Study On The Mechanism Underlying The Role Of EHD2 In Regulating The Intracellular Trafficking Of EGFR

OBJECTIVEEndocytic trafficking is the trafficking of the cell membrane and protein lies in the membrane(including surface receptor such as EGFR),In the early endosome,the receptor is readied for its appropriate cellular fate:recycled back to the plasma membrane,trafficked to the lysosome for degradation,or targeted to another subcellular compartment.It plays a key role in many cell functions such as proliferation?differentiation?polarization?migration and apoptosis,and has an important function in tumor generation and development.EHD2,a member of EHD family(including EHDl,EHD2,EHD3,EHD4),is a novel membrane trafficking regulator protein.believed to serve as regulators of endocytic trafficking including endocytosis and recycling.It participates in membrane trafficking,including endocytosis and transition from early endosome to late endosome and regulate recycling,thereby regulate intracellular signal transduction of memberane protein.Our project is aimed at identifying the role of EHD2 in the intracellular trafficking of EGFR.we constructed EHD2 RNAi plasmid and infected HeLa cell lines to knock-down EHD2 endogenous expression,then investigated it by immunofluorescence and Fluorescence Activated Cell Sorting.METHOD1.We used pGCSIL-Amp/puromycin plasmid to knock down EHD2 expression in HeLa cell lines by means of using lentivirus to infect HeLa.2.We have got several monoclones from HeLa cell by means of limiting dilution assay,and knocked down EHD2 expression in the monoclones,which was identified by Western Blotting analysis.3.We purified the rabbit polyclonal antibody by polypeptide affinity chromatography4.We used laser scanning confocal microscope to observe EHD2 and EGFR colocalization in HeLa cells stimulated by EGF.5.We used laser scanning confocal microscope to observe role of EHD2 in EGFR trafficking in siEHD2/HeLa cells and control cells stimulated by EGF.Meanwhile we labeling some marker proteins in the trafficking such as Rab5(early endosome antigen marker)?EEA1(early endosome antigen marker)?LAMP1(lysosome marker)etc.6.We used Fluorescence Activated Cell Sorting to study the expression and endocytosis of cell surface EGFR and analysed the role of EHD2 in the intracellular trafficking of EGFR.RESULTS1.EHD2 was expressed slightly highly in HeLa cell lines,we have got some monoclones with limiting dilution method,named as HeLaM1,M2...,and EHD2 expression indifferent monoclones had no significant difference.2.We infected HeLa cells and monoclones by lentivirus with the purified effective RNAi plasimid.With limiting dilution method,we got the EHD2 down-regulation clones,named as HeLaM20-LsiEHD2-46.3.We purified anti-EHD2 and detected the specificity in immunofluorescence assay.4.EHD2 and EGFR colocalized in the cellular with EGF stimulation.5.EHD2 interference increased the endocytosis of EGFR.6.EHD2 interference increased the degradation of EGFR.CONCLUSIONshRNA interference of EHD2 expression increased the endocytosis and degradation of EGFR,which indicated that EHD2 inhibited the endocytosis and degradation of EGFR.