Title: Heregulin-dependent Trafficking and Cleavage of ErbB-4
Abstract: Heregulin was shown to promote the proteolytic cleavage of its receptor, ErbB-4, in several cell lines. The growth factor also rapidly promoted the transient translocation of ErbB-4 to a detergent-insoluble fraction, in which the receptor was hyper-tyrosine-phosphorylated compared with the receptor present in the detergent-soluble pool. However, an 80-kDa proteolytic fragment of ErbB-4 was found in the detergent-soluble fraction, but not in the detergent-insoluble fraction. Although the heregulin-induced cleavage of ErbB-4 produced a fragment of ErbB-4 very similar to that induced by 12-O-tetradecanoylphorbol-13-acetate or pervanadate (each of which is blocked by metalloprotease inhibitors), the growth factor-induced cleavage was not sensitive to these inhibitors under the same conditions. The heregulin-induced cleavage of ErbB-4 could be blocked by conditions that prevent clathrin-coated pit formation, suggesting that heregulin-mediated ErbB-4 cleavage occurs subsequent to internalization. When reagents that prevent acidification of endosomes were employed, heregulin-induced ErbB-4 cleavage was sensitive to metalloprotease inhibitors. The results imply that during ligand-dependent receptor trafficking, activated ErbB-4 receptors are subject to proteolytic cleavage involving an intracellular metalloprotease. Heregulin was shown to promote the proteolytic cleavage of its receptor, ErbB-4, in several cell lines. The growth factor also rapidly promoted the transient translocation of ErbB-4 to a detergent-insoluble fraction, in which the receptor was hyper-tyrosine-phosphorylated compared with the receptor present in the detergent-soluble pool. However, an 80-kDa proteolytic fragment of ErbB-4 was found in the detergent-soluble fraction, but not in the detergent-insoluble fraction. Although the heregulin-induced cleavage of ErbB-4 produced a fragment of ErbB-4 very similar to that induced by 12-O-tetradecanoylphorbol-13-acetate or pervanadate (each of which is blocked by metalloprotease inhibitors), the growth factor-induced cleavage was not sensitive to these inhibitors under the same conditions. The heregulin-induced cleavage of ErbB-4 could be blocked by conditions that prevent clathrin-coated pit formation, suggesting that heregulin-mediated ErbB-4 cleavage occurs subsequent to internalization. When reagents that prevent acidification of endosomes were employed, heregulin-induced ErbB-4 cleavage was sensitive to metalloprotease inhibitors. The results imply that during ligand-dependent receptor trafficking, activated ErbB-4 receptors are subject to proteolytic cleavage involving an intracellular metalloprotease. epidermal growth factor 12-O-tetradecanoylphorbol-13-acetate tumor necrosis factor-alpha converting enzyme mitogen-activated protein kinase Dulbecco's modified Eagle's medium bovine serum albumin ErbB-4 is a member of the epidermal growth factor family of receptor tyrosine kinases (1Plowman 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: 17146-17150Crossref Scopus (692) Google Scholar) and is expressed in many normal adult and fetal tissues, particularly nerve and heart (2Srihivasan R. Poulsom R. Hurst H.C. Gullick W.J. J. Pathol. 1998; 185: 126-145Google Scholar). However, its expression in carcinoma cells is limited compared with other ErbB receptors (2Srihivasan R. Poulsom R. Hurst H.C. Gullick W.J. J. Pathol. 1998; 185: 126-145Google Scholar, 3Vogt U. Bielawski K. Schlotter C.M. Bosse U. Falkiewicz B. Podhasjska A.J. Gene (Amst. ). 1998; 223: 375-380Crossref PubMed Scopus (44) Google Scholar, 4Graber H.U. Friess H. Kaufmann B. Willi D. Zimmermann A. Korc M. Büchler W. Int. J. Cancer. 1999; 84: 24-27Crossref PubMed Scopus (61) Google Scholar). Mice lacking ErbB-4 die during mid-embryogenesis from aborted development of heart ventricle myocardial trabeculae and are also deficient in axon guidance during development of the central nervous system (5Gassmann M. Casagranda F. Orioli D. Simon H. Lai C. Klein R. Lemke G. Nature. 