Identification of the peptide sequences within the EIIIA (EDA) segment of fibronectin that mediate integrin alpha9beta1-dependent cellular activities.
Alternative splicing of the fibronectin (FN) gene transcript provides an efficient mechanism for generating functionally appropriate forms of this adhesive glycoprotein in situ. Cellular FNs that include the EIIIA and/or EIIIB FN-III segments are prominently expressed during embryogenesis, wound healing, tumor progression, and inflammation. However, the roles of this domain in altering overall FN protein structure and regulating cellular function remain unclear. We previously reported that two integrins, alpha9beta1 and alpha4beta1, ligate the EIIIA segment ( Liao, Y. F., Gotwals, P. J., Koteliansky, V. E., Sheppard, D., and Van De Water, L. (2002) J. Biol. Chem. 277, 14467-14474 ) and that the epitopes for function-blocking monoclonal antibodies lie within the C-C' loop of EIIIA ( Liao, Y. F., Wieder, K. G., Classen, J. M., and Van De Water, L. (1999) J. Biol. Chem. 274, 17876-17884 ). We have now performed site-directed mutagenesis within the EIIIA segment and carried out cell adhesion assays on these mutant EIIIAs. We find that the Asp(41) and Gly(42) residues within the C-C' loop of EIIIA are necessary for integrin alpha9beta1 binding. Synthetic peptides based on the predicted important amino acid sequence from the C-C' loop encode sufficient information to completely inhibit alpha9beta1-mediated cell adhesion. We also report that EIIIA promotes filopodial formation in alpha9beta1-expressing cells accompanied by Cdc42 activation. Our data provide a cellular activity for the EIIIA segment, evidence for conformational lability, and peptide sequences for probing EIIIA functions in vitro and in vivo.
Shinde
Arti V
AV
Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA.
Bystroff
Christopher
C
Wang
Chunyu
C
Vogelezang
Mariette G
MG
Vincent
Peter A
PA
Hynes
Richard O
RO
Van De Water
Livingston
L
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005353:Fibronectins; D016023:Integrins; D011993:Recombinant Fusion Proteins; C113256:integrin alpha 9 beta 1