Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.

Download Full-text

Characterization of regions of fibronectin besides the arginine-glycine-aspartic acid sequence required for adhesive function of the cell-binding domain using site-directed mutagenesis

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)98498-x ◽

1991 ◽

Vol 266 (24) ◽

pp. 15938-15943 ◽

Cited By ~ 3

Author(s):

S. Aota ◽

T. Nagai ◽

K.M. Yamada

Keyword(s):

Aspartic Acid ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Cell Binding ◽

Arginine Glycine Aspartic Acid ◽

Cell Binding Domain

Download Full-text

C-terminal heparin-binding domain of fibronectin regulates integrin-mediated cell spreading but not the activation of mitogen-activated protein kinase

Biochemical Journal ◽

10.1042/bj3600239 ◽

2001 ◽

Vol 360 (1) ◽

pp. 239-245 ◽

Cited By ~ 7

Author(s):

Jungyean KIM ◽

Innoc HAN ◽

Yeonhee KIM ◽

Seungin KIM ◽

Eok-Soo OH

Keyword(s):

Cell Spreading ◽

Binding Domain ◽

Mitogen Activated Protein Kinase ◽

Heparin Binding ◽

Cell Binding ◽

Rat Embryo ◽

Mapk Activation ◽

Mitogen Activated Protein ◽

Heparin Binding Domain ◽

Cell Binding Domain

Fibronectin (FN) stimulates multiple signalling events including mitogen-activated protein kinase (MAPK) activation. During cell spreading, both the cell-binding domain and the C-terminal heparin-binding domain (HepII) of FN co-operatively regulate cytoskeleton organization. However, in comparison with the large number of studies on the functions of cell-binding domain, there is little information about the role of HepII. We therefore investigated the effect of HepII on integrin-mediated cell spreading and adhesion on FN and MAPK activation. In contrast with cells on FN substrates, rat embryo fibroblasts on FN120, which lacks HepII, were less spread, had weaker adhesion to FN and failed to form focal adhesions and actin stress fibres. Phosphotyrosine was present in the focal contacts of rat embryo fibroblasts on FN within 30min but was absent from cells on FN120. Overall, tyrosine phosphorylation was much less in cell lysates from cells on FN120, with decreased phosphorylation of focal adhesion kinase (‘pp125FAK’) on tyrosine-397, implying additional regulation of tyrosine phosphorylation by HepII. Nevertheless, adhesion-mediated MAPK activity was similar in cells on FN and on FN120. Furthermore, cells spread on FN and on FN120 substrates showed similar MAPK activation in response to treatment with epidermal growth factor and with platelet-derived growth factor. Consistently, overexpression of syndecan-4, which binds to HepII, enhanced cell spreading and adhesion on FN but did not affect integrin-mediated MAPK activation. We therefore conclude that both HepII and syndecan-4 regulate integrin-mediated cell spreading but not MAPK activation.

Download Full-text

TSG-6 binds via its CUB_C domain to the cell-binding domain of fibronectin and increases fibronectin matrix assembly

Matrix Biology ◽

10.1016/j.matbio.2007.10.003 ◽

2008 ◽

Vol 27 (3) ◽

pp. 201-210 ◽

Cited By ~ 30

Author(s):

S KUZNETSOVA ◽

D MAHONEY ◽

G MARTINMANSO ◽

T ALI ◽

H NENTWICH ◽

...

Keyword(s):

Binding Domain ◽

Cell Binding ◽

Matrix Assembly ◽

Fibronectin Matrix ◽

Cell Binding Domain

Download Full-text

Neurite extension of chicken peripheral nervous system neurons on fibronectin: relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment.

The Journal of Cell Biology ◽

10.1083/jcb.106.4.1289 ◽

1988 ◽

Vol 106 (4) ◽

pp. 1289-1297 ◽

Cited By ~ 77

Author(s):

M J Humphries ◽

S K Akiyama ◽

A Komoriya ◽

K Olden ◽

K M Yamada

Keyword(s):

Nervous System ◽

Cell Adhesion ◽

Neurite Outgrowth ◽

Peripheral Nervous System ◽

Binding Domain ◽

Central Cell ◽

Cell Binding ◽

Alternatively Spliced ◽

Cell Binding Domain ◽

Iiics Region

Fibronectin contains at least two domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types, including fibroblasts. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in fibronectin-mediated neurite extension of neurons from chick embryo dorsal root and sympathetic ganglia. In studies using explanted ganglia, both fl3, a 75-kD tryptic fragment of human plasma fibronectin containing the central cell-binding domain, and CS1-IgG, a synthetic peptide-IgG conjugate containing the principal cell adhesion site from the IIICS, supported neurite outgrowth after adsorption onto the substrate. The maximal activities of fl3 and CSl-IgG were 45-55% and 25-30% that of intact fibronectin, respectively. Co-coating of the substrate with f13 and CS1-IgG produced an additive stimulation of neurite outgrowth, the extent of which approached that obtained with fibronectin. Similar results were obtained with purified neuronal cell preparations isolated by tryptic dissociation of dorsal root ganglia. In complementary studies, blockage of the adhesive function of either the central cell-binding domain (with mAb 333, an antiadhesive monoclonal antibody) or the IIICS (with CS1 peptide), resulted in approximately 60 or 30% reduction in fibronectin-mediated neurite outgrowth, respectively. When tested in combination, the inhibitory activities of mAb 333 and CSl were additive. From these results, we conclude that neurons from the peripheral nervous system can extend neurites on both the central cell-binding domain and the IIICS region of fibronectin, and that these cells are therefore the first normal, embryonic cell type shown to adhere to the IIICS. These results suggest that spatiotemporal fluctuations in the alternative mRNA splicing of the IIICS region of fibronectin may be important in regulation of cell adhesive events during development of the peripheral nervous system.

Download Full-text