Matrix Metalloproteinase-3 Releases Active Heparin-binding EGF-like Growth Factor by Cleavage at a Specific Juxtamembrane Site*
- From the Departments of ‡Surgery and ¶Pathology, Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115 and §The Biosignaling Department, National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki 305, Japan
Abstract
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored precursor that is cleaved to release the soluble mature growth factor. The two forms are active as juxtacrine and paracrine/autocrine growth factors, respectively. The enzymes that process the HB-EGF transmembrane form are unknown. Accordingly, an in vitro assay was established using a fusion protein in which alkaline phosphatase (AP) replaced the transmembrane and cytoplasmic domains of HB-EGF (HB-EGF JM-AP). The fusion protein was anchored to agarose beads coated with anti-AP antibodies. Several matrix metalloproteinases (MMPs) were tested for the ability to release soluble HB-EGF in the in vitrosystem. MMP-3 released soluble 12-kDa immunoreactive and mitogenic HB-EGF within 30 min. On the other hand neither MMP-2 nor MMP-9 had any cleavage activities. A non-cleavable mutant was prepared by replacing the juxtamembrane (JM) region of HB-EGF with the JM region of CD4. The mutant HB-EGF, which in its full-length form was as active a juxtacrine growth factor as was the wild type HB-EGF in vivo, was not cleaved by MMP-3 in the in vitro assay. The C-terminal portion of the cleaved HB-EGF JM-AP that remained attached to the anti-AP beads was N-terminally sequenced and the MMP-3 cleavage site was determined to be Glu151-Asn152, a site within the JM domain. MMP-3 treatment also released soluble HB-EGFin vivo from MC2 cells expressing transmembrane HB-EGF precursor, at a level of about 2-fold above control. It was concluded that MMP-3 cleaves HB-EGF at a specific site in the JM domain and that this enzyme might regulate the conversion of HB-EGF from being a juxtacrine to a paracrine/autocrine growth factor.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM 47397 (to M. K.) and American Cancer Society Grant 83821 (to M. M.).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.
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↵‖ To whom correspondence should be addressed: Children’s Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-7503; Fax: 617-355-7291; E-mail: klagsbrun{at}a1.tch.harvard.edu.
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↵1 The abbreviations used are: TGF-α, transforming growth factor-α; EGF, epidermal growth factor; HB-EGF, heparin-binding epidermal growth factor-like growth factor; MMP, matrix metalloproteinase; pro-MMP, precursor of MMP; PMA, phorbol 12-myristate 13-acetate; APMA, p-aminophenylmercuric acetate; FCS, fetal calf serum; GPS, glutamine/penicillin/streptomycin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane; JM, juxtamembrane.
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↵2 M. Suzuki and M. Klagsbrun, unpublished data.
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↵3 M. Suzuki and M. Klagsbrun, unpublished results.
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- Received June 17, 1997.
- Revision received September 23, 1997.
