The Effects Of CD82 Palmitoylation On The Metabolic Pathways Of EGFR And C-Met In Breast Cancer Cells And Their Molecular Mechanisms

Background:As a tumor metastasis suppressor,tetraspanin CD82 is reduced in many malignant tumors and often affects the composition of tumor microenvironment by changing the heterogeneity of cell membrane.The epidermal growth factor receptor(EGFR)and hepatocyte growth factor receptor(c-Met)signaling pathway can regulate the metastasis ability of tumor cells and participate in the formation of tetraspanin web.Palmitoylation modification enables more stable expression of membrane proteins on the cell membrane and regulates the biological function of membrane proteins.The study of CD82 palmitoylation modification and metabolic pathway of tumor related molecules in tumor cells is still incomplete.After CD82 palmitoylation,there is no clear study on the localization of EGFR and c-Met in tumor cells and the molecular mechanism of metabolic pathways in tumor cells.Objective:The expression and distribution of EGFR and c-Met in breast cancer MDA-MB-231 cells after mutation of different palmitoylation sites were studied,and the related metabolic pathways and their molecular mechanisms were explored.Methods:1.The expression and distribution of CD82,EGFR and c-Met in cell membrane proteins and cytoplasmic proteins after detecting palmitoylation mutations at different sites of CD82 by immunoblotting and immunofluorescence methods.2.Detect the expression of CD82 and EGFR in total cell protein after palmitoylation mutations at different sites of CD82 by immunoblotting..3.Use immunofluorescence method and immunoprecipitation method to detect when CD82’s Cys5+Cys74 site is combined with palmitoylation mutation,CD82 and EGFR are respectively associated with lysosomal markers(Lamp1),circulating body markers(Rab7a)and late stage Co-localization and interaction relationship of endosomal markers(Rab11a).4.Co-immunoprecipitation was used to detect whether CD82 and EGFR have a direct interaction relationship when the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation.5.Co-immunoprecipitation was used to detect whether CD82 and EGFR have a direct interaction relationship when the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation.6.Tubulin inhibitors were used to detect the metabolism of EGFR when the Cys5+Cys74 sites of CD82 were combined with palmitoylation mutations.7.The co-immunoprecipitation method detects the interaction between CD82 and FIP2 when the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation.Result:1.After the mutation of different palmitoylation sites of CD82,in the cell membrane protein,when Cys5+Cys74 sites are combined with mutations,the expression of CD82 and EGFR on the cell membrane decreases,and there is no difference in the expression of c-Met between groups.In the cytoplasmic protein,when the Cys5+Cys74 site is combined with mutations,the expression of CD82 and EGFR in the cytoplasm increased,and the expression of c-Met remained no difference among the groups.2.In the total protein,when the Cys83 single-point mutation of CD82 or Cys74+Cys83 combined with palmitoylation mutation,the expression of CD82 increased,but there was no difference in the expression of EGFR among the groups.3.When the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation,the immunofluorescence results show that CD82 and EGFR co-localize with the circulating body marker Rab11 a the most obvious,followed by the lysosomal marker Lamp1,and the late nuclear The body marker Rab7 a has almost no colocalization.Co-immunoprecipitation results showed that EGFR has no direct relationship with Lamp1,Rab11 a and Rab7 a,and CD82 has a direct relationship with Rab11 a,but not with Lamp1 and Rab7 a.4.Co-immunoprecipitation results showed that EGFR and CD82 have a direct effect in the Cys5+Cys74 combined mutation group,WT group and N group.5.When the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation,the circulatory body inhibitor can effectively inhibit the metabolism of EGFR.6.When the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation,the use of tubulin inhibitors can inhibit the internalization process of EGFR,which proves that EGFR internalization requires the assistance of tubulin.7.The immunoprecipitation results show that when the Cys5+Cys74 site of CD82 is combined with palmitoylation mutation,CD82 and FIP2 have a direct interaction relationship.Conclusion:1.CD82 combined with palmitoylation mutation at Cys5+Cys74 can enhance the internalization of EGFR,but has no effect on the expression and location of c-Met.2.When CD82 is combined with palmitoylation mutation at the Cys5+Cys74 site,with the assistance of tubulin,EGFR is internalized and strengthened by direct binding to CD82 and a large number of localizations on the circulatory body.By forming the EGFR/CD82/Rab11a/FIP2 complex,it is metabolized through the circulation pathway,and re-expression of EGFR and CD82 on the cell membrane.