The propeptide domain of membrane type 1 matrix metalloproteinase is required for binding of tissue inhibitor of metalloproteinases and for activation of pro-gelatinase A.
Activation of secreted latent matrix metalloproteinases (MMPs) is accompanied by cleavage of the N-terminal propeptide, thereby liberating the active zinc from binding to the conserved cysteine in the pro-domain. It has been assumed that an analogous mechanism is responsible for the activation of membrane type 1 MMP (MT1-MMP). Using recombinant wild-type MT1-MMP cDNA and mutant cDNAs transfected into COS-1 cells lacking endogenous MT1-MMP, we have examined the function of the propeptide domain of MT1-MMP. MT1-MMP was characterized by immunoblotting, surface biotinylation, gelatin substrate zymography, and 125I-tissue inhibitor of metalloproteinases 2 (TIMP-2) binding. In contrast to wild-type MT1-MMP-transfected COS-1 cells, transfected COS-1 cells containing a deletion of the N-terminal propeptide domain of MT1-MMP or a chimeric construction (substitution of the pro-domain of MT1-MMP with that of collagenase 3) were functionally inactive in terms of binding of 125I-labeled TIMP-2 to the cell surface and initiating the activation of pro-gelatinase A. These results support the concept that in its native plasma membrane-inserted form, the pro-domain of MT1-MMP plays an essential role in TIMP-2 binding and subsequent activation of pro-gelatinase A.
Cao
J
J
Department of Medicine, Schools of Medicine and Dentistry, State University of New York, Stony Brook, New York 11794, USA.
Drews
M
M
Lee
H M
HM
Conner
C
C
Bahou
W F
WF
Zucker
S
S
D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism*; D006801:Humans; D053504:Matrix Metalloproteinases, Membrane-Associated; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*
D004792:Enzyme Precursors; D008565:Membrane Proteins; D011480:Protease Inhibitors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D018093:Gelatinases; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; C069860:progelatinase