Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in celladhesion to fibronectin splice variants

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4 beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesio n of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtaine d for the existence of a novel heparin-binding site in the centre of the II ICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjun ction with mutations in the HepII region, caused a synergistic loss of acti vity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contain s type III repeats 12-15 but lacks the high affinity integrin-binding LDV s equence, the relative roles played by cell-surface proteoglycans and integr ins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were pa rtially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surf ace proteoglycan binding, although integrin made a dominant contribution. A nti-integrin antibodies and exogenous heparin were capable of inhibiting me lanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding. (C) 2001 Elsevier Scie nce B.V./International Society of Matrix Biology. All rights reserved.