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Heregulin Stimulates Mitogenesis and Phosphatidylinositol 3-Kinase in Mouse Fibroblasts Transfected with erbB2/neu and erbB3(∗)

  • Kermit L. Carraway III
    Correspondence
    Recipient of a postdoctoral fellowship from the American Cancer Society. To whom correspondence should be addressed: Harvard Medical School, Warren Alpert Building, 1st Floor, 200 Longwood Ave., Boston, MA 02115 . Tel.: 617-278-3043; Fax: 617-278-3131
    Affiliations
    Department of Cell Biology, Harvard Medical School, and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115
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  • Stephen P. Soltoff
    Affiliations
    Department of Cell Biology, Harvard Medical School, and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115
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  • A. John Diamonti
    Affiliations
    Department of Cell Biology, Harvard Medical School, and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115
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  • Lewis C. Cantley
    Affiliations
    Department of Cell Biology, Harvard Medical School, and Division of Signal Transduction, Beth Israel Hospital, Boston, Massachusetts 02115
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  • Author Footnotes
    ∗ This research was supported in part by National Institutes of Health Grant GM41890 (to L. C. C.) and a grant from the Massachusetts Department of Public Health (to K. L. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:March 31, 1995DOI:https://doi.org/10.1074/jbc.270.13.7111
      Heregulin (HRG) is a pluripotent growth factor that can stimulate the growth of some human mammary tumor cells and the differentiation of others. Two members of the epidermal growth factor receptor family of receptor/tyrosine kinases, p180erbB3 and p180erbB4, serve as receptors for the HRG ligand. While HRG appears to be capable of stimulating the autophosphorylation activity of p180erbB4, the co-expression of p185erbC2erbC2neu with p180erbB3 is necessary for the HRG-stimulated tyrosine phosphorylation of both of these receptors. On the basis of the sequences surrounding their putative tyrosine phosphorylation sites, we predict that the different HRG-responsive receptors couple to different intracellular SH2 domain-containing proteins. Hence, the different receptors may mediate different cellular responses to the HRG ligand. In the present study we show that HRGβ1 is mitogenic for erbB3-transfected DHFR/G8 cells, an NIH3T3 mouse fibroblast derivative that overexpresses p185erbC2neu. HRG stimulated the incorporation of [3H]thymidine into the DNA of these cells with an EC50 of 70 ± 7 pM. HRG was not mitogenic for parental DHFR/G8 cells that do not express the ErbB3 protein. Phosphatidylinositol (PI) 3-kinase, an enzyme believed to be important in cellular growth regulation by growth factors and oncogenes, is predicted to couple to tyrosine-phosphorylated ErbB3. We observed that HRG stimulated the association of PI 3-kinase with both p185erbC2neu and ErbB3 in transfected DHFR/G8 cells, but not in the parental cell line. We conclude that the ErbB3 protein is capable of mediating a proliferative response of fibroblasts to HRG, and that the activation of PI 3-kinase is an integral part of the growth signaling mechanism.

