Cooperative Role for Activated α4β1 Integrin and Chondroitin Sulfate Proteoglycans in Cell Adhesion to the Heparin III Domain of Fibronectin

doi:10.1074/jbc.274.1.135

Cooperative Role for Activated α4β1 Integrin and Chondroitin Sulfate Proteoglycans in Cell Adhesion to the Heparin III Domain of Fibronectin

IDENTIFICATION OF A NOVEL HEPARIN AND CELL BINDING SEQUENCE IN REPEAT III5*

From the ^‡Departamento de Inmunologı́a, Centro de Investigaciones Biológicas, CSIC, 28006 Madrid, Spain, the ^¶Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa, Italy, and the ^‖Departamento de Inmunologı́a y Oncologı́a, Centro Nacional de Biotecnologı́a, CSIC, 28049 Madrid, Spain

Abstract

We recently reported that the heparin (Hep) III domain of fibronectin contains the H2 cell adhesion site in repeat III5 which binds activated α4 integrins. We have now further characterized the heparin and cell binding activities of this domain. A recombinant fragment containing repeats III4-III5 (FN-III4–5) induced Jurkat cell adhesion upon integrin activation with Mn²⁺ or TS2/16 monoclonal antibody (anti-β1). Adhesion of Mn²⁺-treated cells to FN-III4–5 or FN-III5 fragments was inhibited by chondroitinase ABC and ACII but not by the anti-α4 monoclonal antibody HP2/1. In contrast, HP2/1 completely blocked adhesion of TS2/16-treated cells while chondroitinase had a partial (FN-III4–5) or minor (FN-III5) effect. Thus, the role of each receptor depended on the stimulus used to activate α4β1. The combination of HP2/1 and chondroitinase at dilutions which did not inhibit when used individually abolished adhesion of Mn²⁺ or TS2/16-treated cells to both fragments, indicating a cooperative effect between α4β1 and chondroitin sulfate proteoglycans (CSPG). Furthermore, we have identified a 20-amino acid sequence in III5 (HBP/III5) which binds heparin and induces cell adhesion via CSPG exclusively. Although soluble HBP/III5 was a poor inhibitor, when combined with H2, it abolished adhesion to FN-III4–5 and FN-III5 fragments. These results establish that adhesion to the Hep III domain involves the cooperation of activated α4β1 and CSPG and show that HBP/III5 is a novel heparin and CSPG-binding site contributing to cell adhesion to this domain.

Footnotes

↵* This work was supported by Grants SAF97-0064-C03-02 from the Comisión Interministerial de Ciencia y Tecnologı́a (CICYT), 94/0277 from Fondo de Investigaciones Sanitarias (FIS), and partially by funds from the Associazione Italiana per la Ricerca sul Cancro (to L. Z).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Supported by a fellowship from Fondo de Investigaciones Sanitarias.
↵** To whom correspondence should be addressed: Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain. Tel.: 34-91-564-4562 (ext. 4430); Fax: 34-91-562-7518; E-mail: agarciapardo{at}fresno.csic.es.
Abbreviations:

Fn

fibronectin

CS

chondroitin-sulfate

HS

heparan sulfate

PG

proteoglycan

GAG

glycosaminoglycan

HBP/III5

heparin-binding peptide in repeat III5

CSPG

chondroitin sulfate proteoglycan

mAb

monoclonal antibody

KLH

keyhole lympet hemocyanin

Hep

heparin.
- Received June 26, 1998.
- Revision received September 28, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.