Identification of a Region within the ErbB2/HER2 Intracellular Domain That Is Necessary for Ligand-independent Association

Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads toerbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966–968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association. Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads toerbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966–968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association. epidermal growth factor receptor heregulin extracellular domain intracellular domain transmembrane domain glycoprotein D 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic ErbB2 or HER2 is a type 1 receptor tyrosine kinase that belongs to a family of receptors, the ErbBs (EGFR,1 ErbB2, ErbB3, and ErbB4). Ligand-induced activation of these transmembrane growth factor receptors results in the phosphorylation of specific tyrosine residues in the intracellular domains (ICDs) of the receptors themselves and occurs via transphosphorylation of homo- and heteromeric receptor complexes (1Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). The propensity of the ErbBs to form complexes is integral to their function as signal transducers. A distinguishing characteristic of ErbB2 is that unlike the other ErbB receptors, it lacks the ability to bind a specific ligand. ErbB2 instead is indirectly activated in a ligand-dependent manner via formation of heteromeric complexes with other ErbB family members and thereby responds to the respective ligand (2Yarden Y. Sliwkowski M.X. Nature Rev. Mol. Cell. Biol. 2001; 2: 127-137Crossref PubMed Scopus (5772) Google Scholar). In fact, ErbB2 is the preferred heterodimeric partner (3Graus-Porta D. Beerli R.R. Daly J.M. Hynes N.E. EMBO J. 1997; 16: 1647-1655Crossref PubMed Scopus (1317) Google Scholar), and it has been suggested that its normal role is that of an activation partner for the other ErbB family members (4Klapper L.N. Glathe S. Vaisman N. Hynes N.E. Andrews G.C. Sela M. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4995-5000Crossref PubMed Scopus (375) Google Scholar). ErbB2 functions primarily in this context by providing an active tyrosine kinase. This is best exemplified by the most active ErbB heteromer, the ErbB2-ErbB3 complex (5Riese D.J.I. van Raaij T.M. Plowman G.D. Andrews G.C. Stern D.F. Mol. Cell. Biol. 1995; 15: 5770-5776Crossref PubMed Scopus (349) Google Scholar, 6Pinkas-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 (704) Google Scholar), which is extremely potent in transformation assays (7Alimandi M. Romano A. Curia M.C. Muraro R. Fedi P. Aaronson S.A. Di Fiore P.P. Kraus M.H. Oncogene. 1995; 10: 1813-1821PubMed Google Scholar, 8Wallasch C. Weiss F.U. Niederfellner G. Jallal B. Issing W. Ullrich A. EMBO J. 1995; 14: 4267-4275Crossref PubMed Scopus (364) Google Scholar). ErbB2 complements the kinase-deficient ErbB3 (9Guy P.M. Platko J.V. Cantley L.C. Cerione R.A. Carraway III, K.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8132-8136Crossref PubMed Scopus (604) Google Scholar) by providing an active kinase and by enhancing HRG binding to ErbB3 (10Sliwkowski 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. J. Biol. Chem. 1994; 269: 14661-14665Abstract Full Text PDF PubMed Google Scholar, 11Karunagaran 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 (592) Google Scholar). Both of these characteristics contribute to the signal amplification effect of ErbB2 when it is part of a heteromeric complex. Although EGFR and ErbB4 are fully functional kinases, association with ErbB2 also results in receptor complexes that are more responsive to ligand stimulation and have greater and more sustained signaling effects (12Jones J.T. Akita R.W. Sliwkowski M.X. FEBS Lett. 1999; 447: 227-231Crossref PubMed Scopus (330) Google Scholar, 13Worthylake R. Wiley H.S. J. Biol. Chem. 1997; 272: 8594-8601Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Lenferink A.E. Pinkas-Kramarski R. van de Poll M.L. van Vugt M.J. Klapper L.N. Tzahar E. Waterman H. Sela M. van Zoelen E.J. Yarden Y. EMBO J. 1998; 17: 3385-3397Crossref PubMed Scopus (345) Google Scholar, 15Wang Z.X. Zhang L.F. Yeung T.K. Chen X.M. Mol. Biol. Cell. 1999; 10: 1621-1636Crossref PubMed Scopus (157) Google Scholar). Furthermore heteromeric association with ErbB2 increases the signaling repertoire by providing additional SH2 docking sites beyond those present on the partner receptor (16Lemmon M.A. Schlessinger J. Trends Biochem. Sci. 1994; 19: 459-463Abstract Full Text PDF PubMed Scopus (435) Google Scholar). Ligand-independent activation of ErbB2, as the name implies, occurs in the absence of ligand and is unique to the ErbB2 receptor. Activation by this mechanism results when ErbB2 receptors self-associate. It is generally thought that overexpression of ErbB2 results in dimerization/oligomerization. In cell culture models, moderate overexpression of ErbB2 fails to cause transformation in NIH3T3 cells, whereas high overexpression is transforming (17Di Fiore P.P. Pierce J.H. Kraus M.H. Segatto O. King C.R. Aaronson S.A. Science. 