      INTRODUCTION

      The epidermal growth factor (EGF)
      The abbreviations used are: EGF
      epidermal growth factor
      SH2
      src homology-2
      HRG
      heregulin
      PAGE
      polyacrylamide gel electrophoresis
      PI
      phosphatidylinositol
      PDGF
      platelet-derived growth factor
      BS3
      bis(sulfosuccinimidyl)suberate.
      receptor and its relatives p185erbC2neu, p180erbB3, and p180erbB4 interact with specific polypeptide growth factor ligands to regulate cellular growth and differentiation(
      • Ullrich A.
      • Schlessinger J.
      ). In general, growth factor binding to its target receptor stimulates the intrinsic protein tyrosine kinase activity and autophosphorylation of that receptor. Receptor autophosphorylation then mediates the recruitment of specific intracellular proteins that possess src homology-2 (SH2) domains to the activated receptor complex(
      • Pawson T.
      • Schlessinger J.
      ). This in turn triggers cascades of events that propagate the signal to the nucleus, culminating in a biological response(
      • Bishop J.M.
      ).
      Heregulin (HRG) is a recently identified EGF-like growth factor that can regulate the growth of some cultured human mammary tumor cells(
      • Peles E.
      • Bacus S.S.
      • Koski R.A.
      • Lu H.S.
      • Wen D.
      • Ogden S.G.
      • Ben Levy R.
      • Yarden Y.
      ,
      • Holmes 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 W.I.
      • Goeddel D.V.
      • Vandlen R.L.
      ), and may be involved in regulating the growth and differentiation of neuronal tissues(
      • Falls D.L.
      • Rosen K.M.
      • Corfas G.
      • Lane W.S.
      • Fischbach G.D.
      ,
      • Marchionni M.A.
      • Goodearl A.D.J.
      • Chen M.S.
      • Bermingham-McDonogh O.
      • Kirk C.
      • Hendricks M.
      • Danehy F.
      • Misumi D.
      • Sudhalter J.
      • Kobayashi K.
      • Wroblewski D.
      • Lynch C.
      • Baldassare M.
      • Hiles I.
      • Davis J.B.
      • Hsuan J.J.
      • Totty N.F.
      • Otsu M.
      • McBurney R.N.
      • Waterfield M.D.
      • Stroobant P.
      • Gwynne D.
      ). HRG was originally proposed to be a ligand for p185erbC2neu on the basis of its ability to stimulate the tyrosine phosphorylation of that receptor, as well as its ability to become covalently cross-linked to p185erbC2neu in various cancer cells. However, HRG does not interact with p185erbC2neu in all cells that express this receptor(
      • Peles E.
      • Ben-Levy R.
      • Tzahar E.
      • Liu N.
      • Wen D.
      • Yarden Y.
      ).
      We have recently demonstrated that p180erbB3 is a receptor for HRG(
      • Carraway III, K.L.
      • Sliwkowski M.X.
      • Akita R.W.
      • Platko J.V.
      • Guy P.M.
      • Nuijens A.
      • Diamonti A.J.
      • Vandlen R.L.
      • Cantley L.C.
      • Cerione R.A.
      ,
      • Sliwkowski M.X.
      • Schaefer G.
      • Akita R.W.
      • Lofgren J.A.
      • Fitzpatrick V.D.
      • Nuijens A.
      • Fendly B.M.
      • Cerione R.A.
      • Vandlen R.L.
      • Carraway III, K.L.
      ). Interestingly, it appears that p180erbB3 is incapable of mediating transmembrane signaling in response to HRG, probably because it possesses an impaired intrinsic tyrosine kinase activity(
      • Guy P.M.
      • Platko J.V.
      • Cantley L.C.
      • Cerione R.A.
      • Carraway III, K.L.
      ). We have observed that the co-expression of p180erbB3 with p185erbC2neu is necessary for the heregulin-stimulated phosphorylation of both receptors, and for the creation of a high-affinity binding site for HRG (
      • Sliwkowski M.X.
      • Schaefer G.
      • Akita R.W.
      • Lofgren J.A.
      • Fitzpatrick V.D.
      • Nuijens A.
      • Fendly B.M.
      • Cerione R.A.
      • Vandlen R.L.
      • Carraway III, K.L.
      ). On the basis of these observations, we have proposed that a ligand-stimulated receptor heterodimerization event is responsible for transmembrane signaling(
      • Carraway III, K.L.
      • Cantley L.C.
      ). HRG also appears to be a ligand for p180erbB4, although in this case a receptor heterodimerization event is not required to mediate signal transduction(
      • Plowman G.D.
      • Green J.M.
      • Culouscou J.-M.
      • Carlton G.W.
      • Rothwell V.M.
      • Buckley S.
      ,
      • Tzahar E.
      • Levkowitz G.
      • Karunagaran D.
      • Yi L.
      • Peles E.
      • Lavi S.
      • Chang D.
      • Liu N.
      • Yayon A.
      • Wen D.
      • Yarden Y.
      ).
      Previous studies have demonstrated that HRG is capable of stimulating the growth of some human mammary tumor cell lines (
      • Holmes 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 W.I.
      • Goeddel D.V.
      • Vandlen R.L.
      ) and the differentiation of others(
      • Peles E.
      • Bacus S.S.
      • Koski R.A.
      • Lu H.S.
      • Wen D.
      • Ogden S.G.
      • Ben Levy R.
      • Yarden Y.
      ,
      • Culouscou J.-M.
      • Plowman G.D.
      • Carlton G.W.
      • Green J.M.
      • Shoyab M.
      ). The diverse responses elicited by HRG undoubtedly arise from differences in the cellular signaling pathways emanating from activated receptors. Hence, interest has developed in characterizing the intracellular proteins that might participate in cellular responses to HRG. p180erbB3 is unique among the EGF receptor family members in that it possesses several putative phosphorylation sites that fit the consensus for binding phosphatidylinositol (PI) 3-kinase, an enzyme implicated in the regulation of cellular growth and transformation(
      • Kapeller R.
      • Cantley L.C.
      ). It has been observed that the treatment of some cells with EGF results in the tyrosine phosphorylation of p180erbB3 and the recruitment of PI 3-kinase to this receptor(
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ,
      • Kim H.-H.
      • Sierke S.L.
      • Koland J.G.
      ). These observations suggest that p180erbB3 may play a role in coupling PI 3-kinase to the action of EGF-like growth factors, in a manner analogous to that whereby insulin receptor substrate-1 couples PI 3-kinase to insulin action (
      • White M.F.
      • Kahn C.R.
      ).
      The purpose of the studies described in this report was to determine whether ErbB3 mediates HRG-dependent cell growth and whether PI 3-kinase associates with the ErbB3 protein in response to HRG stimulation.

