ErbB-2 Amplification Inhibits Down-regulation and Induces Constitutive Activation of Both ErbB-2 and Epidermal Growth Factor Receptors
1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês
10.1074/jbc.274.13.8865
ISSN1083-351X
AutoresRebecca A. Worthylake, Lee K. Opresko, H Wiley,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoErbB-2/HER2 is an important signaling partner for the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 is also associated with poor prognosis in breast cancer. To investigate how erbB-2 amplification affects its interactions with the EGFR, we used a human mammary epithelial cell system in which erbB-2 expression was increased 7–20-fold by gene transfection. We found that amplification of erbB-2 caused constitutive activation of erbB-2 as well as ligand-independent activation of the EGFR. Overexpression of erbB-2 strongly inhibited erbB-2 down-regulation following transactivation by EGFR. Significantly, down-regulation of activated EGFR was also inhibited by erbB-2 amplification, resulting in enhanced ligand-dependent activation of the EGFR. The rate of EGFR endocytosis was not affected by erbB-2 overexpression, but the rate of lysosomal targeting was significantly reduced. In addition, erbB-2 overexpression promoted rapid recycling of activated EGFR back to the cell surface and decreased ligand dissociation from the EGFR. Our data suggest that overexpression of erbB-2 inhibits both its down-regulation and that of the EGFR. The net effect is increased signaling through the EGFR system. ErbB-2/HER2 is an important signaling partner for the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 is also associated with poor prognosis in breast cancer. To investigate how erbB-2 amplification affects its interactions with the EGFR, we used a human mammary epithelial cell system in which erbB-2 expression was increased 7–20-fold by gene transfection. We found that amplification of erbB-2 caused constitutive activation of erbB-2 as well as ligand-independent activation of the EGFR. Overexpression of erbB-2 strongly inhibited erbB-2 down-regulation following transactivation by EGFR. Significantly, down-regulation of activated EGFR was also inhibited by erbB-2 amplification, resulting in enhanced ligand-dependent activation of the EGFR. The rate of EGFR endocytosis was not affected by erbB-2 overexpression, but the rate of lysosomal targeting was significantly reduced. In addition, erbB-2 overexpression promoted rapid recycling of activated EGFR back to the cell surface and decreased ligand dissociation from the EGFR. Our data suggest that overexpression of erbB-2 inhibits both its down-regulation and that of the EGFR. The net effect is increased signaling through the EGFR system. The epidermal growth factor (EGF) 1The abbreviations used are:EGF, epidermal growth factor; Tyr(P), phosphotyrosine; EGFR, EGF receptor. family of receptors and ligands contains four related receptor tyrosine kinases and seven related ligands (1Carraway III, K.L. Cantley L.C. Cell. 1994; 78: 5-8Abstract Full Text PDF PubMed Scopus (586) Google Scholar, 2Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Crossref PubMed Scopus (655) Google Scholar, 3Chang H. Riese II, D.J. Gilbert W. Stern D.F. McMahan U.J. Nature. 1997; 387: 509-516Crossref PubMed Scopus (257) Google Scholar, 4Carraway K. BioEssays. 1996; 18: 263-266Crossref PubMed Scopus (33) Google Scholar, 5Peles E. Yarden Y. BioEssays. 1993; 15: 815-823Crossref PubMed Scopus (260) Google Scholar). Overexpression of each of the receptors (EGFR, erbB-2, erbB-3, and erbB-4) has been correlated with poor prognosis in breast and other cancers (6Bacus S.S. Zelnick C.R. Plowman G. Yarden Y. Am. J. Clin. Path. 1994; 102: S13-S24PubMed Google Scholar, 7Plowman G.D. Culouscou J.M. Whitney G.S. Green J.M. Carlton G.W. Foy L. Neubauer M.G. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1746-1750Crossref PubMed Scopus (689) Google Scholar, 8Alimandi M. Romano A. Curia M.C. Muraro R. Fedi P. Aaronson S.A. DiFiore P.P. Kraus M.H. Oncogene. 1995; 10: 1813-1821PubMed Google Scholar). Compelling clinical studies on breast cancer reveal that amplification of erbB-2 has a high degree of correlation with disease recurrence and poor survival (9Slamon D.J. Godolphin W. Jones L.A. Holt J.A. Wong S.G. Keith D.E. Levin W.J. Stuart S.G. Udove J. Ullrich A. Press M.F. Science. 