Molecular Mechanisms Study Of EHD2 Mediated EGFR Endocytosis

Objectives:EGFR(epithelial growth factor receptor,EGFR)is a receptor protein presents in the cell membrane,when combined with a ligand such as EGF,various EGFR-mediated signaling pathways will be activated,or the EGFR itself undergo endocytosis and then degradation process,the above of EGFR is closely related to a variety of life activities thus as cell growth,proliferation apoptosis,etc.Many studys found overexpression of EGFR in lung cancer,breast cancer,cervical cancer and other cancers is associated wih the cancer occurs and development.Currently,molecular targeted therapy against EGFR primarily inhibit gene expression at the transcriptional level or by antibodies and small molecule drugs.EHD2(EH-domain containing protein 2)is a mediator involved in endocytosis and endocytic vesicles transportation.The N-terminal of this protein contains an ATP enzyme domain,which is for the hydrolysis of ATP to provide energy for its own activities.EHD2 C-terminus contains an EH domain,which is binding site interactions with other molecules.The issue is to explore whether EHD2 has a regulatory role in EGFR endocytic and transport,in-depth study the important role of ATP enzyme domain of EHD2 in the EGFR endocytosis and transportation process.Exploring new molecular mechanisms regulating EGFR receptors from the perspective of vesicle transport,thus to opened a new path for EGFR targeted therapy of tumors.Methods: 1.By means of Western Blotting and immunofluorescence technologies,validating the influence of knock down or overexpression of EHD2 on EGFR expression levels and their colocalization.2.Under the activation of EGF,analyse the regulation of knockdown,overexpression EHD2 on EGFR endocytosis and transportation.3.By means of immunofluorescence technologies,finding the channels through which EHD2 regulate EGFR endocytosis.4.By means of Western Blotting and immunofluorescence technologies,validating the influence of ATP enzyme domain mutant EHD2 on EGFR expression levels,EGFR endocytosis as well as their colocalization.5.By means of 2D clones formation assay,analyse the influence of EGFR expression level on cell growth and proliferation.6.Under the activation of EGF,make a real-time observation of the transportation of EHD2 and EGFR endocytotic vecicles in living cells.7.Under the activation of EGF,analyse the influence of knockdown,overexpression or mutant EHD2 on microfilament.Results: 1.EGFR expression levels is reduced when knockdown of EHD2,EGFR expression levels is upregulated when EHD2 overexpression,immunofluorescence showed EHD2 and EGFR exist obvious co-localization.2.In Hela cells,under EGF stimulation,decrease expression of EHD2 accelerated endocytosis of EGFR,overexpression EHD2 inhibited endocytosis of EGFR.3.In Hela cells,after EGF stimulation,EHD2 and EGFR mainly co-localized within CAV1,but most of the EGFR co-localized with CLTC,and not co-localize with CLTC.But in MCF7 cells,EHD2,EGFR,CLTC co-localize obviously.4.Overexpression of EHD2-T94 A mutation increased the expression levels of EGFR.Overexpression of EHD2-I157 Q mutation decreased the expression levels of EGFR.Immunofluorescence showed EHD2-T94 A mutation mainly located in the cell membrane,meanwhile make EGFR a increased localization on membrane,EHD2-T94 A and EGFR significantly co-localize on the cell membrane.Immunofluorescence showed EHD2-I157 Q mutation mainly located in the cytoplasm,meanwhile make EGFR a decrease localization on membrane,EHD2-I157 Q with EGFR significantly co-localize on the cell membrane and cytoplasm.5.2D clone experiment showed reduced expression of EHD2 decreased colony formation in Hela,overexpression of EHD2 incresed colony formation in Hela,overexpression of EHD2-T94 A incresed colony formation slightly in Hela and have a lower degree than EHD2 wild type,overexpression of EHD2-I157 Q increased colony formation in Hela,and higher than EHD2-T94 A group,but lower than EHD2 wild type group..6.After EGF stimulation,inhibition of EGFR endocytosis caused intracellular actin structure reorganization,and there was a swelling phenomenon in 293 T cells.7.In Hela cells,knockdown EHD2,overexpression of wild-type and mutant EHD2 make intracellular actin morphological changes: knockdown intracellular EHD2 reduce stress fibers;after overexpressing EHD2 the stress fibers increased.;EHD2-T94 A overexpression increased the stress fibers,and cells were mostly emerged in the form of full expansion;stress fibers in EHD2-I157 Q overexpressing cells was significantly reduced.Conclusions: 1.EHD2 positively correlated with the expression of EGFR,speculating that EHD2 mainly by adjusting the internal EGFR endocytosis to regulate EGFR expression levels.2.Role for EHD2 in regulating EGFR was achieved mainly by regulating caveolia endocytic pathway in Hela cells,but in MCF7 cells it was done mainly by clathrin passway.3.EHD2-T94 A mutant enhanced the ability of EHD2 to inhibit EGFR endocytosis,EHD2-I157 Q mutant reduced the ability of EHD2 to inhibit EGFR endocytosis.4.Clonogenic assay confirmed EHD2 expression levels positively correlated with colony-forming ability,further confirmed the regulation between EHD2 and EGFR.5.Under EGF stimulation,EHD2 inhibit EGFR from endocytosis,EGFR signaling pathway can be excessive amplificated,then actin morphology changed,the specific mechanism needs further study.