TM4SF5 막단백질의 intracellular domain의 기능에 관한 연구

Abstract

TM4SF5는197amino acid로 구성되어 막을 4번 통과하는 transmembrane glycoprotein으로 여러 암종에서 발현이 보고되어온 tetraspanin의 subfamily이다. Integrin과의 cross-talk를 통해 단일 세포 차원에서는 EMT(epithelial-mesenchymal transition)을 유도함으로써 cell의 morphological change 뿐만이 아니라 actin reorganization을 통한 migration과 invasion, 세포 주기 이행 조절을 통한 cell proliferation 촉진이 알려져 있다. 하지만 TM4SF5의 단백질을 매개로 어떻게 intracellular signal로 매개되는지 그 메커니즘에 관해서는 밝혀져 있지 않다. 따라서 본 연구에서는 간암 세포주를 이용하여 TM4SF5 막단백질의 intracellular domain들의 기능에 관한 연구를 수행하게 되었다. TM4SF5의 intra cellular loop domain과 C-terminal domain의 deletion mutant를 통한 연구 결과, TM4SF5에 의한 FAK 활성은 TM4SF5의 intra cellular loop를 통한 FAK과의 직접적인 binding을 필요로 한다는 것을 β1 integrin antibody와 TM4SF5 inhibitor를 통해서 관찰할 수 있었다. C-terminal domain을 통해서는 TM4SF5에 의한 p130Cas의 활성 및 ROS생성에 변화가 관찰되었다. 특히 외부 ECM의 농도변화에 따른 cell replating assay를 통해 TM4SF5에 의한 p130Cas 활성이 달라지는 것을 관찰 할 수 있었다. 이러한 연구들을 통해 다양한 외부환경, 특히 ECM에 대해 TM4SF5에 의한 일련의 FAK 활성 및 mechanosensing과 관련된 p130Cas의 활성화 기능이 TM4SF5의 intracellular domain에 의해 조절되는 기전이 존재함을 제시한다.

Transmembrane4 L six family member5 (TM4SF5), a branch of the tetraspanin superfamily, is a tetratransmembrane glycoprotein that consists of 197 amino acid. Certain tetraspanins are highly expressed in tumor cells from many types of cancers, whereas TM4SF5 is reported to be over-expressed in hepatocarcinoma. The biological functions of TM4SF5 are predicted to be rendered in tetraspain-enriched microdomains (TERM), although direct evidence has not been previously shown. In previous studies, TM4SF5 is known to cross-talk with integrins and induces EMT (epithelial-mesenchymal transition) at the cellular level, resulting in not only morphological elongation change through actin reorganization, but also promotion of cell migration/invasion and cell proliferation. However, the direct roles of TM4SF5 in intracellular signal transduction remain largely unknown. Here we explored the mechanistic roles of TM4SF5 in intracellular signal transduction. In this study, we have investigated the roles of TM4SF5 intracellular domain by using its deletion mutants of the cytoplasmic regions of TM4SF5 in diverse functions human hepatocarcinoma cells. An interesting aspect in TM4SF5 roles was observed with respect to both its intracellular loop domain and C-terminal domain. At first, we found that the functional blocking anti-integrin β1 antibody and TSAHC inhibitor treatment abolished TM4SF5-enhanced FAK signaling activity. Moreover, we identified that the binding between TM4SF5 intracellular loop domain and FAK was essential for the TM4SF5-mediated FAK activation. Further study of TM4SF5 C-terminal deletion mutant, we observed that TM4SF5 regulates p130Cas signaling activity and ROS generation via its C-terminal domain. In addition, we revealed that the involvement of p130Cas activity which is known to mediate mechanosensing is associated with the differential regulation by TM4SF5 C-terminal domain depending on time and different extracellular environment, in particular, extracellular matrix concentration under cell adhesion-dependent condition. It is of further interest to determine the identification of a role for TM4SF5-mediated p130Cas regulation during mechanosensing and or ROS signaling. Altogether, this study suggests the mechanism of TM4SF5-mediated FAK activation and the mechanosensitive p130Cas activation via the TM4SF5 intracellular domains and partially gives a insight into the role of TM4SF5 in liver cancer.