A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells.
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.
Moyano
José V
JV
Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Velázquez 144, 28006 Madrid, Spain.
Maqueda
Alfredo
A
Albar
Juan P
JP
Garcia-Pardo
Angeles
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D019169:Jurkat Cells; D008545:Melanoma; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013314:Stress, Mechanical; D014162:Transfection; D014407:Tumor Cells, Cultured
D005353:Fibronectins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006493:Heparin