1989; 244: 707-712Crossref PubMed Scopus (6261) Google Scholar). Both clinical and basic research studies indicate a role for erbB-2 amplification in initial transformation events and in progression to metastases (10Bouchard L. Lamarre L. Tremblay P.J. Jolicoeur P. Cell. 1989; 57: 931-936Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 11Tan M. Yao J. Yu D. Cancer Res. 1997; 57: 1199-1205PubMed Google Scholar). Despite the correlative evidence between erbB-2 overexpression and breast cancer, however, the mechanism by which erbB-2 facilitates cell transformation is unknown. The EGFR has five known ligands (EGF, transforming growth factor-α, heparin binding EGF-like growth factor, amphiregulin, and betacellulin) that directly bind and activate the receptor (12Massague J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515Crossref PubMed Scopus (600) Google Scholar). Three other ligands, heregulin, heregulin-2, and heregulin-3, bind to erbB-3 and/or erbB-4 (3Chang H. Riese II, D.J. Gilbert W. Stern D.F. McMahan U.J. Nature. 1997; 387: 509-516Crossref PubMed Scopus (257) Google Scholar, 5Peles E. Yarden Y. BioEssays. 1993; 15: 815-823Crossref PubMed Scopus (260) Google Scholar, 13Zhang D. Sliwkowski M.X. Mark M. Frantz G. Akita R. Sun Y. Hillan K. Crowley C. Brush J. Godowske P.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9562-9567Crossref PubMed Scopus (328) Google Scholar). No ligand has been found that binds erbB-2, although both EGF and heregulin can activate erbB-2 in-trans through ligand-induced heterodimerization and subsequent tyrosine phosphorylation of erbB-2 (14Karunagaran D. Tzahar E. Beerli R.R. Chen X. Graus-Porta D. Ratzkin B.J. Seger R. Hynes N.E. Yarden Y. EMBO J. 1996; 15: 254-264Crossref PubMed Scopus (588) Google Scholar, 15Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.J. Wood D.V. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (926) Google Scholar, 16Stern D.F. Kamps M.P. EMBO J. 1988; 7: 995-1001Crossref PubMed Scopus (175) Google Scholar). ErbB-2 can be directly phosphorylated by the activated primary receptor (e.g. EGFR or erbB-4) (17Qian X. LeVea C.M. Freeman J.K. Dougall W.C. Greene M.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1500-1504Crossref PubMed Scopus (141) Google Scholar). Alternately, formation of the heterodimer induces a conformation change that activates the intrinsic tyrosine kinase domain of erbB-2 (18Sasaoka T. Langlois W.J. Bai F. Rose D.W. Leitner J.W. Decker S.J. Saltiel A.R. Gill G.N. Kobayashi M. Draznin B. Olefsky J.M. J. Biol. Chem. 1996; 271: 8338-8344Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 19Wright J.D. Reuter C.W.M. Weber M.J. J. Biol. Chem. 1995; 270: 12085-12093Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 20Worthylake R. Wiley H. J. Biol. Chem. 1997; 272: 8594-8601Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Presumably, extensive interactions between EGF receptor family members allows for diversification of signaling cascades (21Pinkas-Kramarski R. Soussan L. Waterman H. Levkowitz G. Alroy I. Klapper L. Lavi S. Seger R. Ratzkin B.J. Sela M. Yarden Y. EMBO J. 1996; 15: 2452-2467Crossref PubMed Scopus (698) Google Scholar, 22Earp H.S. Dawson T.L. Li X. Yu H. Breast Cancer Res. Treat. 1995; 35: 115-132Crossref PubMed Scopus (355) Google Scholar). The EGFR is negatively regulated by both serine and threonine phosphorylation as well as by intracellular trafficking (23Lund K.A. Wiley H.S. Sibley D. Houslay M. Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification. 3. John Wiley and Sons, Ltd., Sussex1993: 277-303Google Scholar). Rapid internalization and lysosomal degradation result in short-term as well as long-term down-regulation of receptor activity (24Sorkin A. Waters C.M. BioEssays. 1993; 15: 375-382Crossref PubMed Scopus (233) Google Scholar). Proper trafficking of the EGFR is important for regulation of cell growth (25Wells A. Welsh B.J. Lazar C.S. Wiley H.S. Gill G.N. Rosenfeld M.G. Science. 1990; 247: 2751-2760Crossref Scopus (343) Google Scholar). Many signaling complexes are thought to be activated by assembly at the cell surface, and internalization has been proposed to negatively regulate this activity (24Sorkin A. Waters C.M. BioEssays. 