Structural And Biochemical Characterization Of Liprin-?1 Mediated Interactions In Integrin Endocytosis

There are many specialized areas on the cell membrane that perform certain functions,such as the focal adhesion formed by many proteins on the basis of clustering integrin.The dynamics of these specialized regions are related to many important cellular processes,such as the turnover of integrin-mediated focal adhesions,which is critical to the cell migration.And cell migration is essential for tissue remodeling,wound healing,axon growth,and so on.Abnormalities in cell migration,such as abnormal migration and invasion of tumor cell lines,have been observed in many pathological states.Therefore,it is particularly important to investigate the molecular organization and mechanisms behind these plasma membrane specialized regions and their regulation.The turnover of integrin-mediated focal adhesions,is composed of disassembly and recycle of focal adhesions in the rear of migrating cells through endocytosis,and reformation of them on the leading edge of migrating cells.These specialized regions often share a core complex that coordinates complicated biological functions,including the scaffold protein Liprin family,which is mainly divided into Liprin-? and Liprin-?.Liprin-? is an important protein which has been widely studied.It plays a key role in the turnover of adhesions by inducing microtubules to target to the leading edge of migrating cells.However,as a member of Liprin family,Liprin-?1colocalizes with Liprin-? around focal adhesions,its function in focal adhesions has not been well studied.Based on our preliminary data,it was found that Liprin-?1 may interact with some endocytosis related proteins Numb,RUFY1,SH3KBP1,and HIP1,which is unreported before.We firstly detected the interaction between Liprin-?1 and these proteins by Co-IP and Western Blot assay,and confirmed that Liprin-?1 indeed interacts with two of the endocytosis related proteins,Numb and RUFY1,and further confirmed that the N-terminal region of Liprin-?1 is involved in the interaction with Numb and RUFY1.And we successfully expressed and purified the relevant truncated fragments of Liprin-?1,Numb and RUFY1,and the interacting regions were further determined by Fast Protein Liquid Chromatography(FPLC)and Isothermal Titration Calorimeter(ITC): Liprin-?1?H and Numb?Nter;Liprin-?1?H,Liprin-?1?CC and RUFY1?Nter,and preliminary crystal screening of Liprin-?1?H and Numb?Nter complex was carriedout.At the same time,the smaller binding regions of Liprin-?1?H and Numb?Nter;Liprin-?1?H,Liprin-?1?CC and RUFY1?Nter are further detected by Fast Protein Liquid Chromatography(FPLC)and Isothermal Titration Calorimeter(ITC),to do further crystal screening and X-ray diffraction.The structure of the complexes of Liprin-?1 and Numb,RUFY1 will be analyzed to gain a better understanding of their interactions and related regulation and to further explain how Liprin-?1-mediated interactions play a role in integrin endocytosis.In summary,the interactions between Liprin-?1 and endocytosis related proteins Numb,RUFY1 were found and proved for the first time in this paper,and the interaction mechanism between Liprin-?1 and Numb,RUFY1 was further investigated.According to our results,Liprin-?1 interacts with endocytosis related proteins,and may regulate integrin-mediated focal adhesions dynamics by participating in the endocytosis of integrin.This study will provide a new idea for the investigation of the regulatory mechanism of Liprin-?1 in integrin endocytosis and focal adhesion turnover.