Report |
Address correspondence to Zena Werb, Department of Anatomy, HSW 1321, University of California, 513 Parnassus Ave., San Francisco, CA 94143-0452. Tel.: (415) 476-4622. Fax: (415) 476-4565. E-mail: zena{at}itsa.ucsf.edu
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Abstract |
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Key Words: signal crosstalk; bombesin; HB-EGF; shedding; tetraspanin
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Introduction |
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Metalloproteases regulate cell behavior by modifying both the macro- and microenvironment of cells during their normal growth and development (Werb, 1997; Werb and Yan, 1998). Misregulation of metalloprotease activities contributes to many pathological processes, including tumorigenesis (Sternlicht et al., 1999). The integral membrane metalloproteases with a disintegrin domain (ADAMs) cleave various membrane-bound proteins, including ligands, receptors, and ligandreceptor complexes (Black and White, 1998; Blobel, 2000). Among the well-studied ADAMs are tumor necrosis factor (TNF)-converting enzyme (TACE; ADAM17), which not only cleaves TNF-
but also converts pro-TGF
precursors to active TGF
(Peschon et al., 1998), and Kuzbanian (KUZ; ADAM10), which is a key regulator of Notch signaling pathways in Drosophila possibly because of its metalloprotease activity (Pan and Rubin, 1997; Qi et al., 1999). Thus, proteolysis by ADAMs can change the active state of surface molecular complexes, affecting the signaling pathways inside cells.
Metalloprotease-mediated release of EGFR ligands not only contributes to a normal development process, as revealed with the metalloprotease-deficient mice, but also plays important roles in the abnormal growth of tumor cells. Inhibition of metalloprotease-mediated EGFR ligand shedding reduces the proliferation and migration of breast cancer cells (Dong et al., 1999). Moreover, so-called autocrine growth of tumor cells is often EGFR dependent, and GPCR ligands, such as bombesin, also act as growth factors. In PC3 prostate cancer cells, the metalloprotease inhibitor BB94 inhibits bombesin- and phorbol esterinduced EGFR transactivation (Prenzel et al., 1999). Thus, metalloproteases are an integral part of this EGFR-dependent autocrine growth pathway. However, understanding the pathway from GPCR to EGFR activation has been limited by the lack of knowledge about the metalloprotease involved. Several metalloproteases are capable of cleaving EGFR ligands, but there is no evidence that they support the EGFR transactivation by GPCR. For example, ADAM9 cleaves HB-EGF when PKC is activated, but neither wild-type nor dominant-negative ADAM9 affects the EGFR transactivation (Izumi et al., 1998). Here we report that the metalloprotease KUZ (ADAM10), described initially as the regulator of Notch signaling, supports the GPCR-induced transactivation of the EGFR signaling pathway.
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Results and discussion |
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To test whether other ADAMs have the same effect on EGFR transactivation in COS7 cells, we transfected the same amount of plasmids carrying wild-type ADAM15, TACE (ADAM17), and KUZ and similar constructs with a Flag-tagged COOH terminus (Fig. 1, EG). Bombesin induced a fourfold increase of EGFR phosphorylation in vector-transfected cells and an eightfold increase in cells transfected with wild-type KUZ, and increased phosphorylation of all three isoforms of the adaptor Src homology 2 domaincontaining protein (SHC) (Fig. 1, E and G). However, transfection of neither TACE nor ADAM15 increased EGFR phosphorylation; in fact, they gave a reproducible decrease in phosphorylation (Fig. 1 G). The effect on EGFR transactivation did not correlate with the expression level of these ADAMs in COS7 cells. In fact, KUZ was expressed at lower levels than either ADAM17 or ADAM15 (Fig. 1 F). In COS7 cells, transfection of the evolutionarily related ADAM9 or a dominant-negative ADAM9 had no effect on EGFR transactivation (Izumi et al., 1998; Prenzel et al., 1999). Therefore, KUZ appeared to be uniquely effective as a mediator of EGFR transactivation in COS7 cells.
