Possible Involvement of the Interaction of the α5 Subunit of α5β1 Integrin with the Synergistic Region of the Central Cell-Binding Domain of Fibronectin in Cells to Fibronectin Binding

1995 ◽  
Vol 216 (1) ◽  
pp. 273-276 ◽  
Author(s):  
Masanobu Obara ◽  
Katsutoshi Yoshizato
Keyword(s):  
Binding Domain ◽  
Central Cell ◽  
Cell Binding ◽  
Fibronectin Binding ◽  
Α5β1 Integrin ◽  
Cell Binding Domain ◽  
In Cells

scholarly journals 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.

1988 ◽  
Vol 106 (4) ◽  
pp. 1289-1297 ◽  
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.

scholarly journals 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.

1991 ◽  
Vol 114 (6) ◽  
pp. 1295-1305 ◽  
Author(s):  
T Nagai ◽  
N Yamakawa ◽  
S Aota ◽  
S S Yamada ◽  
S K Akiyama ◽  
...  
Keyword(s):  
Cell Spreading ◽  
Binding Domain ◽  
Central Cell ◽  
Site Directed Mutagenesis ◽  
Cell Binding ◽  
Matrix Assembly ◽  
Fibronectin Receptor ◽  
Molar Basis ◽  
Rgd Site ◽  
Cell Binding Domain

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.

scholarly journals Fibronectin mediates Treponema pallidum cytadherence through recognition of fibronectin cell-binding domain.

1985 ◽  
Vol 161 (3) ◽  
pp. 514-525 ◽  
Author(s):  
D D Thomas ◽  
J B Baseman ◽  
J F Alderete
Keyword(s):  
Treponema Pallidum ◽  
Binding Domain ◽  
Host Cells ◽  
Scatchard Analysis ◽  
Cell Surfaces ◽  
Cell Binding ◽  
High Affinity ◽  
Fibronectin Binding ◽  
Affinity Interaction ◽  
Cell Binding Domain

The specificity of the interaction between Treponema pallidum and fibronectin was demonstrated. Treatment of host cells with only antifibronectin sera and not anticollagen or antilaminin sera, inhibited treponemal cytadsorption. Incubation of fibronectin-coated coverslips with monoclonal antibody to the cell-binding domain of fibronectin reduced treponemal attachment to the same extent as antifibronectin serum. Both iodinated fibronectin and iodinated cell-binding domain bound to T. pallidum in a saturable manner. Specificity of the T. pallidum association with the cell-binding domain was the most effective inhibitor of the binding of either radioiodinated cell-binding domain or fibronectin to T. pallidum. Scatchard analysis gave Kd on the order of 10(-7) M for both cell-binding domain and fibronectin binding to T. pallidum, consistent with the high affinity interaction of these organisms with host cell surfaces. Finally, the same level of attachment of treponemes was achieved on coverslips coated with cell-binding domain as that observed for organisms incubated with fibronectin, indicating that the cell-binding domain polypeptide is functionally identical to fibronectin in mediating T. pallidum adherence.

Controlled release of the fibronectin central cell binding domain from polymeric microspheres

2004 ◽  
Vol 95 (3) ◽  
pp. 557-566 ◽  
Author(s):  
Camille Bouissou ◽  
Ursula Potter ◽  
Harri Altroff ◽  
Helen Mardon ◽  
Christopher van der Walle
Keyword(s):  
Controlled Release ◽  
Binding Domain ◽  
Central Cell ◽  
Cell Binding ◽  
Polymeric Microspheres ◽  
Cell Binding Domain

scholarly journals Fine mapping of inhibitory anti-α5 monoclonal antibody epitopes that differentially affect integrin–ligand binding

1999 ◽  
Vol 344 (2) ◽  
pp. 527-533 ◽  
Author(s):  
Louise BURROWS ◽  
Katherine CLARK ◽  
A. Paul MOULD ◽  
Martin J. HUMPHRIES
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Binding Domain ◽  
Central Cell ◽  
Subunit Structure ◽  
Cell Binding ◽  
Α Subunit ◽  
Type Iii ◽  
Integrin Ligand ◽  
Cell Binding Domain

The high-affinity interaction of integrin α5β1 with the central cell-binding domain of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the tenth type III repeat) and a second site Pro-His-Ser-Arg-Asn (PHSRN) in the adjacent ninth type III repeat, which synergizes with RGD. Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel peptidic ligand for α5β1, identified by phage display, which blocks α5β1-mediated cell adhesion to fibronectin. A key question is the location of the binding sites for these ligand sequences within the integrin. In this study we have identified residues that form part of the epitopes of three inhibitory anti-α5 monoclonal antibodies (mAbs): 16, P1D6 and SNAKA52. These mAbs have distinct functional properties. mAb 16 blocks the recognition of RGD and RRETAWA, whereas P1D6 blocks binding to the synergy sequence. The binding of SNAKA52 is inhibited by anti-β1 mAbs, indicating that its epitope is close to the interface between the α and β subunits. Residues in human α5 were replaced with the corresponding residues in mouse α5 by site-directed mutagenesis; wild-type or mutant human α5 was expressed on the surface of α5-deficient Chinese hamster ovary cells. mAb binding was assessed by flow cytometry and by adhesion to the central cell-binding domain of fibronectin or RRETAWA by cell attachment assay. All three epitopes were located to different putative loops in the N-terminal domain of α5. As expected, disruption of these epitopes had no effect on ligand recognition by α5β1. The locations of these epitopes are consistent with the β-propeller model for integrin α-subunit structure and allow us to propose a topological image of the integrin-ligand complex.

scholarly journals Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein.

1991 ◽  
Vol 2 (11) ◽  
pp. 951-964 ◽  
Author(s):  
J L Guan ◽  
J E Trevithick ◽  
R O Hynes
Keyword(s):  
Tyrosine Phosphorylation ◽  
Concanavalin A ◽  
Cell Spreading ◽  
Binding Domain ◽  
Heparin Binding ◽  
Cell Binding ◽  
Heparin Binding Domain ◽  
Coated Surfaces ◽  
Cell Binding Domain ◽  
In Cells

We describe a 120-kDa protein (pp120) that is phosphorylated on tyrosine in cells attached to fibronectin-coated surfaces. The protein appears to be located in focal contacts where it codistributes with beta 1 integrins. pp120 is distinct from the beta 1 subunit of integrins and from vinculin and alpha-actinin. pp120 is rapidly dephosphorylated in cells suspended by trypsinization but becomes rapidly phosphorylated in cells attaching and spreading on fibronectin. Attachment of cells to RGD-containing peptides, polylysine, or concanavalin A is not sufficient to induce phosphorylation of pp120. The 120-kDa cell-binding domain of fibronectin can induce some phosphorylation of pp120, but further phosphorylation occurs in the presence also of the heparin-binding domain of fibronectin. Phosphorylation of pp120 precedes, but is correlated with, subsequent cell spreading. Phosphorylation of pp120 can also be triggered by attachment of cells to anti-integrin antibodies, and this requires the cytoplasmic domain of the integrin beta 1 subunit. Thus interaction of beta 1 integrins with extracellular ligands (fibronectin or antibodies) triggers phosphorylation of an intracellular 120-kDa protein, pp120, that may be involved in the responses of cells to attachment.

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