      EXPERIMENTAL PROCEDURES

      Materials

      Anti-p185erbB2 monoclonal antibody 3E8, rHRGβ1177-244 and iodinated growth factor were the generous gifts of Dr. M. Sliwkowski (Genentech, Inc.). [3H]Thymidine was purchased from Amersham. Anti-p85 (N-SH2) was described previously(
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ). Anti-ErbB3 peptide antibody 3184 was raised to the same peptide as antibody 3185(
      • Carraway III, K.L.
      • Sliwkowski M.X.
      • Akita R.W.
      • Platko J.V.
      • Guy P.M.
      • Nuijens A.
      • Diamonti A.J.
      • Vandlen R.L.
      • Cantley L.C.
      • Cerione R.A.
      ), and was affinity-purified prior to use in immunoprecipitation experiments. Affinity-purified anti-ErbB3 peptide antibody 3183 was prepared as described previously(
      • Guy P.M.
      • Platko J.V.
      • Cantley L.C.
      • Cerione R.A.
      • Carraway III, K.L.
      ).

      Cell Culture and Lines

      Cells were carried in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. D33 cells were prepared by transfecting DHFR/G8 cells with bovine erbB3 under the MMTV promoter using the pMAMneo plasmid (Clontech), as described previously(
      • Carraway III, K.L.
      • Sliwkowski M.X.
      • Akita R.W.
      • Platko J.V.
      • Guy P.M.
      • Nuijens A.
      • Diamonti A.J.
      • Vandlen R.L.
      • Cantley L.C.
      • Cerione R.A.
      ). Stable transfectants were selected in 0.4 mg/ml G418 (Life Technologies, Inc.), and colonies were screened for ErbB3 expression by [125I]rHRGβ1177-244 binding. Briefly, stably transfected and selected cells were grown in six-well dishes to 70-90% confluence. Cells were then simultaneously treated with dexamethasone and serum starved overnight in Dulbecco's modified Eagle's medium with 0.1% calf serum. Serum-starved cells were then treated for 30 min at room temperature with [125I]rHRGβ1177-244 (0.1 nM, 1400 Ci/mmol) in the absence and presence of 50 nM unlabeled rHRGβ1177-244. Wells were washed twice with ice-cold phosphate-buffered saline, and cells solubilized in RIPA buffer. Scatchard analysis and covalent cross-linking of [125I]rHRGβ1177-244 to cells were carried out as described previously(
      • Carraway III, K.L.
      • Sliwkowski M.X.
      • Akita R.W.
      • Platko J.V.
      • Guy P.M.
      • Nuijens A.
      • Diamonti A.J.
      • Vandlen R.L.
      • Cantley L.C.
      • Cerione R.A.
      ).

      Immunoprecipitation Experiments

      100-mm dishes of DHFR/G8 or D33 cells were serum starved and treated overnight with 40 ng/ml dexamethasone, and then treated for 5 min without and with 20 nM rHRGβ1177-244. Cells were washed twice with ice-cold phosphate-buffered saline and lysed in 1 ml of either Nonidet P-40 lysis buffer (20 mM HEPES/Na, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1% Nonidet P-40, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 250 μM phenylmethylsulfonyl chloride) or RIPA buffer (20 mM Tris/HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM Na3VO4, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 5 μg/ml pepstatin A, 250 μM phenylmethylsulfonyl chloride). Lysates were cleared by a 15-min centrifugation at 12,000 × g, and cleared lysates were immunoprecipitated with 1.5 μg of anti-p185erbB2 monoclonal 3E8(
      • Fendly B.M.
      • Winget M.
      • Hudziak R.M.
      • Lipari M.T.
      • Napier M.A.
      • Ullrich A.
      ), 1.5 μg of affinity-purified anti-ErbB3 peptide antibody 3184, or 3 μg of antiphosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Inc.). Immunoprecipitates were washed three times with lysis buffer, and analyzed by 6 or 7% SDS-PAGE followed by immunoblotting with anti-ErbB3 3183, anti-p85 (N-SH2), or antiphosphotyrosine. Lipid kinase assays with immunoprecipitates were carried out as described previously(
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ).