1993; 15: 375-382Crossref PubMed Scopus (233) Google Scholar, 26Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4611) Google Scholar). Once internalized, occupied receptors are sorted in recycling endosomes and subsequently degraded in lysosomes, effectively reducing the number of activated receptors. Overexpression of EGFR has been shown to impair their down-regulation, apparently because of limiting levels of regulatory molecules that mediate rapid endocytosis and lysosomal targeting (24Sorkin A. Waters C.M. BioEssays. 1993; 15: 375-382Crossref PubMed Scopus (233) Google Scholar, 27Wiley H.S. J. Cell Biol. 1988; 107: 801-810Crossref PubMed Scopus (168) Google Scholar, 28French A.R. Sudlow G.P. Wiley H.S. Lauffenburger D.A. J. Biol. Chem. 1994; 269: 15749-15755Abstract Full Text PDF PubMed Google Scholar, 29Kurten R.C. Cadena D.L. Gill G.N. Science. 1996; 272: 1008-1010Crossref PubMed Scopus (317) Google Scholar). Although activation of erbB-2, erbB-3, and erbB-4 has been well characterized, the negative regulation of these receptors has not been extensively studied. There are conflicting reports regarding the trafficking of erbB-2 following transactivation with EGF. We, as well as other investigators, have described efficient down-regulation and lysosomal targeting of erbB-2 in three different cell types (20Worthylake R. Wiley H. J. Biol. Chem. 1997; 272: 8594-8601Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 30Kornilova E.S. Taverna D. Hoeck W. Hynes N.E. Oncogene. 1992; 7: 511-519PubMed Google Scholar). In contrast, chimeric receptors composed of the EGFR extracellular domain and the erbB-2 cytoplasmic domain, were not down-regulated following activation with EGF (31Baulida J. Kraus M.H. Alimandi M. Di Fiore P.P. Carpenter G. J. Biol. Chem. 1996; 271: 5251-5257Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). In another study, down-regulation of erbB-2 was investigated in SKBR-3 cells, which have an amplified erbB-2 gene (32King C.R. Borrello I. Bellot F. Comoglio P. Schlessinger J. EMBO J. 1988; 7: 1647-1651Crossref PubMed Scopus (179) Google Scholar). It was reported that although erbB-2 was transactivated following EGF treatment, there was no measurable change in erbB-2 half-life. One way to reconcile these disparate results is to postulate that although lysosomal targeting is normally involved in erbB-2 down-regulation, it is impaired when erbB-2 is overexpressed. To test this hypothesis, we examined the negative regulation of erbB-2 in mammary epithelial cells that overexpress the protein due to introduction of a transgene. We found that overexpression of erbB-2 severely inhibits its own down-regulation. Intriguingly, we also found that down-regulation of the EGFR was inhibited even though EGFR levels were similar between parental and erbB-2 overexpressing cells. Consistent with the findings of other investigators, we found that overexpression of erbB-2 results in an elevated basal level of activated receptors (33Guy C.T. Webster M.A. Schaller M. Parsons T.J. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10578-10582Crossref PubMed Scopus (1021) Google Scholar, 34Ram T.G. Ethier S.P. Cell Growth Differ. 1996; 7: 551-567PubMed Google Scholar). However, we not only observed constitutive activation of erbB-2, but also of EGFR. These results show that receptor cross-talk is not only important in receptor activation, but also plays a role in negative regulatory processes. N-13 polyclonal antibody directed against the amino-terminal 13 residues of the EGF receptor was a gift from Dr. Debora Cadena. 1917 polyclonal antibody directed against the carboxyl-terminal 18 residues of erbB-2 was provided by Dr. Gordon Gill. Ab5 mouse monoclonal antibody against the extracellular domain of erbB-2 was from Oncogene Sciences. 225 mouse monoclonal antibody against the EGFR was purified from hybridomas obtained from the American Type Culture Collection. RC20 anti-phosphotyrosine/horseradish peroxidase conjugate was purchased from Transduction Laboratories. Antibodies 13A9 against the EGFR and 4D5 against human erbB-2 were gifts from Genentech. These were directly labeled with Alexa 488 and Alexa 594 dyes, respectively, following the manufacture's protocol (Molecular Probes, Inc.). B82 mouse L cells transfected with the gene for human EGF receptor were grown in Dulbecco's modified Eagle's medium containing dialyzed 10% calf serum (HyClone) and 5 μm methotrexate. The human mammary epithelial cell lines MTSV1–7, ce1, and ce2 have been described previously (35D'Souza B. Berdichevsky F. Kyprianou N. Taylor-Papadimitriou J. Oncogene. 