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 α5β1 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 α5β1 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 α5β1 for the progression of the cytoskeletal response and that these include activation of RhoA.
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April 2003
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Research Article|
April 15 2003
A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells
José V. MOYANO;
José V. MOYANO
∗Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Velázquez 144, 28006 Madrid, Spain
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Alfredo MAQUEDA;
Alfredo MAQUEDA
∗Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Velázquez 144, 28006 Madrid, Spain
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Juan P. ALBAR;
Juan P. ALBAR
†Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, CSIC, Cantoblanco, 28049 Madrid, Spain
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Angeles GARCIA-PARDO
Angeles GARCIA-PARDO
1
∗Departamento de Inmunología, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), Velázquez 144, 28006 Madrid, Spain
1To whom correspondence should be addressed (e-mail agarciapardo@cib.csic.es).
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Publisher: Portland Press Ltd
Received:
August 28 2002
Revision Received:
December 12 2002
Accepted:
January 08 2003
Accepted Manuscript online:
January 08 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 371 (2): 565–571.
Article history
Received:
August 28 2002
Revision Received:
December 12 2002
Accepted:
January 08 2003
Accepted Manuscript online:
January 08 2003
Citation
José V. MOYANO, Alfredo MAQUEDA, Juan P. ALBAR, Angeles GARCIA-PARDO; A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells. Biochem J 15 April 2003; 371 (2): 565–571. doi: https://doi.org/10.1042/bj20021344
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