Skelemin in integrin α(IIb)β(3) mediated cell spreading.

Skelemin, a myosin-associated protein in skeletal muscle, has been demonstrated to interact with integrin α(IIb)β(3) in nonmuscle cells during initial stages of cell spreading. The significance of this interaction and the role of skelemin in integrin signaling and cytoskeletal reorganization were investigated in this study. We established a series of Chinese hamster ovary cell lines expressing wild-type or mutant α(IIb)β(3) receptors in which skelemin binding residues at the membrane proximal region of integrin tails were mutated to alanine. Most cells displayed unimpaired adhesive capacity and spreading on immobilized fibrinogen at the early stages of cell spreading. In addition, they formed normal focal adhesions and stress fibers with no indication of impaired cell spreading. R995A/R997A/L1000A, H722A, and K716A exhibited the greatest cell spreading, which was associated with enhanced p-Src activation but was independent of FAK activation. Transfection of the cells with GFP-skelemin, containing only the C2 integrin binding domain, caused wild-type cells to round up, but had no effect on R995A/R997A/L1000A, H722A, and K716A cell spreading. Furthermore, the protrusions of the leading edge of K716A cells showed strong colocalization of talin with α(IIb)β(3) which was associated with a loss in skelemin binding. Thus, we propose that during early stages of cell spreading, skelemin exerts contractile force on cell spreading and modulates the attachment of cytoskeletal proteins and Src to integrin clusters.