1993; 8: 1797-1806PubMed Google Scholar) and were obtained from Dr. Joyce Taylor-Papadimitriou. They were grown in Dulbecco's modified Eagle's medium containing 10% calf serum (HyClone), 1 μm insulin, and 5 μmdexamethasone. Selection for erbB-2 expression was maintained using 500 μg/ml G418. Confluent cultures were lysed in RIPA buffer (36Harlow E. Lane D. Antibodies: A Laboratory Manual. First Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar), debris was removed by centrifugation and samples were brought to 2% SDS, 1% β-mercaptoethanol and heated to 100 °C for 10 min. Equal amounts of protein from each sample was separated on a 5–7.5% polyacrylamide gradient gels and transferred to nitrocellulose. EGFR and erbB-2 were detected by N-13 and 1917 polyclonal sera, respectively, using125I-labeled protein A as described (20Worthylake R. Wiley H. J. Biol. Chem. 1997; 272: 8594-8601Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The concentrations and incubation times with 125I-labeled protein A were in the linear range of the protein load of the gels. The blots were analyzed by storage phosphor plates using the Bio-Rad G250 Molecular Imager. The Bio-Rad Molecular Analyst software package was used to quantify the amount of radioactivity associated with each band. To determine the phosphotyrosine content of erbB-2 and the EGFR, cells were lysed for 10 min at 0 °C in RIPA buffer supplemented with 100 μm Na3VO4. The lysate was clarified by centrifugation for 10 min at 10,000 × g, and receptors were immunoprecipitated with 5 μl of either the 1917 or 4D5 antibodies (erbB-2) or 2 μg of 225 (EGFR) and 100 μl of protein A-Sepharose (50% slurry). The beads were washed several times in lysis buffer and boiled in SDS sample buffer prior to gel electrophoresis and transfer to nitrocellulose. Phosphotyrosine levels were measured by detection with RC20 antibody and the Renaissance ECL kit from NEN Life Science Products Inc. Blots were exposed to film, and analyzed using a Bio-Rad Imaging Densitometer and the Molecular Analyst software package. Distribution of both EGFR and erbB-2 was evaluated using fixed and permeabilized cells as described previously (20Worthylake R. Wiley H. J. Biol. Chem. 1997; 272: 8594-8601Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Cells were labeled for 1 h with a mixture of 13A9 and 4D5 antibodies directly labeled with Alexa 488 and Alexa 594 dyes, respectively, at a final concentration of 1 μg/ml. After rinsing, the cells were mounted in Prolong anti-fade medium (Molecular Probes, Inc.) and viewed with a Nikon inverted fluorescence microscope with a ×60, 1.4 N.A. oil immersion objective. Images were acquired as described below. Specificity of the labeling was verified by using control cells (B82) lacking EGFR and human erbB-2. To follow the transfer of EGFR to lysosomes, the lysosomes were first labeled by incubating cells for 15 min at 37 °C with 5 mg/ml fluorescein-labeled dextran (M r 10,000, anionic, lysine fixable; Molecular Probes, Inc.), then chased for an additional 2 h. During the chase period, cells were pulsed for 15 min with 1.5 × 10−8m EGF-Texas Red-streptavidin complex (Molecular Probes, Inc.) and chased for up to 105 min. Total chase time following the initial fluorescein-labeled dextran treatment was 2 h for all samples. Cells were fixed with 3.6% paraformaldehyde and mounted in Prolong. Coverslips were viewed with a Nikon inverted fluorescence microscope with a ×60, 1.4 N.A. oil immersion objective. Sets of 3 images at 3 different focal planes spaced 0.5 μm apart centered on the perinuclear endosomes were acquired at 520 and 615 nm (for fluorescein and Texas Red, respectively). The images (12 bit, 656 × 517) were acquired using a Princeton Instruments cooled CCD camera attached to a Macintosh workstation running Openlab software (Improvision, Inc). The image triplets were deconvolved using nearest-neighbor subtraction (37Agard D.A. Hiraoka Y. Shaw P. Sedat J.W. Taylor D.L. Wang Y.L. Fluorescence Microscopy of Living Cells in Culture. 