Fig. 7. B5 is a noncompetitive allosteric inhibitor that induces a low-affinity state.

(A) Latency-associated peptide decreases B5 binding to αvβ8-expressing HT1080 cells. β8-expressing HT1080 cells stained with B5 (2 μg/ml) with increasing concentrations of latency-associated peptide (LAP) reported as mean fluorescence intensity (MFI) (n = 6). (B) Lineweaver-Burk plots of solid-phase binding assays of αvβ8-AP binding to latency-associated peptide with two different concentrations of B5 (squares), or RGD peptide (circles) as a competitive inhibitor control, or no inhibitor (triangles-dotted line). B5 plots show similar x intercepts as uninhibited receptor but different slopes and y intercepts consistent with noncompetitive inhibition. RGD plots intersect above the x axis with the uninhibited receptor consistent with a competitive mode of inhibition. Representative of two experiments with similar results. (C to E) B5 induces low-affinity binding sufficient to mediate cell adhesion, but insufficient to support TGF-β activation. (C) β8-expressing 293 cells adhered to latency-associated peptide with saturating concentrations of RGD peptides and B5 (filled squares, solid line) or isotype control (open squares, hashed line). Assays performed as in Fig. 3A. n = 3. **P < 0.01 by unpaired Student’s t test. (D) B5 induces a low-affinity state maximally inhibiting the binding of soluble αvβ8 to latency-associated peptide by 92% in the presence of RGE peptide (filled inverted triangles, hashed lines); remaining binding blocked completely by RGD peptide (filled squares, solid line). Boxed magnified area of the highest B5 and RGD/E concentrations shows small amount of residual binding remaining with B5 and RGE peptide completely blocked by B5+RGD. Isotype with RGD peptide (open squares, solid line) or RGE peptide (open inverted triangles, hashed line). *P = 0.039 by nonlinear regression and F test of the bottom of each data set. (E) B5 blocks TGF-β activation by ~70%, and the addition of RGD peptide completely blocks remaining activation. β8-transfected 293 cells with RGD peptide and saturating concentrations of B5 (filled squares, solid line) or isotype control (open squares, hashed lines) at the indicated concentrations. Mock-transfected 293 cells with RGD peptide and saturating concentrations of B5 (filled diamonds, solid line) or isotype control (open triangles, hashed lines). n = 4. ***P < 0.001, by ANOVA and Tukey’s post-test of the highest concentration of RGD peptide and antibodies.