Cell adhesion to
fibronectin results in formation of actin stress fibres and focal adhesions. In fibroblasts, this response requires two co-operative signals provided by interactions of the RGD sequence with
alpha5beta1 integrin and the
heparin-binding domain II (
Hep II) domain with
syndecan-4. Within
Hep II, this activity was mapped to repeat III13 and to the
peptide FN-C/H-V(WQPPRARITGY, repeat III14). We previously described that the synthetic
heparin-binding
peptide/III5 (HBP/III5) (WTPPRAQITGYRLTVGLTRR, repeat III5) binds
heparin and mediates cell adhesion via
chondroitin sulphate
proteoglycans. We have now studied whether HBP/III5 co-operates with alpha5beta1 and drives a full cytoskeletal response in
melanoma cells. SKMEL-178 cells attached and spread on the RGD-containing FNIII7-FNIII10 (FNIII7-10) fragment, but did not form stress fibres or focal adhesions. Co-immobilization of HBP/III5 with FNIII7-10 or adding soluble HBP/III5 to cells prespread on FNIII7-10, effectively induced these structures. Cell transfection with dominant-negative N19RhoA, a member of the
small GTPase family, abolished the HBP/III5 effect. Both
chondroitinase and
heparitinase diminished focal adhesions, indicating that both types of
proteoglycans bound HBP/III5 in
melanoma cells. We have mapped the active sequence of HBP/III5 to YRLTVGLTRR, which is a novel sequence in
fibronectin with focal-adhesion-promoting activity. The last two
arginine (R) residues of this sequence are required for activity, since their replacement by
alanine completely abrogated the HBP/III5 cytoskeletal effect. Moreover, this sequence is also active in the context of large
fibronectin fragments. Our results establish that the
Hep III region provides co-operative signals to alpha5beta1 for the progression of the cytoskeletal response and that these include activation of RhoA.