CRMP2 Promotes The Trafficking Of Phosphorylated AMPA Receptor GluA2 Subunit

Objective:To investigate the effect of CRMP2 on the trafficking expression of phosphorylated AMPA receptor GluA2 subunit.Methods:In this study,we performed molecular cloning technique,GST-Pull down,Co-IP assays,immunofluorescence staining and whole cell patch clamp recording technique.Firstly,we constructed a phosphorylated mutant of GluA2 S880 and wild-type GluA2 subunit,examining the difference in binding between CRMP2 and GluA2 in different modification states.Then under the condition of overexpressing CRMP2,we simulated exogenous and endogenous phosphorylation of GluA2 S880,observed the expression of GluA2 on the surface of the neuron and the post-synaptic potential,as well as analyzed its I-V curve to study the effects on the trafficking of phosphorylated GluA2.Finally,under the condition of over-expressing PKCζ,we used the si CRMP2 interference fragment to interfere with CRMP2 expression,observed neuron m EPSCs and analyzed the slope of the I-V curve to study the effect of CRMP2 on phosphorylated GluA2 subunit trafficking.Results:1)under the condition of exogenously phosphorylated by phorbol myristate acetate(TPA),overexpressed CRMP2 can promote the surface expression of phosphorylated GluA2 and enhance the amplitude and frequency of neuronal m EPSCs.2)It was found that CRMP2 interacts with GluA2 by GST-Pull down,Co-IP and co-localization experiments,and this interaction effect was related to the modification state of GluA2.The interaction of phosphorylated GluA2(S880D)to CRMP2 was enhanced while it was weakened as for non-phosphorylated GluA2(S880A)to CRMP2.3)Over-expressing CRMP2 in HEK 293 T cells and cultured hippocampal neurons can promote surface expression of wild-type GluA2 and enhance the amplitude and frequency of neuronal m EPSCs,while increasing the slope of its I-V curve.4)Under the condition that GluA2 was endogenously phosphorylated by PKCζ,over-expressing CRMP2 promoted the abundance of GluA2 surface expression and enhanced the amplitude of neuronal m EPSCs,while there is no significant difference in the frequency.The slope of I-V curve increased in the experimental group.Under the same conditions,after interference with CRMP2 expression,the amplitude and frequency of m EPSCs of neurons decreased so as the slope of I-V curve.Conclusions:1)There is a protein interaction between CRMP2 and GluA2,and the interaction effect will enhance when GluA2 S880 is phosphorylated.2)CRMP2 can promote surface expression of wild-type GluA2.3)CRMP2 enhanced its interaction to phosphorylated GluA2(S880D)to promote the re-expression of phosphorylated GluA2 on the neuronal surface.