Ezrin is a 69 kDa polypeptide, which includes586 amino acids. Ezrin can be phosphorylated on threonine and its related molecules are concentrated in surface projections such as microvilli and membrane ruffles where they link the microfilaments to the membrane. The ezrin molecule is composed of three domains.The N-terminal FERM domain associates with the plasma membrane, which is followed by an extended a-helical domain. The C-terminal half of ezrin links it to the cytoskeleton and associates with the actin cytoskeleton.

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  As a member of the ezrin-radirin-moesin (ERM) family, ezrin primarily acts as a connector between the plasma membrane and the cytoskeleton. Ezrin has been implicated in many roles, such as involved in epithelial cell morphogenesis, formation of microvilli, cell adhesion sited and signal transduction. Recent study revealed that ezrin was abnormal expression in many cancers, suggesting that the ezrin played an important role in invasion and metastasis of cancers.









  The searching of interaction protein of ezrin may figure out the function clue. So, after screening human lymphoid cell cDNA library by yeast two-hybrid, an interaction protein was found: the c-terminal peptide of TGF- beta receptor interacting protein-l(TRIP-l). TRIP-1 is a cytoplasmic substrate of the TGF-beta type II receptor kinase and plays a role in TGF-beta signaling.









  So with yeast two-hybrid screening of the human fetal brain cDNA library, we identified TRIP-1 as the potential interaction partner of Ezrin. In this paper, the studies will focus on confirming the interaction between Ezrin and TRIP-1 and researching into the regions involved in the interaction. Main procedures and methods:









  (1) The ezrin gene was inserted into the plasmid pAS1 in yeast two hybrid system to construct bait plasmid pAS1-Ezrin. The vector reconstructed was transformed into yeast strain Y190. The BD-Bait protein was examined for the reporter gene LacZ activation and toxicity to yeast strain.









  (2) The human lymphoid cell cDNA plasmids was introduced into the yeast containing bait plasmid pAS1-Ezrin. Yeasts were plated on containing synthetic dropout nutrient medium. Andβ-galactosidase filter assay was performed for screening positive clones.









  (3)The plasmids DNA in positive yeast colonies were isolated, transformed into E.coli DH5αto obtain AD-Library/DH5αtransformants. AD-Library plasmids were extracted, digested with two restriction enzymes and sorted. AD-Library plasmids were transformed into yeast Y190 containing bait plasmid pAS1-Ezrin to retest protein-protein interaction.









  (4)The AD-Library plasmids in true positive colonies were sequenced and analyzed by bioinformatics techniques. One clone is the c-terminal peptide of TGF- beta receptor interacting protein-1 (TRIP-1) .A TRIP-1 cDNA fragment was obtained by extracting mRNA from NIH3T3 cells and RT-PCR. We transfected the TRIP-1 gene into 293 cells and expressed the relevant protein.









  (5)We inserted the Ezrin gene into the pGEX and expressed the relevant protein successfully. By in vitro GST-pull down assay, interaction between Ezrin and TRIP-1 was verified.









  (6)In order to learn the details of interaction between Ezrin and TRIP-1, the different fragment of Ezrin and TRIP-1 was inserted into expression vector and expressed. A series of GST-pull down experiments revealed that the interaction took place on the C-terminal of both proteins.









  In conclusion, above research showed the interaction between Ezrin and TRIP-1.All the information obtained will be valuable for the future functional study of Ezrin.