1995; 378: 390-394Crossref PubMed Scopus (954) Google Scholar, 6Golding J.P. Trainor P. Krumlauf R. Gassman M. Nat. Cell Biol. 2000; 2: 103-109Crossref PubMed Scopus (140) Google Scholar). Although all the EGF1 receptor family members are able to stimulate cell proliferation, ErbB-4 is also implicated as a positive regulator of the differentiation of certain epithelial and neuronal tissues (7Schroeder J.A. Lee D.C. Cell Growth Differ. 1998; 9: 451-465PubMed Google Scholar, 8Vaskovsky A. Lupowitz Z. Erlich S. Pinkas-Kramarski R. J. Neurochem. 2000; 74: 979-987Crossref PubMed Scopus (82) Google Scholar, 9Jones F.E. Welte T. Fu X.-Y. Stern D.F. J. Cell Biol. 1999; 147: 77-87Crossref PubMed Scopus (159) Google Scholar). Ligands for the ErbB-4 receptor include various heregulin isoforms, which also bind to ErbB-3 (10Jones J.T. Akita R.W. Sliwkowski M.X. FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (325) Google Scholar, 11Beerli R.R. Graus-Porta D. Woods-Cook K. Chen X. Yarden Y. Hynes N.E. Mol. Cell. Biol. 1995; 15: 6496-6505Crossref PubMed Scopus (177) Google Scholar), and betacellulin (10Jones J.T. Akita R.W. Sliwkowski M.X. FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (325) Google Scholar, 12Riese D.J., II Bermingham Y. van Raaij T.M. Buckley S. Plowman G.D. Stern D.F. Oncogene. 1996; 12: 345-353PubMed Google Scholar), which also binds to the EGF receptor (12Riese D.J., II Bermingham Y. van Raaij T.M. Buckley S. Plowman G.D. Stern D.F. Oncogene. 1996; 12: 345-353PubMed Google Scholar). Following ligand stimulation, the ErbB-4 receptor undergoes homodimerization and/or heterodimerization with other receptors of the ErbB family and initiates cellular responses (13Riese D.J., II Stern D.F. Bioessays. 1998; 20: 41-48Crossref PubMed Scopus (698) Google Scholar, 14Alroy I. Yarden Y. FEBS Lett. 1997; 410: 83-86Crossref PubMed Scopus (657) Google Scholar). Desensitization and down-regulation of activated growth factor receptors have an important role in regulating cellular events triggered by ligand binding and determine signaling duration and/or potency. A common pathway for down-regulation of many activated growth factor receptors is clathrin-coated pit-mediated endocytosis (15Sorkin, A. (1998) Front. Biosci. 3, d729–738.Google Scholar). In the case of the EGF receptor, the activated receptor rapidly enters clathrin-coated pits and is internalized and subsequently sorted from endosomes to lysosomes, where the receptor and its ligand are degraded (15Sorkin, A. (1998) Front. Biosci. 3, d729–738.Google Scholar, 16Carpenter G. BioEssays. 2000; 22: 697-707Crossref PubMed Scopus (307) Google Scholar). However, studies with NIH 3T3 cells expressing wild-type ErbB receptors or chimeric receptors and some ErbB-expressing human carcinoma cell lines showed that all other activated ErbB receptors (ErbB-2, ErbB-3, and ErbB-4) are endocytosis-impaired and are inefficiently internalized (17Baulida 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 (377) Google Scholar). Earlier studies from this laboratory showed that the ErbB-4 receptor is slowly and constitutively cleaved, producing a membrane-anchored cytoplasmic domain fragment of 80 kDa (18Vecchi M. Carpenter G. J. Cell Biol. 1997; 139: 995-1003Crossref PubMed Scopus (114) Google Scholar). ErbB-4 receptor cleavage is greatly enhanced by the protein kinase C activator TPA or by pervanadate, a potent phosphotyrosine phosphatase inhibitor (19Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 20Vecchi M. Rudolph-Owen L.A. Brown C.L. Dempsey P.J. Carpenter G. J. Biol. Chem. 1998; 273: 20589-20595Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These results suggest the presence of intracellular signaling pathways that can regulate this cleavage event. Both the TPA- and pervanadate-induced cleavages of ErbB-4 are completely inhibited by the hydroxamate-based metalloprotease inhibitor BB-94. Recently, it has been shown that the tumor necrosis factor-converting enzyme (TACE) is required for TPA- or pervanadate-induced ErbB-4 cleavage (21Rio C. Buxbaum J.D. Peschon J.J. Corfas G. J. Biol. Chem. 2000; 275: 