Mechanism Of The Effect Of EGFR Transmembrane Domain Dimerization Site On ERBB Family Phosphorylation

As the first discovered Receptor Tyrosine Kinase,EGFR is mainly responsible for regulating cell proliferation,survival and differentiation.At the same time,as the most classic drug target,EGFR represents an era of targeted therapy.EGFR-targeted drugs have profoundly changed the way of tumor treatment and brought great benefits to patients.At present,although the research on EGFR-related signaling pathways and mechanisms of action is relatively mature,targeted drugs developed for EGFR and its mutations have also been widely used in the clinical treatment of cancer,and there are already antibodies and small molecule compounds,Various types of EGFR inhibitors are used clinically,but EGFR is still a hot research hotspot.In particular,the current research and development of EGFR inhibitors mainly focuses on the extracellular domain and kinase domain,but these regions are prone to drug resistance mutations,which affect the therapeutic effect,and even make EGFR-targeted drugs completely ineffective for patients.At present,the relevant research on the EGFR transmembrane domain is not complete.As a transmembrane protein,the transmembrane domain of EGFR has anα-helical structure and is mainly composed of hydrophobic amino acids.The transmembrane domain can anchor EGFR on the cell membrane,they can also interact with the transmembrane domains of other proteins,so this domain is a key structure affecting EGFR-related biological functions.The EGFR transmembrane domain has a non-negligible role in the formation of EGFR dimers,the phosphorylation and activation of downstream signaling pathways,and this region is relatively conservative and not prone to mutation,so it is an ideal new target for antitumor drugs.In this paper,on the basis of the previous molecular dynamics simulation results in the laboratory,the key amino acid residues of the binding of the EGFR transmembrane domain were theoretically analyzed,and further,it was further analyzed from an experimental point of view how these sites affect the EGFR transmembrane domain.Key sites for structural binding and function,and how these sites have the potential to become new targets for EGFR-targeted drugs.In this paper,based on CRISPR/Cas9 technology,two EGFR-mutant human colon cancer SW480stably transfected cell lines were constructed first,which were named SW480^646KKKand SW480^666KKKaccording to the design.Then,the possible effects of mutation of key amino acid sites in EGFR transmembrane domain on cell biological function were analyzed by cell proliferation assay,drug sensitivity assay,and cell apoptosis assay.Finally,using dSTORM super-resolution cell imaging technology,phosphorylated antibody chip detection and bioinformatics,the mutation of key amino acid sites in the EGFR transmembrane domain may affect the aggregation state of EGFR on the cell surface,the downstream phosphorylation of the ERBB family,and the difference in signaling pathways.The experimental results showed that（1）EGFR mutant homozygous SW480 stable transfection cell line was successfully constructed by CRISPR/Cas9 technology.（2）The amino acid sites of Leu665,Phe666 and Met667 on the transmembrane domain of EGFR have a significant impact on the proliferation and drug sensitivity of human colon cancer SW480 cell;EGFR classical dimerization inhibitors such as erlotinib and gefitinib are more sensitive to SW480^666KKK;in the apoptosis detection results,no significant difference was found between SW480^wtand SW480^646KKK,and SW480^666KKKwas found in the early apoptotic cell population.There is a significant difference from the late apoptotic cell population.The proportion of the early apoptotic cell population increased from 2.84%in the SW480^wtgroup to 4.93%,and the late apoptotic cell population increased from 2.85%in the SW480^wtgroup to7.81%.（3）In order to study the dimerization and phosphorylation of ERBB signaling pathway,in this paper,cell dSTORM super-resolution cell imaging was used in this paper.The experimental results showed that Leu665,Phe666 and Met667 amino acid sites are key binding sites in the process of EGFR dimerization.After rh EGF stimulation,EGFR on the surface of SW480^666KKKcells did not aggregate,and was relatively dispersed,which was significantly different from SW480^wtand SW480^666KKKcells.In theory,mutations affecting dimerization would further affect phosphorylation of downstream pathways.The EGFR phosphorylation antibody chip detection data verified this speculation.The experimental results showed that after the key amino acid site mutation in the EGFR transmembrane domain,the phosphorylation degree of ten phosphorylated proteins such as p EGFR（Tyr992）remained unchanged in the SW480^666KKKgroup after rh EGF stimulation.remained unchanged or even decreased,especially p EGFR（Ser1046/1047）,p EGFR（Ser1070）,p EGFR（Tyr1173）and p ERBB2（Tyr1248）,which are key phosphorylation sites for drug development,and the inhibition of these phosphorylation sites by SW480^666KKKreflects it Potential to become a new anti-tumor target.（4）Finally,in the bioinformatics analysis,the ERBB signaling pathway was significantly regulated.More phosphorylated proteins were involved in the signal transduction process in the SW480^646KKKgroup,while the phosphorylation in the SW480^666KKKgroup was inhibited to a certain extent.SW480^646KKKis a mutation that enhances EGFR dimerization in theory,and the experimental results are basically consistent with theoretical predictions.This mutation promotes cell proliferation,reduces the sensitivity to EGFR inhibitors,and cluster density on the cell membrane surface is more likely to occur.The associated phosphorylation sites were more up-regulated compared to wild type.In theory,SW480^666KKKis a mutation that inhibits EGFR dimerization.This mutation has a certain inhibitory effect on cell biological function,cell membrane surface aggregation state and activation of related phosphorylation sites,and is more sensitive to drugs.In conclusion,Leu665,Phe666 and Met667 amino acid sites of EGFR transmembrane domain are key binding sites in the dimerization process,and mutations of these sites seriously affect the phosphorylation level of ERBB signaling pathway and its related proliferation,apoptosis and drug sensitivity.These results not only provide more convincing evidence for the binding form of transmembrane domain dimerization of EGFR,but also provide a new potential target for antitumor drugs by studying the effect of phosphorylation on ERBB family.