Figure 1.

The N-terminal domain of TIMP-2 demonstrates enhanced α3β1 integrin-binding activity and retains anti-mitogenic activity. a) Integrin α3β1-binding to Ala+TIMP-2, N-TIMP-2 and C-TIMP-2/GST-fusion proteins. Wells of Maxisorb™ 96 well plates were coated with protein solutions, blocked and probed for integrin binding activity as described in Materials and Methods. Integrin-binding to N-TIMP-2 was two fold greater compared with Ala+TIMP-2 and three fold greater than C-TIMP-2 (* p<0.0001). The results demonstrate that the N-terminal region of the TIMP-2, containing the OB fold has the most potent α3β1-binding activity. α3β1-binding to N-TIMP-2 was 2.5 fold increased over binding to Ala+TIMP-2 when values are normalized for coating concentrations. b) Binding of α3β1 to N-TIMP-2 shows concentration dependence and is linear (R²= 0.9881) over the range of protein coating concentrations tested. Assuming saturation binding for the coating protein on the Maxisorb™ plates, the calculated dissociation constants (K_D) for the α3β1-binding interaction with N-TIMP-2 were 0.35, 0.12 and 0.19 nM for the 1, 5 and 10 µM coating concentrations, respectively, and the mean value was K_D ~0.22 ± 0.12 nM. These values are in excellent agreement with values previously reported for TIMP-2 binding to the cell surface in an α3β1-dependent fashion (K_D=0.9 ± 0.12 nM) and are consistent with the approximate 2.5 fold increase in N-TIMP-2 binding compared with Ala+TIMP-2 binding observed in Figure 1a. c) The N-terminal α3β1-binding domain of TIMP-2 retains anti-mitogenic activity. VEGF-A-stimulated (10 ng/mL) hMVECs demonstrate greater than two-fold increase in cell number after 24 h compared with control (untreated cells). Ala+TIMP-2 and N-TIMP-2 significantly inhibited the mitogenic response to VEGF-A in vitro, * p<0.0001.