30. Academic Press, San Diego, CA1989: 353-377Google Scholar). The deconvolved image of the lysosomes (fluorescein) was then used to generate a binary mask using grayscale values between 700 and 4095. This mask was then applied to the deconvolved image of the EGF (Texas Red) to identify all lysosomal “objects” that contained EGF. The integrated intensity of all of these objects was then taken as the amount of EGF within lysosomal structures. A mask of the EGF image was generated in the same way and applied to the EGF image to determine the total integrated intensity of all EGF-containing objects within the cell. The fraction of all EGF colocalized in lysosomes was then calculated. At each time point, four random fields of cells were analyzed which contained between 100 and 200 vesicles per field. Number of surface-associated erbB-2 molecules was determined by steady state analysis (38Wiley H.S. Cunningham D.D. Cell. 1981; 25: 433-440Abstract Full Text PDF PubMed Scopus (165) Google Scholar). 4D5 antibody was radioiodinated to a specific activity of 4.5 × 106 cpm/pmol (39Wiley H.S. Herbst J.J. Walsh B.J. Lauffenburger D.A. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1991; 266: 11083-11094Abstract Full Text PDF PubMed Google Scholar) and cells were incubated with concentrations from 6.7 × 10−11 to 2 × 10−8m for 3 h at 37 °C. The relative amount of antibody associated with the cell surface was determined by acid stripping (40Lund K.A. Opresko L.K. Starbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Abstract Full Text PDF PubMed Google Scholar) and the data was analyzed as described previously (38Wiley H.S. Cunningham D.D. Cell. 1981; 25: 433-440Abstract Full Text PDF PubMed Scopus (165) Google Scholar). Scatchard analysis of EGF binding to cells used125I-EGF at a specific activity of 1.6 × 106 cpm/pmol and an incubation period of 4.5 h at 0 °C using ligand concentrations from 1.7 × 10−11to 1.7 × 10−8m as described (40Lund K.A. Opresko L.K. Starbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Abstract Full Text PDF PubMed Google Scholar). Specific internalization rates for the EGFR were determined as described (40Lund K.A. Opresko L.K. Starbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Abstract Full Text PDF PubMed Google Scholar). Measurements were made using a ligand concentration of 10 ng/ml, and each rate constant determination was derived from a 5-min incubation period with ligand. Specific internalization rates were determined by plotting the integral surface-associated ligand against the amount internalized, and the slopes were determined by linear regression (40Lund K.A. Opresko L.K. Starbuck C. Walsh B.J. Wiley H.S. J. Biol. Chem. 1990; 265: 15713-15723Abstract Full Text PDF PubMed Google Scholar). Cells were grown to confluence in 35-mm dishes and switched to serum-free Dulbecco's modified Eagle's medium containing 20 mm HEPES (pH 7.4) and no bicarbonate (D/H/B) 12 h before experiments. The cells were incubated at 37 °C in 0.1–30 ng/ml 125I-EGF for 3 h to allow the sorting process to reach a steady state (39Wiley H.S. Herbst J.J. Walsh B.J. Lauffenburger D.A. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1991; 266: 11083-11094Abstract Full Text PDF PubMed Google Scholar, 41Herbst J.J. Opresko L.K. Walsh B.J. Lauffenburger D.A. Wiley H.S. J. Biol. Chem. 1994; 269: 12865-12873Abstract Full Text PDF PubMed Google Scholar). Cells were then washed with acid-strip (50 mm glycine-HCl, 100 mm NaCl, 2 mg/ml polyvinylpyrrolidone, pH 3.0) for 2 min at 0 °C to remove surface-bound ligand (39Wiley H.S. Herbst J.J. Walsh B.J. Lauffenburger D.A. Rosenfeld M.G. Gill G.N. J. Biol. Chem. 1991; 266: 11083-11094Abstract Full Text PDF PubMed Google Scholar), rinsed twice with phosphate-buffered saline, and returned to 37 °C in D/H/B containing 1 μg/ml unlabeled ligand to prevent rebinding and reinternalization of recycled ligand. The medium was collected at 10 min and an aliquot counted for total radioactivity. Cells were solubilized with 2% sodium dodecyl sulfate and the amount of radioactivity remaining was determined. The medium was loaded on a 15% native