Mostafavi-Pour Z, Askari J A, Whittard J D, Humphries M J
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, 2.205 Stopford Building, Oxford Road, M13 9PT, Manchester, UK.
Matrix Biol. 2001 Feb;20(1):63-73. doi: 10.1016/s0945-053x(00)00131-1.
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.
细胞外基质分子纤连蛋白（FN）是一种糖蛋白，其主要功能特性是支持细胞黏附。FN包含至少两个不同的细胞结合结构域：中央细胞结合结构域和HepII/IIICS区域。HepII区域由III型重复序列12 - 14组成，并含有蛋白聚糖结合位点，而可变剪接的IIICS片段拥有主要的α4β1整合素结合位点。细胞表面蛋白聚糖和整合素对于介导细胞与FN这一区域的黏附都很重要。通过比较肝素与HepII/IIICS区域不同重组剪接变体的结合情况，获得了在IIICS中央存在一个新的肝素结合位点的证据。该区域碱性氨基酸序列的定点诱变减少了肝素与重组HepII/IIICS蛋白的结合，并且与HepII区域的突变一起导致活性的协同丧失。使用包含III型重复序列12 - 15和全长IIICS区域的FN的H/120变体，以及包含III型重复序列12 - 15但缺乏高亲和力整合素结合LDV序列的FN的H/95变体，研究了细胞表面蛋白聚糖和整合素在介导细胞黏附中所起的相对作用。这是通过研究抗整合素抗体和外源性肝素对A375黑色素瘤细胞附着于野生型以及H/120和H/95的三种不同突变体（其中潜在的蛋白聚糖结合位点被部分或完全去除）的影响来实现的。发现A375细胞对H/120及其突变体的黏附涉及整合素和细胞表面蛋白聚糖结合的协同作用，尽管整合素起主要作用。抗整合素抗体和外源性肝素能够抑制黑色素瘤细胞对H/95的黏附，在这种情况下，黏附主要是由于细胞表面蛋白聚糖而非整合素结合。
