TIMP-2 is released as an intact molecule following binding to MT1-MMP on the cell surface.
Binding of tissue inhibitor of metalloproteinase-2 (TIMP-2) to pro-MMP-2 and mature membrane type-1 MMP (MT1-MMP) on the cell surface is required for activation of MMP-2. It has been reported that following binding to cell surface receptors, TIMP-2 undergoes endocytosis and extensive degradation in lysosomes. The purpose of this study was to reexamine the fate of TIMP-2 following binding to transfected HT1080 cell surface MT1-MMP at 4 degrees C. Following 37 degrees C incubation, 125I-TIMP-2 release, endocytosis, and degradation were characterized under varying conditions. More than 85% of the total 125I-TIMP-2 bound to cells was released as intact functional molecules; <15% was degraded. Transfection of HT1080 cells with dominant negative mutant dynamin cDNA resulted in delayed endocytosis and release of 125I-TIMP-2 from cells. Pharmacologic agents that induce clustering of cell surface receptors (concanavalin A) and interfere with endosomal/lysosomal function (bafilomycin A(1)) resulted in enhanced binding of 125I-TIMP-2 to cell surface receptors. Abrogation of activation of proMT1-MMP with a furin inhibitor prevented binding and endocytosis of 125I-TIMP-2. Biotinylation of cell surface MT1-MMP followed by Western blotting confirmed the presence of mature MT1-MMP on the cell surface and degraded MT1-MMP in the intracellular compartment. In conclusion, these studies demonstrate that TIMP-2 is released from cells primarily as an intact functional molecule following binding to MT1-MMP on the cell surface.
Zucker
Stanley
S
Department of Research, Veterans Affairs Medical Center, Northport, NY 11768, USA. s_zucker@yahoo.com
Hymowitz
Michelle
M
Conner
Cathleen
C
DeClerck
Yves
Y
Cao
Jian
J
D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003208:Concanavalin A / Q000494:pharmacology; D004705:Endocytosis; D011992:Endosomes / Q000187:drug effects; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005354:Fibrosarcoma / Q000473:pathology; D006801:Humans; D007700:Kinetics; D018942:Macrolides / Q000494:pharmacology; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects; D013696:Temperature; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D018942:Macrolides; D011956:Receptors, Cell Surface; D003208:Concanavalin A; D019716:Tissue Inhibitor of Metalloproteinase-2; C040929:bafilomycin A1; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases