Abstract
Zinc-dependent metalloproteases can mediate the shedding of the extracellular domain of many unrelated transmembrane proteins from the cell surface. In most instances, this process, also known as ectodomain shedding, is regulated via protein kinase C (PKC). The tumor necrosis factor alpha-converting enzyme (TACE) was the first protease involved in regulated protein ectodomain shedding identified. Although TACE belongs to the family of metalloprotease-disintegrins, few members of this family have been shown to participate in regulated ectodomain shedding. In fact, the phenotype of tace-/- cells and that of Chinese hamster ovary cell mutants defective in ectodomain shedding points to the existence of a common PKC-activated ectodomain shedding system, whose proteolytic component is TACE, that acts on a variety of transmembrane proteins. Examples of these proteins include the Alzheimer's disease-related protein beta-amyloid precursor protein (betaAPP) and the transmembrane growth factors protransforming growth factor-alpha (pro-TGF-alpha) and, as shown in this report, proheparin-binding epidermal growth factor-like growth factor (pro-HB-EGF). Here we show that the mercurial compound 4-aminophenylmercuric acetate (APMA), frequently used to activate in vitro recombinant matrix metalloproteases, is an activator of the shedding of betaAPP, pro-HB-EGF, and pro-TGF-alpha. Treatment of tace-/- cells or Chinese hamster ovary shedding-defective mutants with APMA activates the cleavage of pro-TGF-alpha but not that of pro-HB-EGF or betaAPP, indicating that APMA activates TACE and also a previously unacknowledged proteolytic activity specific for pro-TGF-alpha. Characterization of this proteolytic activity indicates that it acts on pro-TGF-alpha located at the cell surface and that it is a metalloprotease active in cells defective in furin activity. In summary, treatment of shedding-defective cell lines with APMA unveils the existence of a metalloprotease activity alternative to TACE with the ability to specifically shed the ectodomain of pro-TGF-alpha.
Highlights
During the last decade protein ectodomain shedding has rapidly evolved from an emerging concept to an important aspect of the cell biology
Chinese hamster ovary (CHO) mutant cell lines (M2) initially isolated for lack of activated pro-TGF-␣ shedding are defective in an unidentified gene that is different from protein kinase C (PKC) or tumor necrosis factor ␣-converting enzyme (TACE) but necessary for the regulated shedding of pro-TNF-␣, L-selectin, interleukin-6 receptor ␣, and APP (14 –16), indicating that the activity of TACE is tightly controlled by a component defective in these mutant cell lines
aminophenylmercuric acetate (APMA) was found to activate the shedding of these molecules from CHO cells with a dose dependence and time course indistinguishable from those found for APP
Summary
Antibodies and Reagents—Anti-HA monoclonal antibodies were from Babco (Richmond, CA), the monoclonal anti-APP antibodies 22C11 and anti-Alz were from Roche Molecular Biochemicals, the polyclonal antibody against the cytoplasmic domain of APP was a gift from Dr Sam Gandy, and the polyclonal antibodies against the cytoplasmic tail of pro-TGF-␣ have been previously described [23]. Metabolic Labeling and Immunoprecipitation—Approximately 3 ϫ 106 cells expressing pro-HA/TGF-␣ or a pro-HA/TGF-␣ C-terminal mutant were metabolically labeled with 500 Ci/ml [35S]cysteine (Biolink 2000, Arlington Heights, IL) for 45 min in cysteine-free medium at 37 °C and chased for variable periods of time in complete medium with or without 1 M PMA, 0.5 mM APMA, or APMA and BB-94 at 37 or 23 °C as indicated. Cells were incubated with PBS containing 5% bovine serum albumin and 5 g/ml anti-HA monoclonal antibodies, washed extensively with DMEM, shifted to 37 °C, and further chased for 20 min in the presence or absence of 0.5 mM APMA. Western Blotting—Approximately 3 ϫ 106 parental HeLa, CHO, M2, or taceϪ/Ϫ cells or the same cells permanently transfected with proHA/HB-EGF were washed with DMEM and treated with 1 ml of DMEM with or without 1 M PMA or 0.5 mM APMA for variable periods of time. Aliquots from conditioned media and from cell lysates were subjected to Western blotting analysis using the monoclonal anti-APP antibodies 22C11 or Alz as recom-
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