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Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis.
Redick SD
,
Settles DL
,
Briscoe G
,
Erickson HP
.
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Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.
Figure 1. Two views of the three-dimensional structure of FN9-10 (PDB ID: 1fnf; Leahy et al. 1996) and a linear representation of the relevant regions. The RGDS segment is in red, and the two residues with the next strongest effect on adhesion (R1445 and R1379) are in magenta. The remainder of the PHSRN site is shown in light blue (mutation of these residues had little effect on adhesion activity (Aota et al. 1994)). The residues shown in yellow contribute to adhesion, but the effects of these mutations are most obvious when combined with the mutation R1379. The C-C′ loop (NSP), which does not contribute to adhesion, is colored green. A linear representation of the relevant sequences is shown below with residue coloring corresponding to that shown above. The amino acid sequence of FN is shown for the seven species from which it has been cloned.
Figure 2. Cell adhesion to proteins with mutations in or adjacent to the PHRSN sequence. ΔRGDS and FN10 were included as controls to indicate adhesion in the absence of RGD or FN9. The average number of cells bound to each protein is given as a percentage of the number of cells attached to wild-type FN7–10. Note that in this experiment, the wells were coated with 20 μg/ml protein, which is the high concentration in later experiments.
Figure 5. Competitive inhibition of cell adhesion by soluble proteins. Just before the addition of cells, soluble FN10, FN7–10, or FN7–10 mutant proteins were added to wells coated with 20 μg/ml FN7–10, and the plates were subjected to the centrifugation assay.
Figure 3. Cell adhesion defects of T1385A and N1386A mutants. We tested cell adhesion to plates coated with two concentrations of wild-type and mutant FN7–10: 20 μg/ml (high FN) or 0.8 μg/ml (low FN). Adhesion to each mutant protein was normalized to the wild type at each coating concentration; adhesion to 0.8 μg/ml wild-type FN7–10 was ∼70% of adhesion to 20 μg/ml. The low concentration of FN gave a more sensitive indication of adhesion defects.
Figure 7. A model for FN9-10 binding to α5β1 integrin. FN residue coloring is as in Fig. 1. The head of the integrin dimer is ∼10–12-nm-wide, based on electron microscopy studies (Carrell et al. 1985; Nermut et al. 1988), and is shown approximately to scale with FN9-10. The contact between the aspartate carboxylate of the RGD and α5 is shown in bold. The contact between the arginine guanidinium and the DDL in β1 is shown as a gray line to indicate that it lies in a plane behind the Asp/α5 contact. Although we have not determined which integrin subunit R1445 contacts, a line illustrates a possible contact with the β subunit. The contacts of the most important synergy residues are shown as solid lines, and the contributory residues are shown as dotted lines.
Figure 4. Arginine residues 1369 and 1371 were examined for their effect on adhesion. Plates were coated with 20 μg/ml (high FN) or 0.8 μg/ml (low FN) of the indicated mutant FN7–10.
Figure 6. Gradient sedimentation analysis of FN7–10 mutant proteins. The indicated proteins were sedimented through 15–40% glycerol gradients, and the fractions were analyzed by SDS-PAGE. Ovalbumin (OVA, 3.5 S) and bovine serum albumin (BSA, 4.6 S) were used as standards. Lanes are labeled as follows: ST, starting material; 5–10, fractions 5–10; and M, markers (with size indicated on the lower left gel). The 30–40% recovery of protein is typical for this type of gradient because of the loss on the walls of the tubes.
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