polyacrylamide slab gel and the intact and degraded EGF was separated by isotachophoresis (41Herbst J.J. Opresko L.K. Walsh B.J. Lauffenburger D.A. Wiley H.S. J. Biol. Chem. 1994; 269: 12865-12873Abstract Full Text PDF PubMed Google Scholar). After drying the gel, the relative amount of radioactivity in the bands corresponding to intact and degraded EGF was quantified using a Bio-Rad G250 Molecular Imager. Cell number per plate was determined by counting parallel plates. Fraction of intact ligand was then plotted as a function of ligand in the cells at the start of the chase (lost into the medium + amount remaining) as described previously (28French A.R. Sudlow G.P. Wiley H.S. Lauffenburger D.A. J. Biol. Chem. 1994; 269: 15749-15755Abstract Full Text PDF PubMed Google Scholar). Cells were labeled to steady state (24 h) with cysteine and methionine-free Dulbecco's modified Eagle's medium (ICN) supplemented with 250 μCi/ml EXPRE35S35S from NEN Life Science Products Inc. which contains both radiolabeled methionine and cysteine. Cultures were rinsed 6 times with normal culture medium and chased with medium with or without 100 ng/ml EGF. At 0, 1, 3, 5, and 7 h chase, cells were lysed in RIPA buffer and equal amounts of protein were subjected to immunoprecipitation with 5 μl of 1917 antibody. Samples were then separated by SDS-gel electrophoresis and the gels were then dried on 3MM paper followed by quantitation of erbB-2 bands using a Bio-Rad Molecular Imager as described above. Ligand binding not only activates the EGFR but also initiates negative regulatory processes. Overexpression of the EGFR, however, has been shown to inhibit this negative regulation. Since erbB-2 acts as a signaling partner of the EGFR, we wanted to determine whether erbB-2 overexpression affected its own negative regulation, or that of the EGFR. We employed a human mammary epithelial cell line (MTSV) and two derivative lines (ce-1 and ce-2) which have been transfected with the gene for erbB-2 (35D'Souza B. Berdichevsky F. Kyprianou N. Taylor-Papadimitriou J. Oncogene. 1993; 8: 1797-1806PubMed Google Scholar, 42Bartek J. Bartkova J. Kyprianou N. Lalani E.-N. Staskova Z. Shearer M. Chang S. Taylor-Papadimitriou J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3520-3524Crossref PubMed Scopus (143) Google Scholar). The parent cell line, derived from human breast aspirates, was immortalized with SV40 large T antigen, but is not tumorigenic. Transfection with the erbB-2 gene alters the growth characteristics of the ce-1 and ce-2 cells in that the transfectants grow in soft agar can be propagated as tumors in nude mice, and show disorganized growth on collagen gels (35D'Souza B. Berdichevsky F. Kyprianou N. Taylor-Papadimitriou J. Oncogene. 1993; 8: 1797-1806PubMed Google Scholar). Western blot analysis and binding of radiolabeled anti-erbB-2 (4D5) at 37 °C was used to quantify the expression levels of erbB-2 in these cells. This approach was taken because equilibrium binding could not be achieved at 0 °C due to the very slow binding of 4D5. Steady state binding at 37 °C also allowed us to estimate the relative distribution of erB-2 between cell surface and intracellular pools (38Wiley H.S. Cunningham D.D. Cell. 1981; 25: 433-440Abstract Full Text PDF PubMed Scopus (165) Google Scholar). Preliminary experiments showed that treating the cells overnight with 4D5 did not appear to alter the cellular distribution of erbB-2 (data not shown). Steady state binding of anti-erbB-2 indicated an accessible pool of 9.8 × 104 and 1.5 × 106 erbB-2 molecules per cell in MTSV and ce2 cells, respectively (Fig.1 A), a 15-fold increase in the transfected cell line. Acid stripping of the bound radiolabeled 4D5 showed that surface expression of erbB-2 was 6.3 × 104 in the case of MTSV cells and 6.4 × 105 for ce2 cells. ErbB-2 expression in the ce-1 line varied between 2- and 6-fold higher than the parental cells (data not shown). Western blot analysis indicated a 23-fold increase in erbB-2 mass in the ce2 versus MTSV lines, somewhat higher than the values derived from the steady state analysis, suggesting that not all cellular erbB-2 readily exchanges with the cell surface. The affinity of the 4D5 antibody for erbB-2 was similar for both the MTSV and ce2 cells at 12 