Modulation of NMDA Receptor Dependent Calcium Influx and Gene Expression Through EphB Receptors

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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
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Background

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

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Does EphrinB2 enhance calcium conductance through
NMDARs?

• Methods
• Cortical neurons from embryonic rats were
  assessed for change in Ca2 with Ca2 indicator
  dye using Fura-2AM
• Treatment
• 1. Glutamate stimulation
• 2. EphrinB

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Results
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Discussion

• Does EphrinB2 enhance calcium conductance
  through NMDARs?
• Yes, EphrinB2 treated cultures enhances Ca2
  influx through NMDARs.
• NMDARs are required because treatment with APV
  reduces Ca2 influx.

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Is the cytoplasmic domain of EphB2 required for
NMDA-mediated Ca2 influx?

• Methods
• Neurons were transfected with
• 1. EphB2WT wild-type
• 2. EphB2TR cytoplasmic domain deleted
• Tagged with GFP visualized with FM
• Glutamate stimulation

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Results
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Discussion

• Is the cytoplasmic domain of EphB2 required for
  NMDA-mediated Ca2 influx?Yes.
• Cytoplasmic domain of EphB2 contains the tyrosine
  kinase domain which is required for
  phosphorylation of Src.
• Src phosphorylation triggers Ca2 influx through
  phosphorylation of NR2B (NMDARs)
• Cytoplasmic domain is required to activate
  downstream events that leads to increase in Ca2
  influx.

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Does phosphorylation of NR2B at 1252, 1336, and
1472 by Src affect Ca2 influx through the NMDARs?

• Methods
• Expression of NR1 with
• 1. Control NR2BWT wild-type
• 2. Mutated NR2B Y1252F Y1336F,Y1472F
• 3. Triple Mutant (TM)Y1252F/Y1336F/Y1472F
• Glutamate stimulation
• Western blot analysis with antibodies recognize
  phosphorylated tyrosine

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Results
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Discussion

• Does phosphorylation of NR2B at 1252, 1336, and
  1472 by Src affect Ca2 influx through the
  NMDARs?
• Yes.
• Phosphorylation of tyrosine residues at the three
  sites enhances Ca2 influx
• Activation of B2WT and NR2B showed a large
  increase in Ca2

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Does the Src family of kinases affect the
phosphorylation of NR2B?

• 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
• Using antibody specific for Phosph-Tyr416, Src
  levels was assessed
• Phosphorylation of Tyr416 is indicative of Src
  activation.

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Results
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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
  Ca2 influx
• PP3, an inactive analog of PP2, did not inhibit
  Src function

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Movie