1987; 237: 178-182Crossref PubMed Scopus (923) Google Scholar, 18Di Marco E. Pierce J.H. Knicley C.L. Di Fiore P.P. Mol. Cell. Biol. 1990; 10: 3247-3252Crossref PubMed Scopus (72) Google Scholar). The clinical significance of this laboratory observation is underscored by the fact that the erbB2 gene is frequently amplified in a subset of women with primary breast cancer (19Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Science. 1987; 235: 177-182Crossref PubMed Scopus (10239) Google Scholar, 20Slamon 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 (6429) Google Scholar). This molecular alteration leads to overexpression and correlates with a particularly aggressive phenotype (21Seshadri R. Firgaira F. Horsfall D. McCaul K. Setlur V. Kitchen P. J. Clin. Oncol. 1993; 10: 1936-1942Crossref Scopus (505) Google Scholar). An alternative mode of ligand-independent homodimerization is also observed when rats are treated with a carcinogen. In this case a mutation in the transmembrane domain, the rat neu mutation (V664E), is generated (22Bargmann C.I. Hung M.C. Weinberg R.A. Cell. 1986; 45: 649-657Abstract Full Text PDF PubMed Scopus (877) Google Scholar, 23Burke C. Stern D. Mol. Cell. Biol. 1998; 18: 5371-7379Crossref PubMed Google Scholar) and results in constitutive receptor activation and the formation of neuroglioblastomas (24Padhy L.C. Shih C. Cowing D. Finkelstein R. Weinberg R.A. Cell. 1982; 28: 865-871Abstract Full Text PDF PubMed Scopus (197) Google Scholar, 25Schechter A.L. Stern D.F. Vaidyanathan L. Decker S.J. Drebin J.A. Greene M.I. Weinberg R.A. Nature. 1984; 312: 513-516Crossref PubMed Scopus (978) Google Scholar). Although the functional contribution of specific domains to overall receptor activation has been studied in detail for the ErbB receptors, the paucity of structural data for this receptor family has made it difficult to elucidate specific activation mechanisms. It has been demonstrated that ligand binding is a prerequisite for activation of ErbB1, ErbB3, and ErbB4 in both homomeric complexes as well as heteromeric complexes in which ErbB2 is the partner receptor. Ligand binding requires extracellular domain (ECD) subdomains I and II for HRG binding to ErbB3 (26Singer E. Landgraf R. Horan T. Slamon D. Eisenberg D. J. Biol. Chem. 2001; 276: 44266-44274Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), whereas subdomains I and III (27Lax I. Bellot F. Honegger A.M. Schmidt A. Ullrich A. Givol D. Schlessinger J. Cell Regul. 1990; 1: 173-188Crossref PubMed Scopus (27) Google Scholar, 28Harte M.T. Gentry L.E. Arch. Biochem. Biophys. 1995; 322: 378-389Crossref PubMed Scopus (11) Google Scholar) are required for EGF binding to EGFR. However, complete receptor activation also requires sequences in the C terminus (13Worthylake R. Wiley H.S. J. Biol. Chem. 1997; 272: 8594-8601Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 29Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,30Chantry A. J. Biol. Chem. 1995; 270: 3068-3073Abstract Full Text Full Text PDF PubMed Google Scholar). Based on the distinctive nature of ErbB2 activation, we have examined a sequence in the ICD that may be important for ligand-independent ErbB2 activation. Specifically, we have targeted a region of the C terminus previously shown to be important for the ErbB2 transactivation capability of the other ErbB family members (29Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Our results suggest that a three-amino acid sequence comprising amino acids 966–968 may be part of a larger dimerization motif. Cells were maintained in Ham's F12/Dulbecco's modified Eagle's medium (50:50) containing 10% fetal bovine serum, 2 mml-glutamine, and 100 units/ml penicillin/streptomycin. K562 cells are hematopoietic cells that do not express ErbB receptors. ErbB2-neu (C18) rabbit polyclonal and ErbB3 (C17) rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology. FLAG mouse monoclonal antibodies were purchased from Sigma, and glycoprotein D (gD) antibodies were generated at Genentech (31Paborsky L.R. Fendly B.M. Fisher K.L. Lawn R.M. Marks B.J. McCray G. Tate K.M. Vehar G.A. Gorman C.M. Protein Eng. 1990; 3: 547-553Crossref PubMed Scopus (37) Google Scholar). Twenty-four hours post-transfection, subconfluent cells were serum-starved in F12/Dulbecco's modified Eagle's medium (50:50) for 2 h at 37 °C. Cells were lysed in RPMI 1640 lysis buffer containing 1% Triton X-100, 1% CHAPS, 25 mm Tris, pH 7.5, protease inhibitor mixture I (Calbiochem), and 1 mm pervanadate. Lysates were