Modulatory Roles for Integrin Activation and the Synergy Site of Fibronectin during Matrix Assembly

Abstract

Initiation of fibronectin (FN) matrix assembly is dependent on specific interactions between FN and cell surface integrin receptors. Here, we show that de novo FN matrix assembly exhibits a slow phase during initiation of fibrillogenesis followed by a more rapid growth phase. Mn2+, which acts by enhancing integrin function, increased the rate of FN fibril growth, but only after the initial lag phase. The RGD cell-binding sequence in type III repeat 10 is an absolute requirement for initiation by alpha5beta1 integrin. To investigate the role of the cell-binding synergy site in the adjacent repeat III9, a full-length recombinant FN containing a synergy mutation, FN(syn-), was tested for its ability to form fibrils. Mutation of this site drastically reduced FN assembly by CHOalpha5 cells. Only sparse short fibrils were formed even after prolonged incubation, indicating that FN(syn-) is defective in progression of the assembly process. These results show that the synergy site is essential for alpha5beta1-mediated accumulation of a FN matrix. However, the incorporation of FN(syn-) into fibrils and the deoxycholate-insoluble matrix could be stimulated by Mn2+. Therefore, exogenous activation of integrin receptors can overcome the requirement for FN's synergy site as well as modulate the rate of FN matrix formation.

Regulation of Matrix Assembly through Rigidity-dependent Fibronectin Conformational Changes

“…Friedland et al (44) discovered that tensioned FN-integrin bonds require the synergy site, and the number of strong FN-␣5␤1 bonds correlates with the stiffness of the substrate. The strength of integrin binding to FN contributes to matrix assembly (17) because recombinant FN with a mutation at the synergy site is not assembled into matrix unless ␣5␤1 is stimulated by treatment with Mn 2ϩ (45). Based on this evidence, we tested how modulating integrin-FN binding affects matrix assembly by activating integrins with Mn 2ϩ treatment.…”

Regulation of urokinase receptor function and pericellular proteolysis by the integrin α5β1

“…Here we have directly investigated the effect of α 5 β 1 on uPAR function, using CHO cells expressing different levels of α 5 β 1 . α5CHO are transfected with the human α 5 -integrin subunit, and have a chimera of human α 5 coupled with endogenous β 1 on their surface (21). B2CHO are essentially α 5 null, containing only 2% of the α 5 β 1 found in parental cells (22).…”

“…Cell lines, antibodies and proteins CHO cells stably expressing the human α 5 -integrin subunit (α5CHO, clone #17) (21) were kindly provided by Prof. Yoshikazu Takada (The Scripps Research Institute, San Diego, CA, USA). α 5 β 1 -deficient B2 variant CHO cells (B2CHO) (22) were kindly provided by Prof. Rudy Juliano (University of North Carolina, Chapel Hill, NC, USA).…”

Extended interaction of β1 integrin subunit-deficient cells (GD25) with surfaces modified with fibronectin-derived peptides: Culture optimization, adhesion and cytokine panel studies

“…RGD, a common integrin-binding motif, is found in many ECM proteins, including collagen, vitronectin, laminin and fibronectin [3]. Unique to fibronectin is the synergy sequence, PHSRN, which is specific to the α5β1 integrin and can lead to an increase in cell adhesion and mobility, as well as enhance fibronectin matrix formation [4][5][6]. Several other integrin pairs are able to bind to fibronectin, including α4β1, α3β1, αvβ6 and αvβ3, the latter binding the RGD motif without employing the synergy sequence [5][6][7].…”

“…Unique to fibronectin is the synergy sequence, PHSRN, which is specific to the α5β1 integrin and can lead to an increase in cell adhesion and mobility, as well as enhance fibronectin matrix formation [4][5][6]. Several other integrin pairs are able to bind to fibronectin, including α4β1, α3β1, αvβ6 and αvβ3, the latter binding the RGD motif without employing the synergy sequence [5][6][7]. Depending on the integrin pair that complexes with fibronectin, a variety of downstream effects can occur within the cell through differential activation of outside-in signaling pathways.…”