To assess the effects of KUZ on signaling pathways downstream of the transactivation of EGFR, we investigated the docking and phosphorylation of proteins known to associate with phosphorylated EGFR. Transfection of wild-type KUZ increased, and of KMP decreased, the bombesin-dependent phosphorylation of SHC (Fig. 2 A) and of another adaptor Gab1 (Fig. 2 B). KUZ, but not K
MP, increased the amount of phosphorylated EGFR that coprecipitated with SHC upon bombesin treatment (unpublished data), indicating that KUZ facilitates the direct recruitment of SHC by activated EGFR. Thus, KUZ increases the activation of signaling components docked on activated EGFR upon stimulation of GPCR.
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Antagonists of bombesin inhibit tumor growth in nude mice seeded with PC3 cells, and their effects are EGFR dependent, suggesting that EGFR is involved in bombesin-induced tumor growth (Plonowski et al., 2000; Heasley, 2001). Interestingly, in preliminary studies, we have found that the expression of KUZ increases with neoplastic progression in transgenic models of mouse prostate cancer (unpublished data). Moreover, ADAM10, the human homologue of KUZ, significantly increases with androgen treatment in PC3 cells, whereas TACE expression is inhibited (McCulloch et al., 2000). To assess whether endogenous KUZ was involved in GPCR transactivation of the EGFR signaling pathway in PC3 prostate cancer cells, we investigated the effect of KUZ on the activation of MAP kinase (Erk1/2). Inhibition of EGFR kinase with AG1478 and neutralization of HB-EGF with CRM197 both reduced Erk1/2 phosphorylation by bombesin, suggesting that transactivation of EGFR is required, at least in part, for bombesin to activate Erk signaling in PC3 cells (Fig. 2 D).
To determine whether PC3 cells require KUZ for this transactivation, we used two methods to inhibit endogenous KUZ and then tested the response of the cells to bombesin. First, transfecting KMP suppressed transactivation of Erk1/2 by bombesin (Fig. 2 E). Second, introduction of the specific antisense morpholino oligonucleotide against ADAM10 inhibited the bombesin-mediated transactivation of Erk1/2, whereas a control oligonucleotide with same nucleotide sequence but in a reverse orientation did not (Fig. 2 F), nor did the antisense oligonucleotides against ADAMs 9 and 15 and TACE (unpublished data). These results show that blocking KUZ inhibits transactivation, implicating endogenous KUZ as a critical player in GPCR transactivation of EGFR signaling.
The effect of KUZ on EGFR transactivation required its metalloprotease activity. KUZ was involved in shedding HB-EGF from COS7 cells. Stimulation of cells with bombesin increased the amount of shed HB-EGF in the medium. KUZ enhanced the ability of bombesin to release HB-EGF into the medium (Fig. 3 A). In contrast, KMP blocked the release of HB-EGF. These data show that KUZ is required to shed HB-EGF. Inhibition of metalloprotease, using the broad-spectrum inhibitor TAPI, also inhibited bombesin-induced EGFR and SHC phosphorylation (Fig. 3 B). A single E385A mutation causes the loss of KUZ metalloprotease activity. When the mutant K(E-A) was introduced into COS7 cells, EGFR and SHC activation did not increase upon bombesin treatment (Fig. 3 C). Therefore, metalloprotease activity of KUZ is responsible for EGFR transactivation.
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For shedding to occur in cis, KUZ would have to bind to its substrate proHB-EGF. We therefore hypothesized that GPCR affects KUZ activity by regulating the formation of such a complex. Indeed, we found that the antibody to CD9, a tetraspan transmembrane protein, precipitated not only HB-EGF, but also KUZ from cell lysates, suggesting that they coexist in the same molecular complex on the cell membrane (Fig. 4). CD9 coprecipitated with two forms of HB-EGF, a long 24-kD form and a short 18-kD form. Whereas CD9 binding of the long form appeared to be constitutive, the short formCD9 complex increased in the presence of transfected KUZ and with bombesin treatment. Therefore, GPCR regulates KUZ-dependent activation of HB-EGF by promoting the binding of KUZ to the molecular complex centered on CD9. Interestingly, the potency of HB-EGF in stimulating cell growth correlated with its binding to CD9. CD9 also associates with ADAM2 and regulates interaction of ADAM2 with 6ß1 integrin (Chen et al., 1999; Shi et al., 2000).
