The PDZ Domain Of L-periaxin Binding To The C-terminal Of EphrinB2

Periaxin is a scaffolding protein which specifically express in Schwann cell and lens fiber cell.Periaxin plays an important role in the formation of PDG complex in myelin,which is the molecure basis of Cajal bands.Mutation or loss of periaxin disrupts compact myelin and causes severe demyelination phenomenon.During the myelination,the location of Periaxin was different,it located in the nucleus in early develepment,then transfered to the membrane.Periaxin contains two PDZ protein isoforms L-periaxin and S-periaxin by alternative splicing.In addition,PDZ domain plays an important role in the location of periaxin and the formation of PDG complex,and the ligands of PDZ domain of L-periaxin were not reported until now.EphrinB2 is a member of the Eph receptor tyrosine kinases.Furthermore,bidirectional signaling occurs which owing to EphrinB2 interaction with Eph receptor binding,and affects axon growth,cell assembly,migration and myelination.EphrinB2 ligand includes a transmembrane region,an extracellular region and an intracellular region,as well as,contains a PDZ binding site in carboxy-terminal.The PDZ-binding motif mediates organization of protein complexes at the plasma membrane.In this paper,the interaction between L-periaxin and EphrinB2 is analyzed.The colocalization of endogenous Periaxin and EphrinB2 in the cell membrane and cytoplasm of RSC96 cells was found by the immunofluorescent.The interaction between L-periaxin,S-periaxin and EphrinB2 were verified by BiFC,renal luciferase complementary experiment,co-immunoprecipitation and GST Pull-down.The results also indicated that periaxin interact with EphrinB2 through the PDZ domain.In addition,when the PDZ binding site is deleted,the interaction between periaxin and EphrinB2 is disrupted.Self-association of L-periaxin occurs via its acidic domain,and NLS2,3 regions.The self-association of L-periaxin in RSC96 cells is remarkably weakened by DRP2 through the interaction between DRP2 and NLS2,3 regions.Self-association of L-periaxin was also obviously weakened by synthetic NLS3 peptide.Membrane localization of L-periaxin in RSC96 increased by disruption of self-association with DRP2 and NLS3 peptide by the immunofluorescent.MTT assay and flow cytometry was used to detect the proliferation of RSC96 cells when the L-periaxin was knock-out by Talens.Results demonstrated that the cell viability decreased,as well as the amount of cells in G1 increased and decreased in S phase in RSC96(L-periaxin-/-)cell by Flow cytometry and MTT assay.