10379-10387Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Also, a 23-residue domain in the extracellular juxtamembrane domain of ErbB-4 is necessary for this cleavage (21Rio C. Buxbaum J.D. Peschon J.J. Corfas G. J. Biol. Chem. 2000; 275: 10379-10387Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 22Elenius K. Corfas G. Paul S. Choi C.J. Rio C. Plowman G.D. Klagsbrun M. J. Biol. Chem. 1997; 272: 26761-26768Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). However, the exact site of cleavage of ErbB-4 by TACE is not known. TACE and other metalloproteases have been shown to be involved in ectodomain shedding of a variety of membrane proteins, including the precursor for transforming growth factor α, an EGF receptor ligand (23Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1374) Google Scholar). In many of these TACE-dependent cases, cleavage can be stimulated by TPA (24Schlöndorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar), as reported for ErbB-4 (19Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). However, studies of the regulated cleavage of cell-surface proteins by hormones, growth factors, and pervanadate indicate that these proteolytic events are protein kinase C-independent and involve signaling pathways that are not well defined. Fan and Derynck (25Fan H. Derynck R. EMBO J. 1999; 18: 6962-6972Crossref PubMed Google Scholar) have shown that shedding of the transforming growth factor can be stimulated by several different growth factors acting through receptor tyrosine kinase activation and the MAPK signaling pathway. In contrast to stimulation by TPA or pervanadate, the proteolytic sensitivity of the ErbB-4 receptor to ligand stimulation has not been reported. It is of particular interest to know if protease cleavage could be a mechanism for down-regulation of activated ErbB-4 receptor in the absence of rapid endocytosis and lysosomal degradation. In this study, we have found that in several tumor cell lines, ligand stimulation of ErbB-4 produces a reduction of the ErbB-4 receptor level and accumulation of an 80-kDa fragment, suggesting that a proteolytic cleavage of the ErbB-4 receptor occurs. Evidence that this cleavage may occur in an intracellular compartment is presented. Heregulin β1 EGF domain peptide, betacellulin, and heregulin were purchased from R&D Systems (Minneapolis, MN). EGF was prepared from mouse submaxillary glands as described previously (26Savage Jr., C.R. Cohen S. J. Biol. Chem. 1972; 247: 7609-7611Abstract Full Text PDF PubMed Google Scholar). Epiregulin was a gift of Dr. Toshi Komurasaki (Taisho Pharmaceutical Co.). Rabbit antiserum raised against carboxyl-terminal sequence 1108–1264 of human ErbB-4 was generously provided by Dr. Matthias Kraus (European Institute of Oncology, Milan, Italy). Polyclonal antibody to an epitope in the carboxyl terminus of ErbB-4 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine polyclonal IgG and horseradish peroxidase-conjugated protein A were purchased from Zymed Laboratories Inc. (South San Francisco, CA). Protein A-Sepharose CL-4B, enhanced chemiluminescence (ECL) reagents, TPA, GF109203X, and chloroquine were obtained from Sigma. The metalloprotease inhibitorsN-d-l-[2-(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl-1-3-(tert-butyl)-alanyl-1-alanine, 2- aminoethylamide and batimastat (BB-94) were generous gifts of Drs. L. Matrisian and P. Dempsey (Vanderbilt University, Nashville, TN). Folimycin was obtained from Calbiochem-Novabiochem. Pervanadate was freshly prepared by mixing 80 μl of 1 m sodium orthovanadate, 70 μl of phosphate-buffered saline, and 10 μl of 30% H2O2. This solution was applied to cell cultures within 20 min at a final concentration of 100 μm. The human ovarian carcinoma cell lines OVCA429 and OVCA432 were obtained from Dr. Robert Bast (M. D. Anderson Hospital), and OVCAR3 cells were a gift from Dr. Roy Jensen (Vanderbilt University). The human lung carcinoma cell lines H661 and H1155 and the human breast carcinoma line T47D were purchased from American Tissue Culture Collection. These cells were routinely grown