Cooperative role for activated alpha 4 beta 1 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

We recently reported that the heparin (Hep) III domain of fibronectin conta ins the H2 cell adhesion site in repeat III5 which binds activated alpha 4 integrins, We have now further characterized the heparin and cell binding a ctivities of this domain. A recombinant fragment containing repeats III4-II I5 (FN-III4-5) induced Jurkat cell adhesion upon integrin activation with M n2+ or TS2/16 monoclonal antibody (anti-beta 1). Adhesion of Mn2+ treated c ells to FN-III4-5 or FN-IIIB fragments was inhibited by chondroitinase ABC and ACII but not by the anti-alpha 4 monoclonal antibody HP2/1, In contrast , HP2/1 completely blocked adhesion of TS2/16-treated cells while chondroit inase had a partial (FN-III4-5) or minor (FN-III5) effect. Thus, the role o f each receptor depended on the stimulus used to activate alpha 4 beta 1, T he combination of HP2/1 and chondroitinase at dilutions which did not inhib it when used individually abolished adhesion of Mn2+ or TS2/16-treated cell s to both fragments, indicating a cooperative effect between alpha 4 beta 1 and chondroitin sulfate proteoglycans (CSPG). Furthermore, we have identif ied a 20-amino acid sequence in III5 (HBP/III5) which binds heparin and ind uces cell adhesion via CSPG exclusively. Although soluble HBP/III5 was a po or inhibitor, when combined with H2, it abolished adhesion to FN-III4-5 and FN-III5 fragments. These results establish that adhesion to the Rep III do main involves the cooperation of activated alpha 4 beta 1 and CSPG and show that HBP/III5 is a novel heparin and CSPG-binding site contributing to cel l adhesion to this domain.