EGFR Signaling Upregulates NMDA Receptor Subunit GluN2B And Contributes To LTP In The Hippocampus

Background:N-methyl-D-aspartate receptors (NMDARs) are ligand and voltage gated glutamate receptors widely expressed in the brain, intimately associated with synaptic plasticity and play pivitol roles in the pathology of a variety of neurological disorders. The NMDAR consists of two obligatory GluNl subunits and two regulatory GluN2or GluN3subunits, which contains many serine and tyrosine phosphorylation sites at their endoplasmic domains. Modulation of NMDARs by phosphorylation may affect channel properties or trafficking and is therefore crucial to the regulation of NMDAR function. Various studies have described the phosphorylation of GluN2B subunits by receptor tyrosine kinases (RTKs) such as TrkB, ErbB4and EphB receptors. EGFR, an important member of the RTK family is richly expressed in the brain, and participates in the development of the central nervous system (CNS). Exogenous application of EGF facilitates LTP in the hippocampus of adult mice and enhances calcium influx through NMDARs in cultured hippocampal neurons. Moreover, LTP is impaired in HB-EGF knockout mice suggesting a possible role for EGFR signaling in regulating synaptic plasticity through altering NMDAR functions. However, the mechanism by which EGFR regulates NMDARs remains to be elucidated. Objective:Our goal is to investigate the regulation of NMDAR GluN2B subunit by EGFR signaling and its physiological significance during synaptic plasticity.Methods:The distribution of endogenous EGFR in the hippocampus of adult mice was determined using immunohistochemistry. Subcellular distribution of EGFR was determined using immunocytochemistry and western blotting. Whole-cell patch-clamp recording and immunocytochemistry was used to investigate the effect of EGFR activation on GluN2B-containing NMDARs. Western blotting was used to investigate regulation of GluN2B subunits by EGFR signaling. Electrophysiologic recording of field potential in hippocampal slices combined with Western blotting was used to investigate the physiological significance of EGFR signaling during LTP induction.Results:EGFR is widely distributed in the hippocampus of adult mice with robust expression in the non-synaptic region and low expression in synapses. Treating cultured hippocampal neurons with exogenous EGF increases surface expression of GluN2B-containing NMDARs. Activation of EGFR in hippocampal neurons phosphorylates GluN2B at Y1472through Src family kinases (SFKs). Blocking EGFR signaling inhibits GluN2B accumulation in the post-synaptic density during LTP induction and impairs LTP induction. Blocking EGFR activation during contexual fear conditioning inhibits GluN2B Y1472phosphorylation, but does not affect24hour contexual memory recall.Conclusion:EGFR signaling may regulate NMDAR function through phosphorylation of its GluN2B subunit and participates in synaptic plasticity.