in 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and penicillin/streptomycin (Life Technologies, Inc.). The rat glioma cell line C6 was kindly provided by Dr. Mary Ann Thompson (Vanderbilt University), and the cells were grown in medium F-10 containing 15% equine serum and penicillin/streptomycin. SK-Br-3 cells were obtained from Dr. Matthias Kraus (European Institute of Oncology) and were grown in McCoy's medium containing 10% fetal bovine serum. T47-14 cells, transfected NIH 3T3 cells that overexpress human ErbB-4, have been described elsewhere (17Baulida 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 (377) Google Scholar). The growth medium for T47-14 cells was DMEM containing 10% calf serum and penicillin/streptomycin. Atrial tumor myocytes (AT-1 cells) derived from T antigen transgenic mice (27Steinhelper M.E. Lanson Jr., N.A. Dresdner K.P. Del Carpio J.B. Wit A.L. Claycomb W.C. Field L.J. Am. J. Physiol. 1990; 259: H1826-H1834PubMed Google Scholar, 28Del Carpio J.B. Lanson Jr., N.A. Field L.J. Claycomb W.C. Circ. Res. 1991; 69: 1591-1600Crossref PubMed Scopus (69) Google Scholar) were generously provided by D. M. Roden (Vanderbilt University) and were prepared and grown in culture as described previously (29Yang T. Wathen M.S. Felipe A. Tamkun M.M. Snyders D.J. Roden D.M. Circ. Res. 1994; 75: 870-878Crossref PubMed Scopus (72) Google Scholar). Experimental cultures were generally grown in 60- or 100-mm diameter culture dishes until nearly confluent. Cell lysates were obtained essentially as described previously (18Vecchi M. Carpenter G. J. Cell Biol. 1997; 139: 995-1003Crossref PubMed Scopus (114) Google Scholar). Briefly, newly confluent cell monolayers in 60-mm culture dishes were incubated overnight in DMEM containing 0.5% serum. Cells were then rinsed once with DMEM and incubated with the indicated additions for the indicated times at 37 °C in DMEM supplemented with 0.1% BSA and 20 mmHepes (pH 7.2). Next, cells were washed with Ca2+/Mg2+-free phosphate-buffered saline and incubated on ice for 30 min in 400 μl of TGH lysis buffer (1% Triton X-100, 10% glycerol, 20 mm Hepes (pH 7.2), 100 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mmNa3VO4, and 50 mm NaF). Lysates were then centrifuged at 14,000 × g for 10 min at 4 °C. The resulting supernatant was termed the Triton X-100-soluble fraction. The pellet was dissolved in TGH buffer supplemented with 0.5% SDS in a volume equal to its supernatant or as indicated. The solution was then passed through a syringe with a 23-gauge needle five to eight times to shear DNA and was termed the Triton X-100-insoluble fraction. Prior to immunoprecipitation, the SDS concentration was adjusted to 0.1%. For ErbB-4 immunoprecipitation, 1 mg of cell lysate from the Triton X-100-soluble fraction or an equal volume of the Triton X-100-insoluble fraction was incubated with 3 μg of anti-ErbB-4 polyclonal antibody for 3 h at 4 °C before incubation with protein A-Sepharose for 1 h at 4 °C. Subsequently, the immunocomplexes were washed three times with TGH buffer and resuspended in Laemmli buffer. After boiling, proteins in the samples were electrophoretically separated on a reducing SDS-7.5% polyacrylamide gel and transferred to nitrocellulose membranes for Western blotting. Membranes were blocked with 5% milk in TBST buffer (50 mm Tris (pH 7.4), 150 mm NaCl, and 0.05% Tween) for 1 h. For anti-phosphotyrosine blotting, membranes were blocked with 3% BSA in TBST buffer for 1 h. The membranes were then incubated with either ErbB-4 antiserum or anti-phosphotyrosine polyclonal antibody in TBST buffer containing 1% BSA for 2 h at room temperature. After the antibody incubation, the membranes were washed three times with TBST buffer for 30 min and incubated with horseradish peroxidase-conjugated protein A in TBST buffer containing 1% BSA at room temperature. Bound antibodies were detected by ECL. For direct Western blots, a lysate aliquot of ∼100 μg was loaded onto SDS-7.5% polyacrylamide gels, electrophoresed, transferred to nitrocellulose membranes, and blotted as described above. The use of