and 10 nm, respectively. The percent of 4D5 antibody found internalized at steady state was also similar at 41% (±4%) and 55% (±14%) for MTSV and ce2 cells, respectively. The number of EGFR in these cells was determined by Scatchard analysis conducted at 0 °C to prevent receptor down-regulation. As shown in Fig. 1 B, both MTSV and ce2 cells displayed similar numbers of surface EGFR (5.7 × 105 and 9.2 × 105 per cell, respectively) of predominantly a single affinity class. The affinity of these receptors, 1.4 nm, is similar to what has been described previously for fibroblasts (43Wiley H.S. Walsh B.J. Lund K.A. J. Biol. Chem. 1989; 264: 18912-18920Abstract Full Text PDF PubMed Google Scholar). Western blots of detergent extracts of MTSV, ce1, and ce2 cells confirm that they all express a similar number of EGFR (data not shown). Analysis of the Western blots using a molecular imager indicated that relative to cell protein content, ce2 cells express approximately 10% higher EGFR levels whereas the levels of EGFR in ce1 cells was indistinguishable from the parental MTSV cells. These data indicate that the ratio of EGFR to erbB-2 at the cell surface of MTSV cells is approximately 9:1 whereas the ratio in ce2 cells is approximately 1:1. To characterize erbB-2 and EGFR distribution in these cells, sparse cultures were fixed, permeabilized, and stained using directly labeled erbB-2 and EGFR antibodies. As shown in Fig. 2, erbB-2 was found at both the cell surface and in a collection of intracellular vesicles. The EGFR showed a very similar distribution pattern with EGFR and erbB-2 both colocalized at the cell surface and in intracellular vesicles (arrows in Fig. 2). These data suggest that overexpression of erbB-2 is not accompanied by any striking alteration in either its cellular distribution or affinity for antibodies. In addition, the number, distribution, and affinity of EGFR do not appear to be greatly altered as a result of erbB-2 overexpression. Because the EGFR transactivates erbB-2, changing the ratio of erbB-2 to EGFR may alter EGF-mediated erbB-2 phosphorylation. Both parental MTSV cells and overexpressing ce2 cells were treated with EGF for 5 and 10 min. ErbB-2 and EGFR were then immunoprecipitated followed by Western blot analysis for phosphotyrosine (Tyr(P)), to monitor receptor activation. As shown in Fig. 3, addition of EGF increased Tyr(P) levels of both EGFR and erbB-2. In the case of the parental MTSV cells, very little receptor phosphorylation was observed in the absence of EGF addition. Surprisingly, in ce2 cells, there was a substantial amount of phosphorylation of both erbB-2 and the EGFR in the absence of EGF. Although increased basal activation of erbB-2 as a result of overexpression has been documented by other investigators (34Ram T.G. Ethier S.P. Cell Growth Differ. 1996; 7: 551-567PubMed Google Scholar, 35D'Souza B. Berdichevsky F. Kyprianou N. Taylor-Papadimitriou J. Oncogene. 1993; 8: 1797-1806PubMed Google Scholar, 44Samanta A. LeVea C.M. Dougall W.C. Qian X. Greene M.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1711-1715Crossref PubMed Scopus (90) Google Scholar), higher basal activation of the EGFR has not previously been reported. Addition of EGF further increased the level of erbB-2 phosphorylation approximately 3-fold in ce2 cells as compared with 9-fold in the MTSV cells (average of three experiments), indicating that the EGFR was capable of transactivating erbB-2 in both cell types. It seemed possible that constitutive activation of both the EGFR and erbB-2 could be due to autocrine production of EGF-like ligands. We tested this idea by blocking EGFR activation using antagonistic EGFR antibodies 225 and 13A9 (45Gill G.N. Kawamoto T. Cochet C. Le A. Sato J.D. Masui H. McLeod C. Mendelsohn J. J. Biol. Chem. 1984; 259: 7755-7760Abstract Full Text PDF PubMed Google Scholar, 46Carraway III, K.L. Cerione R.A. J. Biol. Chem. 1993; 268: 23860-23867Abstract Full Text PDF PubMed Google Scholar). Neither antibody affected the constitutive activation of either the EGFR or erbB-2 (Fig. 3), suggesting that overexpression of erbB-2 alone was responsible. To determine whether overexpression of erbB-2 affects its down-regulation, we transacti
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