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*
- From the Departments of ‡Medicine and ¶Oral Biology, Schools of Medicine and Dentistry, State University of New York, Stony Brook, New York 11794 and §Department of Veterans Affairs Medical Center, Northport, New York 11768
Abstract
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, and125I-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.
Footnotes
-
↵* This research was supported in part by a merit review grant from the Department of Veterans Affairs and the Schermerhorn Foundation (to S. Z.), National Institutes of Health Grants HL-02431 and HL-49149 (to W. F. B.), and a research grant (to W. B.) and postdoctoral fellowship (to J. C.) from the American Heart Association, New York Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‖ To whom correspondence and should be addressed: Mail Code 151, Veterans Affairs Medical Center, Northport, NY 11768. Tel.: 516-261-4400 (ext. 2861); Fax: 516-544-5317.
-
↵2 The numbering of amino acids of all proteins includes signal peptide sequence.
-
↵3 J. Cao and S. Zucker, manuscript in preparation.
- Abbreviations:
- MMP
-
matrix metalloproteinase
- MT-MMP
-
membrane-type matrix metalloproteinase
- TIMP
-
tissue inhibitor of metalloproteinases
- MTΔpro
-
propeptide-deleted MT1-MMP
- PCR
-
polymerase chain reaction
- PBS
-
phosphate-buffered saline
- Col-3/MT
-
collagenase 3 and MT1-MMP
- PAGE
-
polyacrylamide gel electrophoresis
- wtMT1-MMP
-
wild-type MT1-MMP.
-
- Received March 4, 1998.
- Revision received October 15, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