      [3H]Thymidine Uptake Experiments

      8 × 104 cells were plated in 6-well dishes, grown for 24 h, and then serum starved in Dulbecco's modified Eagle's medium with 0.1% calf serum for another 48 h. Cells were then simultaneously treated with 40 ng/ml dexamethasone and growth factors (in triplicate) for 12 h. 0.5 μCi of [3H]thymidine (85 Ci/mmol) was then added to each well, and cells were incubated another 6 h. Cells in wells were washed twice with ice-cold phosphate-buffered saline, extracted with 5% trichloroacetic acid, and solubilized in 0.1 M NaOH. Trichloroacetic acid-insoluble radioactivity was measured by scintillation counting. For experiments including wortmannin, either Me2SO (final concentration 0.1%) or wortmannin (Sigma) in Me2SO was added 30 min prior to the addition of growth factors.

      RESULTS

      Expression of ErbB3 in Fibroblasts That Overexpress p185erbB2/neu

      Since the presence of p185erbB2neu is required for ErbB3 to mediate a biochemical response to HRG(
      • Sliwkowski M.X.
      • Schaefer G.
      • Akita R.W.
      • Lofgren J.A.
      • Fitzpatrick V.D.
      • Nuijens A.
      • Fendly B.M.
      • Cerione R.A.
      • Vandlen R.L.
      • Carraway III, K.L.
      ), we sought to express ErbB3 in NIH3T3 cells that overexpress p185erbB2neu. DHFR/G8 is a clone of NIH3T3 cells that was originally stably transfected with rat erbB2/neu cDNA(
      • Hung M.-C.
      • Schechter A.L.
      • Chevray P.-Y.M.
      • Stern D.F.
      • Weinberg R.A.
      ). These cells express high levels of p185erbB2neu (3 × 105 molecules per cell), but are not phenotypically transformed.
      DHFR/G8 cells were transfected with bovine erbB3 cDNA under the MMTV promoter, and stably transfected clones were selected by G418 resistance. As an initial screen for ErbB3 expression, clones were analyzed for their ability to specifically bind [125I]rHRGβ1177-244, the bacterially-expressed EGF-like domain of human HRG(
      • Holmes 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 W.I.
      • Goeddel D.V.
      • Vandlen R.L.
      ,
      • Carraway III, K.L.
      • Sliwkowski M.X.
      • Akita R.W.
      • Platko J.V.
      • Guy P.M.
      • Nuijens A.
      • Diamonti A.J.
      • Vandlen R.L.
      • Cantley L.C.
      • Cerione R.A.
      ,
      • Sliwkowski M.X.
      • Schaefer G.
      • Akita R.W.
      • Lofgren J.A.
      • Fitzpatrick V.D.
      • Nuijens A.
      • Fendly B.M.
      • Cerione R.A.
      • Vandlen R.L.
      • Carraway III, K.L.
      ). Fig. 1 shows the binding of 0.1 nM [125I]rHRGβ1177-244 to several of the selected cell lines, in the presence and absence of an excess of unlabeled rHRGβ1177-244. The DHFR/G8 parental cells had no detectable affinity for [125I]rHRGβ1177-244, indicating that these cells do not express significant levels of either ErbB3 or ErbB4. However, many of the selected cell lines were capable of specifically binding the iodinated growth factor. Clone D33 was selected for further analysis. Scatchard analysis revealed that these cells bind the labeled growth factor with a Kd of ∼20 pM, and express modest levels (2-3 × 104 receptors/cell) of HRG receptors.
      R. W. Akita and M. X. Sliwkowski, unpublished observations.
      Figure thumbnail gr1
      Figure 1[125I]rHRGβ1177-244 binding by erbB3-transfected DHFR/G8 cells. DHFR/G8 cells and G418-resistant transfectants were incubated with 0.1 nM [125I]rHRGβ1177-244 in the presence and absence of 50 nM unlabeled rHRGβ1177-244, as indicated, and the cell-associated radioactivity determined.
      Fig. 2A shows the covalent cross-linking of [125I]rHRGβ1177-244 to the surface of DHFR/G8 parental cells and D33 transfectants. We observed that the 7-8 kDa iodinated growth factor cross-linked to three protein species in D33 cells, producing radiolabeled bands at ∼175, ∼190, and >300 kDa (right lane). The appearance of these bands could be blocked with 50 nM unlabeled rHRGβ1177-244 (middle lane), indicating that the labeled bands represent receptors for HRG. In addition, no labeled bands were observed after cross-linking [125I]rHRGβ1177-244 to the parental DHFR/G8 cells (left lane), indicating that ErbB3 expression is required for [125I]rHRGβ1177-244 binding activity. All three bands could be immunoprecipitated from RIPA lysates of D33 cells with anti-ErbB3 (not shown). These observations together with our previous results (
