We have defined the molecular basis of cell adhesion to fibrillin-1, the major structural component of extracellular microfibrils that are associated with elastic fibres. Using human dermal fibroblasts, and recombinant domain swap fragments containing the Arg-Gly-Asp motif, we have demonstrated a requirement for upstream domains for integrin-α5β1-mediated cell adhesion and migration. An adjacent heparin-binding site, which supports focal adhesion formation, was mapped to the fibrillin-1 TB5 motif. Site-directed mutagenesis revealed two arginine residues that are crucial for heparin binding, and confirmed their role in focal adhesion formation. These integrin and syndecan adhesion motifs juxtaposed on fibrillin-1 are evolutionarily conserved and reminiscent of similar functional elements on fibronectin, highlighting their crucial functional importance.

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Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III 12-14 region of fibronectin (HepII domain) through its HS chains. The fine structure of HS is critical to growth factor responses, and whether this extends to matrix ligands is unknown but is suggested from in vitro experiments. Cell attachment to HepII showed that heparin oligosaccharides of >14 sugar residues were required for optimal inhibition. The presence of N-sulfated glucosamine in the HS was essential, whereas 2-O-sulfation of uronic acid or 6-O-sulfation of glucosamine had marginal effects. In the more complex response of focal adhesion formation through syndecan-4, N-sulfates were again required and also glucosamine 6-O-sulfate. The significance of polymer N-sulfation and sulfated domains in HS was confirmed by studies with mutant Chinese hamster ovary cells where heparan sulfation was compromised. Finally, focal adhesion formation was absent in fibroblasts synthesizing short HS chains resulting from a gene trap mutation in one of the two major glucosaminoglycan polymerases (EXT1). Several separate, specific properties of cell surface HS are therefore required in cell adhesion responses to the fibronectin HepII domain.Interactions involving both the integrin-and heparin-binding domains of fibronectin contribute to a complete adhesion response in normal fibroblasts (1) and in melanoma (2) and neuroblastoma cells (3). The primary receptor for adhesion to fibronectin commonly involves the RGD motif of repeat III 10 through integrins such as ␣5␤1 (4), but this integrin-ligand interaction is only sufficient for attachment and spreading. Additional signaling through the cell surface proteoglycan syndecan-4 is required for focal adhesion formation and rearrangement of the actin cytoskeleton into bundled stress fibers (5, 6). Focal adhesions are stable points of contacts that occur between cells and the extracellular matrix. Signaling from focal adhesions contributes not only to cell adhesion but to dynamic changes in gene expression, apoptosis regulation, and control of the cell cycle (7). Syndecan-4 is a ubiquitous vertebrate transmembrane heparan sulfate proteoglycan (HSPG), 2 which localizes to focal adhesions and acts as a co-receptor with ␣5␤1 integrin (1, 8, 9). Syndecan-4 interacts with the HepII domain of fibronectin through its heparan sulfate chains and contributes to additional signals by activating protein kinase C-␣ (10) in the presence of inositol phospholipids (reviewed in Refs. 11 and 12).Heparan sulfate binding occurs primarily via the HepII domain (containing the FN type III repeats 12-14) in the C-terminal region of fibronectin. Solution interaction studies have shown that this domain may contain two distinct HS/heparinbinding sites (13) of which the III 13 module is the main binding site for the initiation of focal adhe...

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