Identification of a novel heparin-binding site in the alternatively spliced IIICS region of fibronectin: roles of integrins and proteoglycans in cell adhesion 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 alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. 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 contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins 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 partially 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-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, 2.205 Stopford Building, Oxford Road, M13 9PT, Manchester, UK.
Z
Z
Mostafavi-Pour
J A
JA
Askari
J D
JD
Whittard
M J
MJ
Humphries
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism / Q000502:physiology; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism / Q000502:physiology; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D039441:Integrin alpha4; D039041:Integrin alpha4beta1; D019012:Integrin beta1 / Q000378:metabolism / Q000502:physiology; D016023:Integrins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism / Q000502:physiology*; D017469:Receptors, Fibronectin / Q000378:metabolism / Q000502:physiology; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism / Q000502:physiology; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D005353:Fibronectins; D006025:Glycosaminoglycans; D039041:Integrin alpha4beta1; D019012:Integrin beta1; D016023:Integrins; D020033:Protein Isoforms; D011509:Proteoglycans; D017469:Receptors, Fibronectin; D016201:Receptors, Lymphocyte Homing; D039441:Integrin alpha4; D006493:Heparin