      • Carraway III, K.L.
      • Sliwkowski M.X.
      • Akita R.W.
      • Platko J.V.
      • Guy P.M.
      • Nuijens A.
      • Diamonti A.J.
      • Vandlen R.L.
      • Cantley L.C.
      • Cerione R.A.
      ,
      • Sliwkowski M.X.
      • Schaefer G.
      • Akita R.W.
      • Lofgren J.A.
      • Fitzpatrick V.D.
      • Nuijens A.
      • Fendly B.M.
      • Cerione R.A.
      • Vandlen R.L.
      • Carraway III, K.L.
      ) suggest that the 175- and 190-kDa bands represent [125I]rHRGβ1177-244 cross-linked to monomeric ErbB3 (see below), while the >300-kDa band represents a ligand-bound heterodimer of the ErbB3 with p185erbB2neu.
      Figure thumbnail gr2
      Figure 2Expression of ErbB3 in DHFR/G8 cells. A, cross-linking of [125I]rHRGβ1177-244 to transfectants. DHFR/G8 (left lane) and D33 cells (right two lanes) were treated with 0.1 nM [125I]rHRGβ1177-244, in the presence and absence of 50 nM unlabeled rHRGβ1177-244, as indicated, and cell surface proteins were cross-linked with 1 mM BS3. Lysates from cells were resolved by 7% SDS-PAGE and visualized by autoradiography. B, DHFR/G8 and D33 cells were treated without and with HRGβ1177-244, and RIPA lysates were immunoprecipitated with anti-ErbB3(3184). Precipitates were analyzed by 6% SDS-PAGE followed by immunoblotting with anti-ErbB3(3183). C, precipitates similar to those described for B were resolved by 7% SDS-PAGE and immunoblotted with antiphosphotyrosine. The data presented are representative of at least three experiments.
      To further demonstrate the expression of ErbB3 in transfected cells, we analyzed anti-ErbB3 immunoprecipitates from DHFR/G8 and D33 cells by immunoblotting with anti-ErbB3 antibodies. RIPA lysates were immunoprecipitated with anti-ErbB3 antibody 3184, precipitated proteins were resolved by SDS-PAGE, and blotted with anti-ErbB3 antibody 3183. As shown in Fig. 2B, a strong band of ∼170 kDa and a weaker band of ∼185 kDa was observed, but only in the D33 transfectants. Interestingly, treatment of D33 cells with HRG caused a shift in the mobility of both bands, consistent with a stimulation of ErbB3 phosphorylation. Immunoblotting of similar anti-ErbB3(3184) immunoprecipitates with antiphosphotyrosine revealed that the tyrosine phosphorylation of both bands was markedly increased after treatment of D33 cells with rHRGβ1177-244 (Fig. 2C).
      Taken together, the cross-linking and immunoblotting data suggest that ErbB3 is expressed in transfected DHFR/G8 cells as a doublet. A similar doublet was observed when wild type NIH3T3 cells were transfected with bovine erbB3(
      • Carraway III, K.L.
      • Sliwkowski M.X.
      • Akita R.W.
      • Platko J.V.
      • Guy P.M.
      • Nuijens A.
      • Diamonti A.J.
      • Vandlen R.L.
      • Cantley L.C.
      • Cerione R.A.
      ). However, previous studies indicate that ErbB3 in human cancer cells migrates as a single ∼180-kDa species (
      • Kraus M.H.
      • Pedi P.
      • Starks V.
      • Muraro R.
      • Aaronson S.A.
      ), and when we expressed this same bovine erbB3 clone in COS cells, a single 180-kDa species was also observed(
      • Sliwkowski M.X.
      • Schaefer G.
      • Akita R.W.
      • Lofgren J.A.
      • Fitzpatrick V.D.
      • Nuijens A.
      • Fendly B.M.
      • Cerione R.A.
      • Vandlen R.L.
      • Carraway III, K.L.
      ). Since the presence of the 171-kDa band varied in extent from one experiment to the next, we suspect that NIH3T3 cells and their derivatives may partially proteolyze or only partially glycosylate the ErbB3 protein. We have observed that both bands may be immunoprecipitated and immunoblotted with an antibody made to a peptide corresponding to the carboxyl-terminal 17 amino acids of ErbB3 (not shown). This suggests that the faster migrating species is not deleted in its tail region, and that all the putative tyrosine phosphorylation sites are present in this form.