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Materials and methods |
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EGFR activation assay
Transfected cells were treated with GPCR agonists (LPA, 10 µM; bombesin, 200 nM) for 5 min at 37°C before being washed with cold PBS and lysed in the lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, plus protease inhibitors). Cell lysates were cleared by spinning at 15,000 rpm for 10 min and diluted with an equal volume of dilution buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride). EGFRs were precipitated from lysates with a polyclonal antibody (Upstate Biotechnology) overnight at 4°C. The precipitates were blotted with an antiphosphotyrosine antibody (4G10) to detect the activated EGFR, and a polyclonal antibody against EGFR (Santa Cruz Biotechnology, Inc.) to detect the quantities of EGFR. In some experiments, cells treated with CRM197 (10 µg/ml), AG1487 (250 nM), or DMSO for 20 min were then stimulated with agonist for 5 min. Metalloprotease inhibitor TAPI was applied at 50 µM in DMSO for 40 min at 37°C before stimulation with bombesin.
Assay for SHC, Gab1, Ras, and Erk1/2 activation
COS7 cells transfected with KUZ, KMP, and vector for 24 h were serum starved for an additional 24 h before stimulation with bombesin for 5 min at 37°C. Adaptor protein SHC or Gab1 was immunoprecipitated with anti-SHC or anti-Gab1 polyclonal antibody (Transduction Lab). Activation of SHC or Gab1 was detected in immunoblots as the phosphorylation of SHC or Gab1 with antiphosphotyrosine antibody 4G10. The activated Ras was precipitated from the same set of cell lysates with the Ras-binding domain of Raf1 supplied in a reagent kit (Upstate Biotechnology). The amount of activated Ras in the cells was detected in the immunoblot with anti-Ras antibody. Phosphorylation of Erk1/2 was detected with the antiphospho-ERK1/2 antibody (New England Biolabs, Inc.) in cell lysates.
Antisense experiment
Morpholino antisense oligonucleotide to human ADAM10 (AATTAACACTCTCAGCAACACCATC) and control morpholino oligonucleotide that has the same base composition but in reverse sequence (CTACCACAACGACTCTCACAATTAA) were synthesized by GeneTools, LLC. Oligos were introduced into PC3 cells with ethoxylated polyethylenimine as the delivery reagent according to the procedure recommended by the manufacturer. PC3 cells in a six-well plate were incubated with 1.5 ml of 1 µM oligo mixture per well for 3 h at 37°C. Cells were washed and incubated in serum-free medium for an additional 36 h before stimulation with bombesin and assaying for Erk1/2 phosphorylation. The endogenous ADAM10 levels in antisense oligotreated cells were detected with a polyclonal antibody to ADAM10 (KUZ; Chemicon International, Inc.).
Shedding of HB-EGF
COS7 cells were transfected with vector, KUZFlag, KMPFlag, and hemagglutinin (HA)-tagged HB-EGF (a gift from M. Klagsburn, Children's Hospital, Boston, MA). After bombesin treatment for 20 min, serum-free media was collected and cleared by spinning at 15,000 rpm for 10 min. Heparin-binding proteins were precipitated from the medium with heparin-agarose at 4°C overnight and immunoblotted with an anti-HA antibody (12CA5) to detect HB-EGF.
Immunoprecipitation of CD9
COS7 cells were transfected with KUZFlag and HA-tagged HB-EGF with Fugene 6. After stimulating cells with bombesin for 5 min, cell lysates were prepared in a buffer containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM CHAPS, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, plus protease inhibitors. CD9 in cell lysates was precipitated with a polyclonal anti-CD9 antibody (4 µg/ml, Santa Cruz Biotechnology, Inc.) overnight, and blotted with monoclonal antibodies against CD9 (BD Biosciences) or HA epitope (12CA5; Roche).
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Footnotes |
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K. Shirakabe's present address is Department of Molecular Cell Biology, Medical Research Institute, Tokyo, 10-0062, Japan.
