A novel RGD-independent fibronectin assembly pathway initiated by alpha 4 beta 1 integrin binding to the alternatively spliced V region

Fibronectin (FN) matrix assembly is a multi-step process that involves bind ing to integrin receptors, FN-FN interactions and connections to the actin cytoskeleton, Ultimately, FN is converted into stable matrix fibrils that a re detergent-insoluble. RGD-binding integrins such as alpha 5 beta 1 play a major role in the assembly of fibrillar FN, Here we show that alpha 4 beta 1 binding to the alternatively spliced V (IIICS) region of FN initiates an alternative assembly pathway. Activation of alpha 4 beta 1 with exogenous agents such as Mn2+ or a beta 1-stimulatory antibody TS2/16 was sufficient to induce initiation of FN fibrillogenesis by Ramos B lymphoma cells and by CHO(B2)alpha 4 cells. Using recombinant FNs lacking specific sequences, we show that assembly is independent of the RGD sequence but requires the V25 /CS-1 segment, Previously, we have characterized an activated recombinant F N (FN Delta III1-7) that rapidly forms detergent-insoluble multimers upon b inding to alpha 5 beta 1 integrin, alpha 4 beta 1 also formed FN Delta III1 -7. I multimers without the aid of exogenous stimulants, suggesting that an activated form of FN can override the need for activation of the integrin, In contrast to assembly by alpha 5 beta 1, actin filaments remained largel y cortical and no change in cell growth rate was observed with alpha 4 beta 1-mediated assembly. These results show that binding sites on FN other tha n the RGD sequence/synergy site and distant from the cell binding domain ca n promote FN assembly, Thus, there appear to be multiple, integrin-specific mechanisms for assembly of FN matrix.