potassium depletion conditions to prevent internalization by clathrin-coated pits has been described elsewhere in detail (30Larkin J.M. Donzell W.C. Anderson R.G.W. J. Cell Biol. 1986; 103: 2619-2627Crossref PubMed Scopus (110) Google Scholar, 31Heuser J.E. Anderson R.G.W. J. Cell Biol. 1989; 108: 389-400Crossref PubMed Scopus (776) Google Scholar). In brief, cell monolayers incubated overnight in low serum were washed once with depletion buffer (50 mm Hepes (pH 7.4), 100 mm NaCl, 1 mm CaCl2, and 1 mmMgCl2) and incubated in DMEM/H2O (1:1) for 5 min. Cells were then washed with depletion buffer once again and incubated in depletion buffer for 2 h at 37 °C. After incubation in depletion buffer, cells were incubated with heregulin β1 or TPA in either depletion buffer containing 0.1% BSA or in regular medium (DMEM containing 0.1% BSA, and 20 mm Hepes) as the control. To determine whether growth factor binding to the ErbB-4 receptor tyrosine kinase initiates proteolytic cleavage of this receptor, as previously reported for TPA (19Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and pervanadate (20Vecchi M. Rudolph-Owen L.A. Brown C.L. Dempsey P.J. Carpenter G. J. Biol. Chem. 1998; 273: 20589-20595Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), several cell lines were surveyed. These included two breast carcinoma cell lines (T47D and SK-Br-3), three ovarian carcinoma cell lines (OVCAR3, OVCA432, and OVCA429), two lung carcinoma cell lines (H661 and H1155), one glioma cell line (C6), a mouse cardiomyocyte cell line (AT-1), and the NIH 3T3-derived cell line T47-14. These cells were chosen on the basis of literature reports indicating that they express moderate to high levels of ErbB-4, which is not commonly expressed in cell lines. The presence of ErbB-4 in these lines was confirmed by Western blotting, and each was tested for heregulin-induced cleavage of the ErbB-4 receptor using an antibody to a cytoplasmic domain epitope. As shown in Fig.1 A, heregulin-induced the accumulation of an 80-kDa ErbB-4 fragment in three cell lines: T47D, OVCAR3, and OVCA432. A similar-sized fragment of ErbB-4 was produced by treatment of these cells with TPA, as reported previously for other cell lines (19Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In the other cell lines tested, no heregulin-induced fragment was detectable, although TPA-induced ErbB-4 cleavage was always detectable, and heregulin did induce ErbB-4 tyrosine phosphorylation (data not shown). Subsequent experiments were conducted with T47D cells. The time course of heregulin-induced accumulation of the 80-kDa fragment in T47D cells is shown in Fig. 1 B. Increased levels of the 80-kDa fragment were detectable at 15 min after the addition of the growth factor and were maximal at 60 min. Thereafter, the amount of the 80-kDa ErbB-4 fragment decreased. Beginning at 60 min after heregulin addition, there was also a decrease in the level of the native form of ErbB-4. This time course is similar to that reported previously for TPA- or pervanadate-induced cleavage (19Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 20Vecchi M. Rudolph-Owen L.A. Brown C.L. Dempsey P.J. Carpenter G. J. Biol. Chem. 1998; 273: 20589-20595Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). From the data in Fig. 1 A, it is clear that TPA produces a more substantial level of ErbB-4 cleavage than does heregulin. Scanning densitometry indicates that at 60 min, the amount of the 80-kDa fragment in cells treated with heregulin is equal to 20% of the total cellular content of ErbB-4. Several ligands have been reported to bind to ErbB-4 and to act as activators of this receptor. These include the α and β forms of heregulin (10Jones J.T. Akita R.W. Sliwkowski M.X. FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (325) Google Scholar, 11Beerli R.R. Graus-Porta D. Woods-Cook K. Chen X. Yarden Y. Hynes N.E. Mol. Cell. Biol. 1995; 15: 6496-6505Crossref PubMed Scopus (177) Google Scholar), betacellulin (12Riese D.J., II Bermingham Y. van Raaij T.M. Buckley S. Plowman G.D. Stern D.F. Oncogene. 