* Abbreviations used in this paper: ADAM, metalloprotease with a disintegrin domain; EGFR, EGF receptor; GPCR, G proteincoupled receptors; HA, hemagglutinin; HB-EGF, heparin-binding EGF; KUZ, Kuzbanian; K
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Acknowledgments |
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This work was supported by funds from the DOD Breast Cancer Program (DAMD17-9818193 to Y. Yan), the National Cancer Institute (CA72006 to Z. Werb), and the University of California San Francisco Prostate Cancer Center Development Program (to Z. Werb).
Submitted: 7 December 2001
Revised: 6 May 2002
Accepted: 21 May 2002
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References |
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![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blobel, C.P. 2000. Remarkable roles of proteolysis on and beyond the cell surface. Curr. Opin. Cell Biol. 12:606612.[CrossRef][Medline]
Chen, M.S., K.S. Tung, S.A. Coonrod, Y. Takahashi, D. Bigler, A. Chang, Y. Yamashita, P.W. Kincade, J.C. Herr, and J.M. White. 1999. Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin 6ß1: implications for murine fertilization. Proc. Natl. Acad. Sci. USA. 96:1183011835.
Dong, J., L.K. Opresko, P.J. Dempsey, D.A. Lauffenburger, R.J. Coffey, and H.S. Wiley. 1999. Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor. Proc. Natl. Acad. Sci. USA. 96:62356240.
Fan, H., and R. Derynck. 1999. Ectodomain shedding of TGF- and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades. EMBO J. 18:69626972.
Hattori, M., M. Osterfield, and J.G. Flanagan. 2000. Regulated cleavage of a contact-mediated axon repellent. Science. 289:13601365.
Izumi, Y., M. Hirata, H. Hasuwa, R. Iwamoto, T. Umata, K. Miyado, Y. Tamai, T. Kurisaki, A. Sehara-Fujisawa, S. Ohno, and E. Mekada. 1998. A metalloprotease-disintegrin, MDC9/meltrin-/ADAM9 and PKC
are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J. 17:72607272.
Nath, D., N.J. Williamson, R. Jarvis, and G. Murphy. 2001. Shedding of c-Met is regulated by crosstalk between a G-protein coupled receptor and the EGF receptor and is mediated by a TIMP-3 sensitive metalloproteinase. J. Cell Sci. 114: 12131220.
Peschon, J.J., J.L. Slack, P. Reddy, K.L. Stocking, S.W. Sunnarborg, D.C. Lee, W.E. Russell, B.J. Castner, R.S. Johnson, J.N. Fitzner, et al. 1998. An essential role for ectodomain shedding in mammalian development. Science. 282:12811284.
Pierce, K.L., A. Tohgo, S. Ahn, M.E. Field, L.M. Luttrell, and R.J. Lefkowitz. 2001b. Epidermal growth factor receptor-dependent ERK activation by G protein-coupled receptors: a co-culture system for identifying intermediates upstream and downstream of HB-EGF shedding. J. Biol. Chem. 276:2315523160.
Prenzel, N., E. Zwick, H. Daub, M. Leserer, R. Abraham, C. Wallasch, and A. Ullrich. 1999. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature. 402:884888.[CrossRef][Medline]
Qi, H., M.D. Rand, X. Wu, N. Sestan, W. Wang, P. Rakic, T. Xu, and S. Artavanis-Tsakonas. 1999. Processing of the notch ligand by the metalloprotease Kuzbanian. Science. 283:9194.
Reddy, P., J.L. Slack, R. Davis, D.P. Cerretti, C.J. Kozlosky, R.A. Blanton, D. Shows, J.J. Peschon, and R.A. Black. 2000. Functional analysis of the domain structure of tumor necrosis factor- converting enzyme. J. Biol. Chem. 275:1460814614.
Shi, W., H. Fan, L. Shum, and R. Derynck. 2000. The tetraspanin CD9 associates with transmembrane TGF- and regulates TGF-
induced EGF receptor activation and cell proliferation. J. Cell Biol. 148:591602.
Werb, Z. 1997. ECM and cell surface proteolysis: regulating cellular ecology. Cell. 91:439442.[Medline]
Werb, Z., and Y. Yan. 1998. A cellular striptease act. Science. 282:12791280.