iso-DGR sequences do not mediate binding of fibronectin N-terminal modules to adherent fibronectin-null fibroblasts.
Fibronectin (FN) without an RGD sequence (FN-RGE), and thus lacking the principal binding site for alpha5beta1 integrin, is deposited into the extracellular matrix of mouse embryos. Spontaneous conversion of (263)NGR and/or (501)NGR to iso-DGR possibly explains this enigma, i.e. ligation of iso-DGR by alphavbeta3 integrin may allow cells to assemble FN. Partial modification of (263)NGR to DGR or iso-DGR was detected in purified plasma FN by mass spectrometry. To test functions of the conversion, one or both NGR sequences were mutated to QGR in recombinant N-terminal 70-kDa construct of FN (70K), full-length FN, or FN-RGE. The mutations did not affect the binding of soluble 70K to already adherent fibroblasts or the ability of soluble 70K to compete with non-mutant FN or FN-RGE for binding to FN assembly sites. Non-mutant FN and FN-N263Q/N501Q with both NGRs mutated to QGRs were assembled equally well by adherent fibroblasts. FN-RGE and FN-RGE-N263Q/N501Q were also assembled equally well. Although substrate-bound 70K mediated cell adhesion in the presence of 1 mm Mn(2+) by a mechanism that was inhibited by cyclic RGD peptide, the peptide did not inhibit 70K binding to cell surface. Mutations of the NGR sequences had no effect on Mn(2+)-enhanced cell adhesion to adsorbed 70K but caused a decrease in cell adhesion to reduced and alkylated 70K. These results demonstrate that iso-DGR sequences spontaneously converted from NGR are cryptic and do not mediate the interaction of the 70K region of FN with the cell surface during FN assembly.
Xu
Jielin
J
Department of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, Wisconsin 53706, USA.
Maurer
Lisa M
LM
Hoffmann
Brian R
BR
Annis
Douglas S
DS
Mosher
Deane F
DF
D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019096:Vitronectin / Q000737:chemistry
D005353:Fibronectins; D009842:Oligopeptides; D011994:Recombinant Proteins; D019096:Vitronectin; C047981:arginyl-glycyl-aspartic acid