Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP).
It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2.MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated.
Zucker
S
S
Department of Medicine, Department of Veterans Affairs Medical Center, Northport, New York 11768, USA.
Drews
M
M
Conner
C
C
Foda
H D
HD
DeClerck
Y A
YA
Langley
K E
KE
Bahou
W F
WF
Docherty
A J
AJ
Cao
J
J
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002478:Cells, Cultured; D018076:DNA, Complementary; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D019716:Tissue Inhibitor of Metalloproteinase-2; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases