Related Experiment Videos
Cell surface binding of TIMP-2 and pro-MMP-2/TIMP-2 complex
M R Emmert-Buck1, H P Emonard, M L Corcoran
1Laboratory of Pathology, National Cancer Institute, Bethesda, MD 20892, USA.
Abstract:
Tissue inhibitor of metalloproteinases (TIMP-2) is a low molecular weight proteinase inhibitor capable of inhibiting activated matrix metalloproteinases (MMPs). TIMP-2 is found both free and in a 1:1 stoichiometric complex with the pro-enzyme form of MMP-2 (pro-MMP-2/TIMP-2 complex). We have measured the binding of recombinant TIMP-2 to intact HT-1080 and MCF-7 cells. HT-1080 cells in suspension bound 125I-labeled rTIMP-2 with a Kd of 2.5 nM and 30,000 sites/cell. Monolayers of MCF-7 cells were similarly found to bind [125I]rTIMP-2 with a Kd of 1.6 nM and 25,000 sites/cell. Specific binding of MMP-2 alone to HT-1080 cells was not observed; however, pro-MMP-2/TIMP-2 complex was capable of binding to the surface of HT-1080 cells in a TIMP-2-dependent manner. Binding of rTIMP-2 was not competed by the presence of TIMP-1. These results suggest that rTIMP-2 alone binds directly to the cell surface of HT-1080 and MCF-7 cell lines, and TIMP-2 is capable of localizing MMP-2 to the surface of HT-1080 cells via interaction with a specific binding site.
Insights
Tissue inhibitor of metalloproteinases-2 (TIMP-2) directly binds to HT-1080 and MCF-7 cells. TIMP-2 also facilitates the localization of matrix metalloproteinase-2 (MMP-2) to cell surfaces.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a proteinase inhibitor.
- TIMP-2 inhibits activated matrix metalloproteinases (MMPs).
- TIMP-2 exists freely or complexed with pro-matrix metalloproteinase-2 (pro-MMP-2).
Purpose of the Study:
- To investigate the binding characteristics of recombinant TIMP-2 (rTIMP-2) to HT-1080 and MCF-7 cell lines.
- To determine if TIMP-2 binds directly to cell surfaces.
- To assess TIMP-2's role in localizing MMP-2 to the cell surface.
Main Methods:
- Radiolabeling of recombinant TIMP-2 with iodine-125 ([125I]rTIMP-2).
- Incubation of [125I]rTIMP-2 with suspended HT-1080 cells and MCF-7 cell monolayers.
- Measurement of binding affinity (Kd) and receptor site density.
- Competition assays using TIMP-1 and pro-MMP-2/TIMP-2 complex.
Main Results:
- HT-1080 cells bound [125I]rTIMP-2 with a Kd of 2.5 nM (30,000 sites/cell).
- MCF-7 cells bound [125I]rTIMP-2 with a Kd of 1.6 nM (25,000 sites/cell).
- Pro-MMP-2/TIMP-2 complex bound to HT-1080 cells in a TIMP-2-dependent manner, while MMP-2 alone did not.
- TIMP-1 did not compete for rTIMP-2 binding.
Conclusions:
- Recombinant TIMP-2 directly binds to the cell surfaces of HT-1080 and MCF-7 cells.
- TIMP-2 possesses specific binding sites on these cell lines.
- TIMP-2 mediates the localization of MMP-2 to the cell surface through these interactions.