      HRG-stimulated [3H]Thymidine Uptake in Transfected Cells

      To test whether ErbB3 is capable of mediating mitogenesis in NIH3T3 derivatives, we examined the uptake of [3H]thymidine by serum-starved DHFR/G8 and D33 cells after HRG, PDGF, or serum treatment. Fig. 3A shows the results of a representative experiment. While some variation was observed from one experiment to the next, 10% calf serum reproducibly stimulated the uptake of [3H]thymidine in both DHFR/G8 and D33 cells by 4-15-fold. 10 ng/ml PDGF consistently stimulated thymidine uptake by both cell lines to an extent of 40-60% of that observed with serum. 5 nM rHRGβ1177-244 also stimulated the thymidine uptake to an extent of 40-60% of that observed with serum, but only in the D33 transfectants. A dose-response analysis for rHRGβ1177-244-stimulated [3H]thymidine uptake by D33 cells yielded a half-maximal response at 70 nM (Fig. 3B), in rough agreement with the observed dissociation constant. These observations indicate that ErbB3 is capable of mediating a mitogenic response to HRG when expressed in rodent fibroblasts.
      Figure thumbnail gr3
      Figure 3Stimulation of [3H]thymidine uptake by HRG. A, DHFR/G8 and D33 cells were treated without stimulus, or with 5 nM HRG, 10 ng/ml PDGF, or 10% calf serum, and the incorporation of [3H]thymidine into the trichloroacetic acid-insoluble cellular fraction was determined after 18 h. The data were normalized to the extent of [3H]thymidine incorporation observed in unstimulated cells. B, HRG dose-response of [3H]thymidine uptake by DHFR/G8 (open squares) and D33 (closed squares) cells. Data are plotted as the fraction of the response to serum. The curve through the data points for the D33 cells represents the nonlinear least squares fit to a single class of HRG binding sites. Error bars in each panel represent the standard error of triplicate samples, and the data presented are representative of at least four experiments.

      Activation of PI 3-Kinase Activity by HRG in Transfected Cells

      ErbB3 possesses six putative tyrosine phosphorylation sites in its carboxyl-terminal tail region that fit the YXXM motif known to mediate associations with the SH2 domains of PI 3-kinase(
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ). Other members of the class I subfamily do not contain this phosphorylated motif, suggesting that ErbB3 may be unique in its ability to directly associate with PI 3-kinase. Indeed, we and others have observed that treatment of some human cancer cells with EGF stimulates the association of PI 3-kinase with ErbB3 but not EGF receptor or p185erbB2neu(
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ,
      • Kim H.-H.
      • Sierke S.L.
      • Koland J.G.
      ).
      To test the involvement of PI 3-kinase in HRG signaling, we first examined stringently washed antireceptor immunoprecipitates for lipid kinase activity. In the experiment shown in Fig. 4A, DHFR/G8 and D33 cells were treated without and with rHRGβ1177-244 and then Nonidet P-40 lysates were prepared. Antiphosphotyrosine, anti-p185erbB2neu, and anti-ErbB3 immunoprecipitates from lysates were assayed for PI 3-kinase activity, and the stimulation of lipid kinase activity associated with the various immunoprecipitates by rHRGβ1177-244 was determined for the two cell lines. We observed that HRG stimulated the association of PI 3-kinase with antiphosphotyrosine and anti-ErbB3 immunoprecipitates in the D33 transfectants by ∼5-fold, and its association with anti-p185erbB2neu immunoprecipitates by ∼2-fold. No stimulation was observed in any of the immunoprecipitations from the DHFR/G8 parental cells.
      Figure thumbnail gr4
      Figure 4HRG stimulation of PI 3-kinase. DHFR/G8 and D33 cells were treated without and with HRG, and Nonidet P-40 lysates were immunoprecipitated with antiphosphotyrosine (α-pY), anti-p185erbC2, or anti-ErbB3 antibodies. A, precipitates were assayed for PI 3-kinase activity, and the fold stimulation observed with HRG treatment was plotted for the two cell lines. Error bars represent the standard error of at least three determinations for each immunoprecipitating antibody. B, precipitates were analyzed by immunoblotting with anti-p85. Data are representative of at least three separate experiments.
      The stimulation of associated PI 3-kinase activity with immunoprecipitates correlated with a stimulation of association with the 85-kDa regulatory subunit of PI 3-kinase. In the experiment shown in Fig. 4B, antiphosphotyrosine, anti-p185erbB2neu, and anti-ErbB3 immunoprecipitates from DHFR/G8 and D33 cells were analyzed for the presence of p85 by immunoblotting. We observed an increased level of p85 in the various immunoprecipitates only in the D33 cells that had been treated with rHRGβ1177-244. Since immunoblotting with antireceptor antibodies revealed identical levels of receptor in the anti-p185erbB2neu and anti-ErbB3 immunoprecipitates (not shown), these results indicate that PI 3-kinase is recruited to both the ErbB3 and p185erbB2neu receptors upon activation with HRG (see “Discussion”).
      To determine whether PI 3-kinase might contribute to HRG-stimulated mitogenesis, we examined the effect of wortmannin on [3H]thymidine uptake in D33 cells. Wortmannin is a fungal metabolite that has been demonstrated to covalently bind to the 110-kDa catalytic subunit of PI 3-kinase. At a concentration of 100 nM wortmannin specifically targets PI 3-kinase, and inhibits >90% of its lipid kinase activity.
      B. C. Duckworth, L. Rameh, A. Kazlauskas, and L. C. Cantley, manuscript in preparation.
      We treated D33 cells for 30 min without or with 100 nM wortmannin prior to the initiation of a thymidine uptake assay. (The conditions of the assay were identical to those used in the experiment illustrated in Fig. 3A.) We observed that wortmannin only modestly inhibited the basal and serum-stimulated [3H]thymidine uptake in D33 cells. However, wortmannin reproducibly inhibited PDGF-stimulated thymidine uptake by ∼75% and rHRGβ1177-244-stimulated uptake by ∼45% (Table 1). These results suggest that the activation of PI 3-kinase activity by HRG significantly contributes to the mitogenic response of erbB2- and erbB3-transfected fibroblasts to this growth factor.

