IGD motifs, which are required for migration stimulatory activity of fibronectin type I modules, do not mediate binding in matrix assembly

Abstract

Picomolar concentrations of proteins comprising only the N-terminal 70-kDa region (70K) of fibronectin (FN) stimulate cell migration into collagen gels. The Ile-Gly-Asp (IGD) motifs in four of the nine FN type 1 (FNI) modules in 70K are important for such migratory stimulating activity. The 70K region mediates binding of nanomolar concentrations of intact FN to cell-surface sites where FN is assembled. Using baculovirus, we expressed wildtype 70K and 70K with Ile-to-Ala mutations in (3)FNI and (5)FNI; (7)FNI and (9)FNI; or (3)FNI, (5)FNI, (7)FNI, and (9)FNI. Wildtype 70K and 70K with Ile-to-Ala mutations were equally active in binding to assembly sites of FN-null fibroblasts. This finding indicates that IGD motifs do not mediate the interaction between 70K and the cell-surface that is important for FN assembly. Further, FN fragment N-(3)FNIII, which does not stimulate migration, binds to assembly sites on FN-null fibroblast. The Ile-to-Ala mutations had effects on the structure of FNI modules as evidenced by decreases in abilities of 70K with Ile-to-Ala mutations to bind to monoclonal antibody 5C3, which recognizes an epitope in (9)FNI, or to bind to FUD, a polypeptide based on the F1 adhesin of Streptococcus pyogenes that interacts with 70K by the β-zipper mechanism. These results suggest that the picomolar interactions of 70K with cells that stimulate cell migration require different conformations of FNI modules than the nanomolar interactions required for assembly.

Figure 1. Diagram of FN and FN fragments and location of IGD motifs in 70K.

(A) The EDA+, V89 splice variant subunit of FN is shown consisting of 12 FNI modules (ovals), two FNII modules (diamonds), and 16 FNIII modules (squares). Plasma FN lacks EDA and one subunit contains a variable region and the other subunit lacks it. Modules are numbered to facilitate naming recombinant proteins according to modular content. MSF is the N-terminus through the sequence encoded by the ¹FNIIIa exon and 10 intronic amino acids . FNI modules containing IGD motifs are indicated with an *. (B) Sequence of FUD with presumptive binding sites for FNI modules in bold and underlined and N- and C-terminal tails in lower case.

Figure 2. 70K with Ile-to-Ala mutations bind to FN^−/− fibroblasts.

(A) FN^−/− fibroblasts adherent to FN-coated coverslips were provided 40 nM FITC-70K, FITC-70K I150/242A, FITC-70K I480/572A, or FITC-70K I150/242/480/572A for 1 h before fluorescent microscopic imaging of the FITC fluorochrome. Results are representative of multiple fields of each condition examined in four separate experiments. Bar, 10 µm. (B) Western blot of cell lysates from cells provided FITC-70K (1), FITC-70K I150/242A (2), FITC-70K I480/572A (3), or FITC-70K I150/242/480/572A (4). The asterisk denotes the FITC-70K band. The Western blot was probed with rabbit anti-FITC followed by peroxidase-conjugated donkey anti-rabbit IgG.

Figure 3. N-³FNIII binds to FN^−/− fibroblasts.

FN^−/− fibroblasts adherent to ¹FNIII-C EDA+-coated coverslips were provided 30 nM N-³FNIII or FN subunit (15 nM dimeric FN) in the presence of 200 nM LPA for 3 h, washed, fixed, and immunostained with the mAb 5C3 followed by rhodamine labeled secondary antibody. Bar, 10 µm.

Figure 4. Structural alterations in 70K with IGD mutations.

Binding relative to no competitor of 1∶30,000 5C3 ascites (A) or 1∶50,000 7D5 ascites (B) to coated FN in the presence of increasing concentration of 70K (□), 70K I150/242A (▵), 70K I480/572A (▾), or 70K I150/242/480/572A (♦). Values are mean plus/minus standard deviation of 3 experiments.

Figure 5. 70K with Ile-to-Ala mutations compete less well for binding of b-FUD to adsorbed FN.

(A) Binding relative to no competitor of 0.3 nM b-FUD to coated FN in the presence of increasing concentrations of 70K (□), 70K I150/242A (▵), 70K I480/572A (▾), or 70K I150/242/480/572A (♦). (B) Binding relative to no competitor of 0.3 nM b-FUD to FN in the presence of increasing concentrations of human 70K (□) or rat 70K (▿). Values are mean plus/minus standard deviation of 3 experiments.