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. 1997 Mar 7;272(10):6159-66.
doi: 10.1074/jbc.272.10.6159.

Structural requirements for biological activity of the ninth and tenth FIII domains of human fibronectin

R P Grant  1 , C Spitzfaden, H Altroff, I D Campbell, H J Mardon
Affiliations

Structural requirements for biological activity of the ninth and tenth FIII domains of human fibronectin

R P Grant et al. J Biol Chem. .

Abstract

The ninth and tenth type III domains of fibronectin each contain specific cell binding sequences, RGD in FIII10 and PHSRN in FIII9, that act synergistically in mediating cell adhesion. We investigated the relationship between domain-domain orientation and synergistic adhesive activity of the FIII9 and FIII10 pair of domains. The interdomain interaction of the FIII9-10 pair was perturbed by introduction of short flexible linkers between the FIII9 and FIII10 domains. Incremental extensions of the interdomain link between FIII9 and FIII10 reduced the initial cell attachment, but had a much more pronounced effect on the downstream cell adhesion events of spreading and phosphorylation of focal adhesion kinase. The extent of disruption of cell adhesion depended upon the length of the interdomain linker. Nuclear magnetic resonance spectroscopy of the wild type and mutant FIII9-10 proteins demonstrated that the structure of the RGD-containing loop is unaffected by domain-domain interactions. We conclude that integrin-mediated cell adhesion to the central cell binding domain of fibronectin depends not only upon specific interaction sites, but also on the relative orientation of these sites. These data have implications for the molecular mechanisms by which integrin-ligand interactions are achieved.

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Figures

Fig. 1
Fig. 1. Domain structure of fibronectin
The diagram illustrates the organization of domains within a FN monomer. Binding regions for other ECM components are indicated below the molecule and the alternatively spliced EDIIIA, EDIIIB, and IIICS regions above. The central cell binding domain (CCBD). The three types of FN structural domains are represented by symbols: ▪, FN type I domain; ○, FN type II domain; open box with number inside, FN type III domain. The site of the disulfide bridges linking two monomers is indicated by SS.
Fig. 2
Fig. 2. Crystal structure of the FIII9–10 pair showing linker insertion point
Ribbon structure of FIII9–10 pair from Leahy et al. (44). Linkers were inserted immediately before valine 1416 (arrow), the first valine of FIII10. The sequences GRGDS and PHSRN are displayed in ball-and-stick format.
Fig. 3
Fig. 3. NMR spectroscopy of the RGD site of FIII10 and FIII9–10
For each residue a pair of strips from 1H,1H NOESY planes of three-dimensional 15N-correlated NOESY spectra of uniformly 15N-labeled FIII10 and FIII9–10 is shown. Strips were taken at the 15N frequency of the 15N,1H direct correlation peak. Left- and right-hand strips of each pair correspond to the spectra of FIII10 and FIII9–10, respectively. The positions of NH and CαH cross-peaks are indicated by rectangles and circles, respectively.
Fig. 4
Fig. 4. Inhibition of cell attachment by function-blocking anti-integrin antibodies
Adhesion of BHK cells to 100 μg ml−1 FN (shaded columns)- or GFIII9–10 (white columns)-coated plastic in the presence of 10 μg ml−1 anti-α2, anti-α3, anti-α5, or anti-αv antibody.
Fig. 5
Fig. 5. Cell attachment to wild type and mutant linker FIII9–10 proteins
BHK (A) and hESF (B) cell attachment at 100 μg ml−1 (shaded columns) and 6.25 μg ml−1 (white columns) coating concentration. Cell attachment to each of the test proteins was assessed by measurement of crystal violet incorporation at A570 nm and expressed relative to attachment on 100 μg ml−1 FN. The data shown represent the mean ± S.E. of at least four experiments.
Fig. 6
Fig. 6. BHK cell adhesion to wild type and mutant linker proteins
Phase contrast microscopy of BHK cells plated on FN (A) GFIII9–10 (B), GFIII9-PG-10 (C ), GFIII9-P[G]5-10 (D), GFIII10 (E), or GST (F) coated at 100 μg ml−1 protein. Bar = 25 μm.
Fig. 7
Fig. 7. Cell spreading on wild type and mutant linker proteins
BHK cells (A, C) and hESF cells (B, D) were assayed for spreading on FN (•), GFIII9–10 (○), GFIII10 (×), GFIII9-PG-10 (▪), GFIII9-P[G]5-10 (□), or GST (▴) on doubling dilutions of each test protein (A and B). The percentage of cells spread on proteins coated at 100 μg ml−1 (shaded columns), and 6.25 μg ml−1 (white columns) is shown in C and D. The data shown represent the mean ± S.E. of at least four experiments.
Fig. 8
Fig. 8. Inhibition of BHK cell adhesion by wild type and mutant FIII9–10 domains
Attachment (A) and spreading (B) of BHK cells to FN (coated at 10 μg ml−1) was assessed in the presence of doubling dilutions of FIII10 (×), FIII9–10 (○), FIII9-PG-10 (▪), FIII9-P[G]5-10 (□), or GRGDS peptide (▵).
Fig. 9
Fig. 9. Phosphorylation of FAK in BHK cells on wild type and mutant FIII9–10 fusion proteins
Total FAK and phosphorylated FAK (coprecipitated with anti-FAK antibodies) from BHK cells plated on plastic coated with 100 μg ml−1 fusion proteins were detected by Western blotting (A). The percentage of phosphorylation of FAK on the mutant proteins relative to phosphorylation on GFIII9–10 (100%) was determined by densitometric analyses of the Western blots in A (B).
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