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

by Mari A. Takasu, Matthew B. Dalva, Richard E. by Mari A. Takasu, Matthew B. Dalva, Richard E. Zigmond, Michael E. GreenbergZigmond, Michael E. Greenberg

BIPN 148BIPN 148Group 7Group 7

D. Lam, M. Pineda, Y. M. Kim, K. Mapalad, D. Lam, M. Pineda, Y. M. Kim, K. Mapalad, B. Lyle, J. Langber, T. McGill, T. LauB. Lyle, J. Langber, T. McGill, T. Lau

BackgroundBackground

NMDA receptors: multi-protein complexNMDA receptors: multi-protein complex Phosphorylation of Tyrosine on NR2 Phosphorylation of Tyrosine on NR2

subunit by a protein of the Src familysubunit by a protein of the Src family Occurs during development and plasticityOccurs during development and plasticity EphB receptors: EphB receptors:

- activated by ephrinB ligands- activated by ephrinB ligands

- interacts with NMDARs- interacts with NMDARs

Does EphrinB2 enhance calcium Does EphrinB2 enhance calcium conductance through NMDARs?conductance through NMDARs?

MethodsMethods Cortical neurons from embryonic Cortical neurons from embryonic

rats were assessed for change in rats were assessed for change in [Ca[Ca2+2+] with Ca] with Ca2+2+ indicator dye using indicator dye using Fura-2AMFura-2AM

Treatment:Treatment:

1. Glutamate stimulation1. Glutamate stimulation

2. EphrinB2. EphrinB

Results Results

DiscussionDiscussion

Does EphrinB2 enhance calcium Does EphrinB2 enhance calcium conductance through NMDARs?conductance through NMDARs?

Yes, EphrinB2 treated cultures Yes, EphrinB2 treated cultures enhances [Caenhances [Ca2+2+] influx through ] influx through NMDARs.NMDARs.

NMDARs are required because NMDARs are required because treatment with APV reduces [Catreatment with APV reduces [Ca2+2+] ] influx.influx.

IIs the cytoplasmic domain of s the cytoplasmic domain of EphB2 required for NMDA-EphB2 required for NMDA-

mediated Camediated Ca2+2+ influx? influx?MethodsMethods Neurons were transfected with:Neurons were transfected with:

1. EphB2WT: wild-type1. EphB2WT: wild-type

2. EphB2TR: cytoplasmic domain 2. EphB2TR: cytoplasmic domain deleteddeleted

Tagged with GFP visualized with FM Tagged with GFP visualized with FM Glutamate stimulationGlutamate stimulation

ResultsResults

Discussion Discussion

IIs the cytoplasmic domain of EphB2 s the cytoplasmic domain of EphB2 required for NMDA-mediated Carequired for NMDA-mediated Ca2+2+ influx?influx?Yes.Yes.

Cytoplasmic domain of EphB2 contains the Cytoplasmic domain of EphB2 contains the tyrosine kinase domain which is required for tyrosine kinase domain which is required for phosphorylation of Src. phosphorylation of Src.

Src phosphorylation triggers CaSrc phosphorylation triggers Ca2+2+ influx influx through phosphorylation of NR2B (NMDARs)through phosphorylation of NR2B (NMDARs)

Cytoplasmic domain is required to activate Cytoplasmic domain is required to activate downstream events that leads to increase in downstream events that leads to increase in CaCa2+2+ influx. influx.

Does phosphorylation of NR2B Does phosphorylation of NR2B at 1252, 1336, and 1472 by Src at 1252, 1336, and 1472 by Src affect Caaffect Ca2+2+ influx through the influx through the

NMDARs?NMDARs?MethodsMethods

Expression of NR1 with:Expression of NR1 with:1. Control: NR2BWT: wild-type1. Control: NR2BWT: wild-type2. Mutated NR2B Y1252F 2. Mutated NR2B Y1252F Y1336F,Y1472F Y1336F,Y1472F 3. 3. Triple Mutant [(TM)Y1252F/Y1336F/Y1472F]Triple Mutant [(TM)Y1252F/Y1336F/Y1472F]

Glutamate stimulationGlutamate stimulation Western blot analysis with antibodies Western blot analysis with antibodies

recognize phosphorylated tyrosine recognize phosphorylated tyrosine

ResultsResults

Discussion Discussion

Does phosphorylation of NR2B at Does phosphorylation of NR2B at 1252, 1336, and 1472 by Src affect 1252, 1336, and 1472 by Src affect CaCa2+2+ influx through the NMDARs? influx through the NMDARs?Yes.Yes.

Phosphorylation of tyrosine residues Phosphorylation of tyrosine residues at the three sites enhances Caat the three sites enhances Ca2+2+ influx influx

Activation of B2WT and NR2B showed Activation of B2WT and NR2B showed a large increase in [Caa large increase in [Ca2+2+]]

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

NR2B?NR2B? Treatment of neurons:Treatment of neurons:

1. PP2: Src family inhibitor1. PP2: Src family inhibitor

2. PP3: inactive analog of PP2 (control)2. PP3: inactive analog of PP2 (control)

3. SU 6656: Src family inhibitor3. SU 6656: Src family inhibitor Cortical neurons were treated with Cortical neurons were treated with

aggregated ephrinB2-Fc, or BDNFaggregated ephrinB2-Fc, or BDNF Using antibody specific for Phosph-Tyr416, Using antibody specific for Phosph-Tyr416,

Src levels was assessedSrc levels was assessedPhosphorylation of Tyr416 is indicative of Src Phosphorylation of Tyr416 is indicative of Src activation. activation.

ResultsResults

DiscussionDiscussion

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

Src family inhibitor (i.e. PP2, SU6656) Src family inhibitor (i.e. PP2, SU6656) reduces NR2B Tyr phosphorylation reduces NR2B Tyr phosphorylation leading to low levels of Caleading to low levels of Ca2+2+ influx influx

PP3, an inactive analog of PP2, did not PP3, an inactive analog of PP2, did not inhibit Src functioninhibit Src function

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