A Biochemical Research About The Intracellular Location Of EHD2

Objectives:EHD2 is a new membrane transport regulatory protein,with a function of regulating the endocytosis transport of cellular receptor.A recently study found that cellular endocytosis regulatory protein can function both in nuclear and at membrane,and its function in nuclear often expressed as the regulation of gene transcription.Our previous results found EHD2 specific expressed in the nuclear of the breast myoepithelial cell,and disappeared from the nucleus and increased outside nuclear,with decreased in total in the cancer tissue.Based on the above results,this study will research in the following aspect.In order to learn more about the sequence factors that affects the nuclear import and export abilities of EHD2,we used cervical cancer cell Hela as model to investigate the factors that may influence the nuclear import and export function of EHD2,except to provide a basis for the study of transcriptional regulation function of EHD2,and furthermore to provide a new way and a new target for the treatment of malignant tumors.Methods:1.Build the EHD2 wild type and different mutant plasmid,which were connected a HA tag at the C-terminal.2.We used 293T cells to generate EHD2 wt lentivirus and mutant lentivirus.3.We used the lentivirus of EHD2wt and mutant to infect Hela cell,and used immunofluorescence to detect the localization of EHD2.Results:1.With the Mutation of the nuclear receptor sequence(184-188aa)of EHD2,EHD2 mainly localized in the cytoplasm.EHD2 deletion mutation EHD2-1-183aa-HA was mainly localized in the cytoplasm,but after extending to EHD2-1-188aa-HA(with 184-188aa),EHD2 was not transport into the nucleus,and continued to be extended to EHD2-1-221aa-HA,EHD2 mainly localized in the nucleus.2.EHD2 contains two NLS sequence:293 to 299 and 535 to 539 amino acids.With the mutation of 293?299 or mutation of 535?539 amino acids,EHD2 mainly localized in the cytoplasm.3.EHD2 contains a nuclear export related sequence:315-329 amino acids.The mutation of 315 to 316 or 324 or 327 to 329 amino acids,EHD2 are mainly located in the nuclear;and the 315aa is a SUMOylation site.4.With the mutation of 65 amino acid,EHD2 can not bind to the membrane,and distribute in the cytoplasm evenly.Conclusions:1.184-188 amino acids in EHD2 is a nuclear receptor binding sequence,affecting nuclear import capabilities of EHD2,but its function may be affected by the presence of nearby sequences.2.EHD2 contains two NLS sequence:293 to 299 and 535 to 539 amino acids.3.EHD2 contains a nuclear export related sequence:315?329 amino acids.4.The 65 amino acid site affects the membrane-binding ability of EHD2.