      DISCUSSION

      A number of reports suggest that members of the HRG family are mitogenic for a variety of cell types, including cultured Schwann cells (
      • Marchionni M.A.
      • Goodearl A.D.J.
      • Chen M.S.
      • Bermingham-McDonogh O.
      • Kirk C.
      • Hendricks M.
      • Danehy F.
      • Misumi D.
      • Sudhalter J.
      • Kobayashi K.
      • Wroblewski D.
      • Lynch C.
      • Baldassare M.
      • Hiles I.
      • Davis J.B.
      • Hsuan J.J.
      • Totty N.F.
      • Otsu M.
      • McBurney R.N.
      • Waterfield M.D.
      • Stroobant P.
      • Gwynne D.
      ) and some human mammary carcinoma cells such as the SKBR3 line(
      • Holmes 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 W.I.
      • Goeddel D.V.
      • Vandlen R.L.
      ). In contrast, HRG appears to elicit a differentiative response in certain other breast cancer cells such as the AU547 and MDA-MB-453 lines(
      • Peles E.
      • Bacus S.S.
      • Koski R.A.
      • Lu H.S.
      • Wen D.
      • Ogden S.G.
      • Ben Levy R.
      • Yarden Y.
      ,
      • Culouscou J.-M.
      • Plowman G.D.
      • Carlton G.W.
      • Green J.M.
      • Shoyab M.
      ). The observed differences in cellular responses to the HRGs must arise from differences in the signaling pathways engaged by the growth factor in the different cells. An obvious point of divergence concerns the receptors present at the surfaces of HRG-responsive cells.
      Members of the HRG family can bind to either p180erbB3 or p180erbB4(
      • Carraway III, K.L.
      • Sliwkowski M.X.
      • Akita R.W.
      • Platko J.V.
      • Guy P.M.
      • Nuijens A.
      • Diamonti A.J.
      • Vandlen R.L.
      • Cantley L.C.
      • Cerione R.A.
      ,
      • Plowman G.D.
      • Green J.M.
      • Culouscou J.-M.
      • Carlton G.W.
      • Rothwell V.M.
      • Buckley S.
      ). However, the mechanisms of receptor activation by the growth factor appears to differ for the two receptors (
      • Carraway III, K.L.
      • Cantley L.C.
      ). HRG stimulation of p180erbB4 is similar to that of the activation of EGF receptor by EGF; ligand binding stimulates receptor autophosphorylation. On the other hand, although HRG binds with relatively high affinity to p180erbB3, interaction of the ligand with this receptor appears not to be sufficient for the stimulation of p180erbB3 tyrosine phosphorylation, probably because p180erbB3 has an impaired kinase activity(
      • Guy P.M.
      • Platko J.V.
      • Cantley L.C.
      • Cerione R.A.
      • Carraway III, K.L.
      ). It appears that the presence of p185erbB2neu is necessary for p180erbB3 to respond to HRG(
      • Sliwkowski M.X.
      • Schaefer G.
      • Akita R.W.
      • Lofgren J.A.
      • Fitzpatrick V.D.
      • Nuijens A.
      • Fendly B.M.
      • Cerione R.A.
      • Vandlen R.L.
      • Carraway III, K.L.
      ), probably the outcome of a ligand-stimulated receptor heterodimerization event(
      • Carraway III, K.L.
      • Cantley L.C.
      ).
      The purpose of the current study was to determine whether the ErbB2-ErbB3 heterodimeric complex is capable of mediating a proliferative response to HRG. To address this question, we employed the DHFR/G8 cell line, an NIH3T3 mouse fibroblast derivative that expresses high levels of p185erbB2neu(
      • Hung M.-C.
      • Schechter A.L.
      • Chevray P.-Y.M.
      • Stern D.F.
      • Weinberg R.A.
      ). Although the constitutive tyrosine phosphorylation of p185erbB2neu is very high, these cells are not transformed and their growth responses to serum and PDGF are quite similar to wild-type NIH3T3 cells (not shown). These observations suggest that the abundance of p185erbB2neu and its basal phosphorylation do not significantly affect the growth properties of the cells. Hence, these cells provide an ideal system for examining cellular responses to HRG after introduction of the ErbB3 receptor.
