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A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells.

Abstract
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.
AuthorsJosé V Moyano, Alfredo Maqueda, Juan P Albar, Angeles Garcia-Pardo
JournalThe Biochemical journal (Biochem J) Vol. 371 Issue Pt 2 Pg. 565-71 (Apr 15 2003) ISSN: 0264-6021 [Print] England
PMID12519080 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Fibronectins
  • Peptide Fragments
  • Recombinant Proteins
  • Heparin
Topics
  • Amino Acid Sequence
  • Binding Sites
  • Cell Adhesion (drug effects, physiology)
  • Fibronectins (chemistry, metabolism)
  • Heparin (metabolism, physiology)
  • Humans
  • Jurkat Cells
  • Melanoma
  • Molecular Sequence Data
  • Peptide Fragments (chemistry, metabolism, pharmacology)
  • Recombinant Proteins (chemistry, metabolism)
  • Stress, Mechanical
  • Transfection
  • Tumor Cells, Cultured

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