1996; 12: 345-353PubMed Google Scholar), and epiregulin (32Shelly M. Pinkas-Kramarski R. Guarino B.C. Waterman H. Wang L.-M. Lyass L. Alimandi M. Kuo A. Bacus S.S. Pierce J.H. Andrews G.C. Yarden Y. J. Biol. Chem. 1998; 273: 10496-10505Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The heregulins also recognize ErbB-3, whereas betacellulin and epiregulin also bind to the EGF receptor. Therefore, we tested these ligands for their capacity to stimulate ErbB-4 cleavage. As shown in Fig.2 A, accumulation of the 80-kDa ErbB-4 fragment was enhanced by treatment of the cells with heregulin β, betacellulin, or heregulin α, in the approximate order of efficacy. As shown in Fig. 2 B, each of these ligands also stimulated ErbB-4 tyrosine phosphorylation in the same cells. Epiregulin or EGF, a negative control, did not alter the basal level of this fragment, and as shown in Fig. 2 B, neither of these two growth factors stimulated ErbB-4 autophosphorylation. ErbB-1 (EGF) receptors have been reported to be present in both the detergent (Triton X-100)-soluble and -insoluble fractions of fibroblasts (33Mineo C. Gill G.N. Anderson R.G.W. J. Biol. Chem. 1999; 274: 30636-30643Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), A-431 cells (34Waugh M.G. Lawson D. Hsuan J.J. Biochem. J. 1999; 337: 591-597Crossref PubMed Scopus (130) Google Scholar), and PC12 cells (35Huang C. Zhou J. Feng A.K. Lynch C.C. Klumperman J. DeArmond S.J. Mobley W.C. J. Biol. Chem. 1999; 274: 36707-36714Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), whereas ErbB-4 presence in both of these fractions has been reported for cardiomyocytes (ErbB-4) (36Zhao Y.-Y. Feron O. Dessy C. Han X. Marchionni M.A. Kelly R.A. Circ. Res. 1999; 84: 1380-1387Crossref PubMed Scopus (64) Google Scholar). The addition of ligand has been reported to promote the migration of ErbB-1 (34Waugh M.G. Lawson D. Hsuan J.J. Biochem. J. 1999; 337: 591-597Crossref PubMed Scopus (130) Google Scholar) and ErbB-4 (36Zhao Y.-Y. Feron O. Dessy C. Han X. Marchionni M.A. Kelly R.A. Circ. Res. 1999; 84: 1380-1387Crossref PubMed Scopus (64) Google Scholar) out of these detergent-insoluble domains. The exact nature of the detergent-insoluble domains is unclear, and in some cases, they are referred to as caveolae (37Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1733) Google Scholar). We have examined ErbB-4 movement between these different fractions to determine whether heregulin-induced ErbB-4 cleavage is associated with these trafficking events. The results, shown in Fig.3 A, demonstrate that in the absence of heregulin, all detectable ErbB-4 molecules were present in the detergent-soluble fraction (compare lanes 1 and7). When heregulin was added to T47D cells, there was a rapid and transient increase in the amount of ErbB-4 in the detergent-insoluble fraction; however, no 80-kDa ErbB-4 fragment was detectable in this fraction (lanes 2–6). In contrast, the 80-kDa fragment transiently accumulated in the detergent-soluble fraction following ligand addition (lanes 8–12). The data shown in Fig. 3 A were quantitated to show the influence of time on the amount of native ErbB-4 that appeared in the detergent-insoluble fraction and the amount of the 80-kDa fragment that was found in the detergent-soluble fraction (Fig. 3 C). Clearly, the native 180-kDa molecule appeared in the detergent-insoluble fraction more rapidly than the 80-kDa fragment was found in the detergent-soluble fraction following heregulin addition. These data do not suggest that translocation of ErbB-4 to the detergent-insoluble fraction is the mechanism by which proteolytic fragmentation occurs. It is possible, however, that the 80-kDa fragment is produced in the detergent-insoluble fraction and very rapidly migrates out of this fraction. To test this, we also determined whether TPA or pervanadate treatment of T47D cells, each of which provokes more extensive fragmentation of ErbB-4 than heregulin, induced translocation of ErbB-4 to the detergent-insoluble fraction. As shown in Fig.4, neither TPA nor pervanadate, in contrast to heregulin, brought about