      We observed that the treatment of erbB3-transfected DHFR/G8 cells with HRGβ1 resulted in a marked stimulation of ErbB3 tyrosine phosphorylation. However, because of its high degree of basal autophosphorylation it was impossible to discern an effect of HRG treatment on the tyrosine phosphorylation of p185erbB2neu (not shown). Our results indicate that ErbB3 mediates a mitogenic response of rodent fibroblasts to HRGβ1 by acting as a receptor for the HRG ligand. Although ErbB3 expression in this system is necessary, it appears that it is not sufficient to mediate the mitogenic response; the presence of p185erbB2neu also appears to be required for growth signaling. We have observed that clones of erbB3-transfected NIH3T3 cells give a very weak and often undetectable mitogenic response to HRGβ1,
      K. L. Carraway III, S. P. Soltoff, A. J. Diamonti, and L. C. Cantley, unpublished observations.
      consistent with the observation that there is a relatively low amount of endogenous p185erbB2neu in these cells.
      Since the intrinsic tyrosine kinase activity of the ErbB3 receptor appears to be impaired relative to other members of the class I receptor family(
      • Guy P.M.
      • Platko J.V.
      • Cantley L.C.
      • Cerione R.A.
      • Carraway III, K.L.
      ), and since p185erbB2neu is already heavily tyrosine phosphorylated in these cells, it is possible that the mitogenic effect of HRG is due exclusively to the cross-phosphorylation of ErbB3 by p185erbB2neu(
      • Carraway III, K.L.
      • Cantley L.C.
      ). On the basis of peptide selection experiments with various SH2 domains(
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ,
      • Songyang Z.
      • Shoelson S.E.
      • McGlade J.
      • Olivier P.
      • Pawson T.
      • Bustelo X.R.
      • Barbacid M.
      • Sabe H.
      • Hanafusa H.
      • Yi T.
      • Ren R.
      • Baltimore D.
      • Ratnofsky S.
      • Feldman R.A.
      • Cantley L.C.
      ), it appears that p180erbB3 has the potential to interact with a different subset of intracellular signaling proteins than the other three members of the EGF receptor family(
      • Carraway III, K.L.
      • Cantley L.C.
      ). Among the proteins predicted to interact uniquely with the p180erbB3 receptor is p85, the 85-kDa regulatory subunit of PI 3-kinase(
      • Carraway III, K.L.
      • Cantley L.C.
      ,
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ).
      We have observed that both p85 and PI 3-kinase activity associate with ErbB3 in HRG-treated D33 cells. The observation that wortmannin significantly inhibits HRG-stimulated [3H]thymidine uptake in D33 cells strongly suggests that PI 3-kinase is a major contributor to the HRG/ErbB3 mitogenic pathway. Interestingly, PI 3-kinase also appears to associate with p185erbB2neu in these cells, even though no p85 binding sites exist in this receptor. We propose that the observed association of PI 3-kinase with p185erbB2neu results from the co-immunoprecipitation of a HRG-stimulated heterodimeric complex of ErbB3 with p185erbB2neu. This notion is supported by our very recent observations that HRG promotes the co-immunoprecipitation of p185erbB2neu with anti-ErbB3 and vice versa.
      S. P. Soltoff, K. L. Carraway III, and L. C. Cantley, manuscript in preparation.
      On the basis of the observations presented here, we conclude that the ErbB2-ErbB3 receptor complex is capable of mediating a mitogenic response in rodent fibroblasts, and that PI 3-kinase is involved in this response. Future experiments will be aimed at determining the role of the p180erbB4 receptor in mediating cellular responses to HRG.

      ACKNOWLEDGEMENTS

      We thank Mark Sliwkowski and Robert Akita for providing unlabeled and iodinated HRG. We gratefully acknowledge the expert technical assistance of Caryn Adley and Margaret Lubkin.

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