-2-Amino-5-phosphonovaleric acid (APV) and 200 nm tetrodotoxin (TTX). Membrane currents were recorded at a holding potential of -70 mv using a MultiClamp 700B amplifier (Molecular Devices). Patch pipettes (WPI) with a resistance of MΩ were filled with an internal solution containing (in mm): 150 CsGluc; 10 HEPES; 2 MgATP; 0.2 EGTA; 5 QX314; 8 NaCl, 290 mosm, ph 7.2. Signals were filtered at 2 khz and digitized at 5 khz via a Digidata 1440A digitizer (Axon Instruments, Molecular Devices). Data were collected using Clampex 10.1 and analyzed with Clampfit 10.1 software (Axon Instruments, Molecular Devices). Only cells with a series resistance lower than 25 MΩ and a noise level lower than 10 pa were analyzed. 80 events per cell were recorded. Cells with very high mepsc frequencies were excluded. REFERENCES 1. Bruckner, K., Pasquale, E. B. & Klein, R. Tyrosine phosphorylation of transmembrane ligands for Eph receptors. Science (New York, N.Y 275, (1997). 2. Grunwald, I. C. et al. Kinase-independent requirement of EphB2 receptors in hippocampal synaptic plasticity. Neuron 32, (2001).

10 3. Lauterbach, J. & Klein, R. Release of full-length EphB2 receptors from hippocampal neurons to cocultured glial cells. J Neurosci 26, (2006). 4 Palmer, A. et al. EphrinB phosphorylation and reverse signaling: regulation by Src kinases and PTP-BL phosphatase. Mol Cell. 9, (2002). 5. Brückner, K. et al. EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains. Neuron 22, (1999). 6 Dalva, M. B. et al. EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 103, (2000). 7 Brambilla, R. et al. Membrane-bound LERK2 ligand can signal through three different Eph-related receptor tyrosine kinases. The EMBO journal 14, (1995). 10