detectable levels of ErbB-4 in the detergent-insoluble fraction. This ligand-dependent distribution of ErbB-4 between the detergent-soluble and -insoluble fractions was also examined for the tyrosine phosphorylation of ErbB-4, as shown in Fig. 3 B. Following heregulin addition, tyrosine-phosphorylated ErbB-4 rapidly appeared in both the detergent-insoluble and -soluble fractions. Tyrosine phosphorylation of the 80-kDa fragment was not detectable in this system. We have used scanning densitometry to compare the level of tyrosine phosphorylation at each time point in both fractions with the amount of ErbB-4 present (Fig. 3 D). These results indicate that the ErbB-4 present in the detergent-insoluble fraction is more highly phosphorylated (∼5-fold) than the ErbB-4 present in the detergent-soluble fraction. The basal level of cleavage of ErbB-4 in untreated cells (18Vecchi M. Carpenter G. J. Cell Biol. 1997; 139: 995-1003Crossref PubMed Scopus (114) Google Scholar) as well as the TPA-induced (19Vecchi M. Baulida J. Carpenter G. J. Biol. Chem. 1996; 271: 18989-18995Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) or pervanadate-induced (20Vecchi M. Rudolph-Owen L.A. Brown C.L. Dempsey P.J. Carpenter G. J. Biol. Chem. 1998; 273: 20589-20595Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) cleavage of this receptor are all sensitive to BB-94, a broad-spectrum metalloprotease inhibitor that blocks the ectodomain cleavage of a number of cell-surface proteins. However, the heregulin-induced cleavage of ErbB-4 was not diminished by preincubation of the cells with BB-94, as shown in Fig.5 A. In this experiment, BB-94 did prevent ErbB-4 cleavage induced by TPA. Similar results were obtained with the metalloprotease inhibitorN-d-l-[2-(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl-1-3-(tert-butyl)-alanyl-1-alanine, 2-aminoethylamide (data not shown) for both heregulin- and pervanadate-induced ErbB-4 cleavages. Also, other metalloprotease inhibitors such as EDTA, BB-2116, BB-3105, and 1,10-phenanthroline did not influence heregulin-induced ErbB-4 cleavage under these conditions. Since the protein kinase C activator TPA is a potent stimulator of ErbB-4 cleavage as well as other cell-surface proteolytic events and is likely activated by heregulin binding to ErbB-4, we used the protein kinase C inhibitor GF109203X to test whether protein kinase C is required for heregulin-induced ErbB-4 cleavage. As shown in Fig.5 B, GF109203X blocked TPA-induced ErbB-4 cleavage, but did not alter heregulin-induced fragmentation of the receptor. The results with metalloprotease inhibitors were surprising in view of the similar-sized ErbB-4 fragment produced by heregulin, TPA, or pervanadate. This suggested either that the heregulin-induced cleavage is metalloprotease-independent or that perhaps receptor trafficking, induced uniquely by the growth factor, might modify the sensitivity of the cleavage to the metalloprotease inhibitors. Following ligand binding, ErbB-4 receptors are slowly endocytosed compared with occupied EGF receptors (17Baulida 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 (377) Google Scholar). However, this slowed rate of internalization might be sufficient to accommodate the rate and extent of heregulin-induced cleavage of ErbB-4. A widely employed technique to prevent internalization of receptors is the K+ depletion method, which prevents the formation of clathrin-coated pits (30Larkin J.M. Donzell W.C. Anderson R.G.W. J. Cell Biol. 1986; 103: 2619-2627Crossref PubMed Scopus (110) Google Scholar, 31Heuser J.E. Anderson R.G.W. J. Cell Biol. 1989; 108: 389-400Crossref PubMed Scopus (776) Google Scholar). The data presented in Fig. 6 show that K+ depletion effectively prevented heregulin-induced ErbB-4 cleavage (panel A), but did not prevent cleavage induced by TPA (panel B). The data obtained with K+ depletion implicate the endocytic pathway as a means by which ErbB-4 trafficking may be required for heregulin-induced cleavage of the receptor. Therefore, we assessed several inhibitors that prevent the acidification of endocytic vesic