The Roles Of The NITY Motif Of Integrin β3 Cytoplasmic Tail In Regulating αⅡbβ3 Related Cell Functions And Its Molecular Mechanism

Integrin αIIbβ3(GPⅡb-Ⅲa), the main membrane receptor in platelets, mediates platelet adhesion, spreading and aggregation by bi-directional signal transduction. Integrin β3 cytoplasmic domain plays a critical role in β3 bi-directional signaling pathways. There are many signal moleculars(such as talin, kindlin, etc.) interacting with β3 specific amino acid sequence in cytoplasm regulating integrin αIIbβ3 bi-directional signal transduction. The roles of the NITY motif of integrin β3 cytoplasmic tail in regulating αIIbβ3 related cell functions and its molecular mechanism have not been fully elucidated. In cell model, Stable Chinese hamster ovary(CHO) cell lines that co-express human wild type integrin αIIb and wild type β3 or mutant β3(β3 deleting cytoplasmic NITY motif) were established in this study. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. Co-immunoprecipitation was used to detect protein interactions. In mouse model, we established the transgenic mouse models of vector, Wild-type β3, and β3 deleting cytoplasmic NITY motif by retrovirus-infected hematopoietic stem cells(HSCs) transplantation and investigate the role of the integrin β3 cytoplasmic NITY motif in regulating αIIbβ3 related platelete functions. Integrin αIIbβ3 inside-out signaling was tested by soluble fibrinogen binding assay. Integrin αIIbβ3 outside-in signaling was tested by spreading assay on immobilized fibrinogen. Results showed that CHO-αIIbβ3,CHO-αIIbβ3?NITY cells were successfully established. The CHO cells transfected with full length αIIbβ3 acquired the ability of adhesion and spreading. Compared with CHO-αIIbβ3 cells, CHO-αIIbβ3?NITY cells showed an impaired capacity of adhesion while no significant difference in spreading of adhered cells. Co-immunoprecipitation showed that kindlin-2 associates with wild type integrin β3. The β3?NITY mutation substantially reduces kindlin-2 association, while src binding with integrin β3 has no significant difference between CHO-αIIbβ3 cells and CHO-αIIbβ3?NITY cells. We also successfully established three transgenic mouse models including vector, wild-type β3, and β3ΔNITY. Compared with β3 wild-type transgenic platelets, β3ΔNITY transgenic platelets bound less soluble fibrinogen with no effect on spreading on immobilized fibrinogen. Conclusion: In β3ΔNITY mutation, inside-out signaling was partially affected, but outside-in signaling was intact. The deletion mutation can suppress kindlin binding to integrin β3, and partially inhibit the integrin αIIbβ3 inside-out signaling.