1. Modulation of NMDA Receptor Dependent Calcium Influx and Gene Expression Through EphB Receptors by Mari A. Takasu, Matthew B. Dalva, Richard E. Zigmond, Michael E. Greenberg BIPN 148 Group 7 D. Lam, M. Pineda, Y. M. Kim, K. Mapalad, B. Lyle, J. Langber, T. McGill, T. Lau

2. Background NMDA receptors: multi-protein complex NMDA receptors: multi-protein complex Phosphorylation of Tyrosine on NR2 subunit by a protein of the Src family Phosphorylation of Tyrosine on NR2 subunit by a protein of the Src family Occurs during development and plasticity Occurs during development and plasticity EphB receptors: EphB receptors: - activated by ephrinB ligands - interacts with NMDARs

3. Does EphrinB2 enhance calcium conductance through NMDARs? Methods Cortical neurons from embryonic rats were assessed for change in [Ca 2+ ] with Ca 2+ indicator dye using Fura-2AM Cortical neurons from embryonic rats were assessed for change in [Ca 2+ ] with Ca 2+ indicator dye using Fura-2AM Treatment: Treatment: 1. Glutamate stimulation 2. EphrinB

4. Results

5. Discussion Does EphrinB2 enhance calcium conductance through NMDARs? Yes, EphrinB2 treated cultures enhances [Ca 2+ ] influx through NMDARs. Yes, EphrinB2 treated cultures enhances [Ca 2+ ] influx through NMDARs. NMDARs are required because treatment with APV reduces [Ca 2+ ] influx. NMDARs are required because treatment with APV reduces [Ca 2+ ] influx.

6. Is the cytoplasmic domain of EphB2 required for NMDA-mediated Ca 2+ influx? Methods Neurons were transfected with: Neurons were transfected with: 1. EphB2WT: wild-type 2. EphB2TR: cytoplasmic domain deleted Tagged with GFP visualized with FM Tagged with GFP visualized with FM Glutamate stimulation Glutamate stimulation

7. Results

8. Discussion Is the cytoplasmic domain of EphB2 required for NMDA-mediated Ca 2+ influx? Yes. Cytoplasmic domain of EphB2 contains the tyrosine kinase domain which is required for phosphorylation of Src. Cytoplasmic domain of EphB2 contains the tyrosine kinase domain which is required for phosphorylation of Src. Src phosphorylation triggers Ca 2+ influx through phosphorylation of NR2B (NMDARs) Src phosphorylation triggers Ca 2+ influx through phosphorylation of NR2B (NMDARs) Cytoplasmic domain is required to activate downstream events that leads to increase in Ca 2+ influx. Cytoplasmic domain is required to activate downstream events that leads to increase in Ca 2+ influx.

9. Does phosphorylation of NR2B at 1252, 1336, and 1472 by Src affect Ca 2+ influx through the NMDARs? Methods Expression of NR1 with: Expression of NR1 with: 1. Control: NR2BWT: wild-type 2. Mutated NR2B Y1252F Y1336F,Y1472F 3. Triple Mutant [(TM)Y1252F/Y1336F/Y1472F] Glutamate stimulation Glutamate stimulation Western blot analysis with antibodies recognize phosphorylated tyrosine Western blot analysis with antibodies recognize phosphorylated tyrosine

10. Results

11. Discussion Does phosphorylation of NR2B at 1252, 1336, and 1472 by Src affect Ca 2+ influx through the NMDARs? Yes. Phosphorylation of tyrosine residues at the three sites enhances Ca 2+ influx Phosphorylation of tyrosine residues at the three sites enhances Ca 2+ influx Activation of B2WT and NR2B showed a large increase in [Ca 2+ ] Activation of B2WT and NR2B showed a large increase in [Ca 2+ ]

12. Does the Src family of kinases affect the phosphorylation of NR2B? Treatment of neurons: Treatment of neurons: 1. PP2: Src family inhibitor 2. PP3: inactive analog of PP2 (control) 3. SU 6656: Src family inhibitor Cortical neurons were treated with aggregated ephrinB2-Fc, or BDNF Cortical neurons were treated with aggregated ephrinB2-Fc, or BDNF Using antibody specific for Phosph-Tyr416, Src levels was assessed Using antibody specific for Phosph-Tyr416, Src levels was assessed Phosphorylation of Tyr416 is indicative of Src activation.

13. Results

14. Discussion Does the Src family of kinases affect the phosphorylation of NR2B? Yes! Src family inhibitor (i.e. PP2, SU6656) reduces NR2B Tyr phosphorylation leading to low levels of Ca 2+ influx Src family inhibitor (i.e. PP2, SU6656) reduces NR2B Tyr phosphorylation leading to low levels of Ca 2+ influx PP3, an inactive analog of PP2, did not inhibit Src function PP3, an inactive analog of PP2, did not inhibit Src function

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