Cell Surface Ectodomain Cleavage of Human Amphiregulin Precursor Is Sensitive to a Metalloprotease Inhibitor
RELEASE OF A PREDOMINANT N-GLYCOSYLATED 43-kDa SOLUBLE FORM*
- Christa L. Brown‡,
- Katherine S. Meise§,
- Gregory D. Plowman¶,
- Robert J. Coffey‡§‖** and
- Peter J. Dempsey‡‖
- From the Departments of ‡Cell Biology and‖Medicine, Vanderbilt University School of Medicine and§Veterans Affairs Medical Center, Nashville, Tennessee 37232-2279 and ¶Sugen, Inc., Redwood City, California 94063-4720
Abstract
Biosynthesis and processing of amphiregulin (AR) have been investigated in human colorectal (HCA-7, Caco-2) and mammary (MCF-7) cancer cell lines, as well as in Madin-Darby canine kidney cells stably expressing various human AR precursor (pro-AR) forms. Both cells expressing endogenous and transfected AR produce multiple cellular and soluble forms of AR with an N-glycosylated 50-kDa pro-AR form being predominant. Our results demonstrate that sequential proteolytic cleavage within the ectodomain of the 50-kDa pro-AR form leads to release of a predominantN-glycosylated 43-kDa soluble AR, as well as the appearance of other cellular and soluble AR forms. Cell surface biotinylation studies using a C-terminal epitope-tagged pro-AR indicate that all cell surface forms are membrane-anchored and support that AR is released by ectodomain cleavage of pro-AR at the plasma membrane. We also show that pro-AR ectodomain cleavage is a regulated process, which can be stimulated by phorbol 12-myristate 13-acetate and inhibited by the metalloprotease inhibitor, batimastat. In addition, we provide evidence that high molecular mass AR forms may retain the full-length N-terminal pro-region, which may influence the biological activities of these forms.
Footnotes
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↵* This work was supported by National Institutes of Health Grant CA46413 (to R. J. C.).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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↵** Veterans Administration Clinical Investigator. To whom correspondence should be addressed: GI Division, Depts. of Medicine and Cell Biology, Vanderbilt University Medical Center, CC2218 Medical Center North, Nashville, TN 37232. Tel.: 615-343-0171; Fax: 615-343-1591; E-mail: robert.coffey{at}mcmail.vanderbilt.edu.
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↵1 The abbreviations used are: AR, amphiregulin; EGFR/HER-1, epidermal growth factor receptor; PMA, phorbol 12-myristate 13-acetate; HER, human epidermal growth factor receptor; MDCK cells, Madin-Darby canine kidney cells; pro-AR, amphiregulin precursor; pro-EGF, epidermal growth factor precursor; pro-TGFα, transforming growth factor α precursor; pro-HB-EGF, heparin-binding epidermal growth factor precursor; HRP, horseradish peroxidase; DMEM, Dulbecco’s modified Eagle’s medium; endo H, endoglycosidase H; mAb, monoclonal antibody; CHO, Chinese hamster ovary; SA, streptavidin; PBS, phosphate-buffered saline; DME−,l-cysteine/l-methionine-free DMEM; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; GAG, glycosaminoglycans.
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- Received February 2, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
