Identification of the Heparin-binding Determinants within Fibronectin Repeat III1

Abstract

Fibronectins are high molecular mass glycoproteins that circulate as soluble molecules in the blood, and are also found in an insoluble, multimeric form in extracellular matrices throughout the body. Soluble fibronectins are polymerized into insoluble extracellular matrix (ECM) fibrils via a cell-dependent process. Recent studies indicate that the interaction of cells with the ECM form of fibronectin promotes actin organization and cell contractility, increases cell growth and migration, and enhances the tensile strength of artificial tissue constructs; ligation of integrins alone is insufficient to trigger these responses. Evidence suggests that the effect of ECM fibronectin on cell function is mediated in part by a matricryptic heparin-binding site within the first III 1 repeat (FNIII 1 ). In this study, we localized the heparin-binding activity of FNIII 1 to a cluster of basic amino acids, Arg 613 , Trp 614 , Arg 615 , and Lys 617 . Site-directed mutagenesis of a recombinant fibronectin construct engineered to mimic the ECM form of fibronectin demonstrates that these residues are also critical for stimulating cell spreading and increasing cell proliferation. Cell proliferation has been tightly correlated with cell area. Using integrin-and heparin-binding fibronectin mutants, we found a positive correlation between cell spreading and growth when cells were submaximally spread on ECM protein-coated surfaces at the time of treatment. However, cells maximally spread on vitronectin or fibronectin still responded to the fibronectin matrix mimetic with an increase in growth, indicating that an absolute change in cell area is not required for the increase in cell proliferation induced by the matricryptic site of FNIII 1 .

Extracellular Matrix Fibronectin Mechanically Couples Skeletal Muscle Contraction With Local Vasodilation

“…A portion of the 9D2 epitope maps directly to the matricryptic site in FNIII-1. 17 Thus, these data support a model in which skeletal muscle contraction opens matricryptic FNIII-1 sites in the surrounding connective tissue matrix that, in turn, interact with cell surface receptors to induce arteriolar dilation.…”

“…Treatment of cultured cells with GST/III1H,8-10 stimulates cell spreading, growth, contractility, and migration to a similar extent as ECM fibronectin. [15][16][17] As such, this fibronectin matrix mimetic effectively bypasses the requirement for soluble, protomeric fibronectin to undergo a conformational change to initiate ECM fibronectin-specific signals. Therefore, if matricryptic FNIII-1 sites in the vicinity of the vasculature play a role in contraction-induced vasodilation, then infusing the fibronectin matrix mimetic, GST/III1H,8-10, directly onto tissues would be expected to stimulate vasodilation locally in the absence of skeletal muscle contraction.…”

Fibronectin fibrillogenesis regulates three-dimensional neovessel formation

“…Second, Fn matrix assembly allows for an increase in the local concentration and order of potential ␣ 5 ␤ 1 integrin-binding sites that would serve as a structural platform for cell spreading (Geiger et al 2001;Mao and Schwarzbauer 2005). Unfolded Fn molecules could further alter cell function by displaying matricryptic heparin-binding sites (Gui et al 2006). As neither mechanically soft matrices displaying high adhesivity nor rigid matrices decorated with suboptimal levels of proadhesive binding sites will induce changes in cytoskeletal architecture and intracellular stiffness (Discher et al 2005;Solon et al 2007), endothelial cells apparently use Fn matrix assembly to purposefully deposit an elastic layer of proadhesive binding sites whose concentration and topology support neovessel formation.…”

N-cadherin Cell-Cell Adhesion Complexes Are Regulated by Fibronectin Matrix Assembly

“…The blots were stripped, reblocked, and reprobed, as described previously (15 2 , allowed to adhere for 1 h, and then processed for immunofluorescence microscopy. In some experiments, adherent cells were treated with either fibronectin or the vehicle control, PBS, for an additional 2 h. Cell area was quantified as described previously (13). Briefly, phase contrast images of fixed cells from triplicate wells were obtained using a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI).…”

“…Soluble protomeric fibronectin circulates in the plasma at a high concentration and is subsequently deposited into the ECM in a fibrillar form by a tightly regulated, cell-dependent process (11). The ECM form of fibronectin stimulates changes in cell growth, migration, and cytoskeletal organization that are distinct from the effects of soluble fibronectin (12)(13)(14)(15)(16)(17)(18). Fibronectin has been implicated in regulating the localization, composition, and function of C-, E-, and VE-cadherin-containing …”