then clarified for 10 min at 10,000 rpm at 4 °C, separated on 4–12% SDS-PAGE, and transferred to a nitrocellulose membrane (Bio-Rad) for Western blot analysis. The membrane was blocked with TBST (10 mmTris, 150 mm NaCl, pH 8, and 0.2% Tween 20) containing 2% bovine serum albumin for 3 h. Membranes were probed with anti-ErbB antibodies overnight at 4 °C, washed three times with TBST, incubated with anti-rabbit horseradish peroxidase antibody diluted 1:20,000 for 1 h at room temperature, and washed an additional five times. Membranes were visualized by ECL (Amersham Biosciences) and exposed to Kodak-X-Omat LS film (Kodak). ErbB2 and mutant were a as previously by Schaefer (29Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The signal sequence of ErbB2 was with the signal sequence of for L.A. D.J. 1984; 3: PubMed Scopus Google Scholar). cells were to the K562 cells were to the Twenty-four hours post-transfection, subconfluent cells were with buffer mm NaCl, 25 mm was to cells at a of 1 mm and incubated for 2 h at room the cells were with and lysed in RPMI 1640 lysis A of the ErbB3 ICD that a three-amino acid is for ErbB2 transactivation (29Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). of an segment in EGFR or ErbB4 the role of the sequence in ErbB2 these residues were to The effect of this mutation on ligand-independent activation was by the tyrosine phosphorylation of the mutant ErbB2 with that of the receptor. Although overexpression of ErbB2 in cells the characteristic phosphorylation of constitutive ErbB2 activation, overexpression of fails to phosphorylation These results that mutation of the sequence the ligand-independent activation that is characteristic of ErbB2 overexpression. An alternative mechanism of ligand-independent ErbB2 activation is the characteristic rat (24Padhy L.C. Shih C. Cowing D. Finkelstein R. Weinberg R.A. Cell. 1982; 28: 865-871Abstract Full Text PDF PubMed Scopus (197) Google Scholar). In this activation mutation occurs within the region at the valine is by The in human is mutation of the ICD sequence activation by a these three amino acids were to in the context of the mutation. ErbB2, or each were in cells, and the tyrosine phosphorylation was by the was to a greater phosphorylation was blocked by the providing additional to the that the sequence is for ligand-independent ErbB2 activation. The constitutive activation of ErbB2 that results from overexpression or mutation of the transmembrane domain is a of constitutive on to mutation of the ICD sequence disrupts ErbB2 activation by the mutation the formation of an active were on cells ErbB2, or shown in cells ErbB2 or by that complexes are However, the the of these receptors complexes 2 of these complexes ErbB2 antibodies and Western with a specific ErbB2 antibody demonstrated that the absence of activated complexes is to the to 2 These results that mutation of the sequence disrupts the ability of ErbB2 to of the association is by ErbB2 overexpression or in the transmembrane domain. The of homomeric association not be to the of receptor at the cell of cells by and that and mutant receptors were at at the cell not These results suggest that sequences in the transmembrane domain are important for homomeric the or more likely a region that this sequence in the may be more that and self-association that results from overexpression or in the transmembrane domain, we to the role that this region in ErbB2 association. Specifically, we to the of receptor association be to the sequence or is an is required for ErbB2 an ErbB2 kinase was examined for its ability to self-associate. The at the binding was to and this mutant has been cells were with ErbB2, or and were the formation of complexes was by ErbB2 and Western shown in ErbB2 and ErbB2 The in formation observed with the mutant has been previously X. C.M. Greene M.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar) and is likely to the increased of this In the not form These results suggest that of association be to the absence of as the ErbB2 Because ErbB2 can be activated by both ligand-independent and we the ErbB2 sequence a role in ligand-dependent activation. activation of ErbB2 by HRG and ErbB3 has been well receptor in the ErbB2-ErbB3 complex is Activation of ErbB2 requires the ligand and ErbB3 requires the active ErbB2 tyrosine kinase to signal the sequence is required for ligand-dependent ErbB2 activation we the effect of mutation of the sequence of ErbB2 on activation. ErbB2 or mutant ErbB2 were with ErbB3 in hematopoietic cells to express ErbB3 Specifically, the effect on receptor phosphorylation in to the ErbB3 was with ErbB2, a kinase-deficient mutant of ErbB2, or the to the results with the cells, was of when it was in the of the of phosphorylation was to a that was of a the ErbB3 was by ErbB2 in to ErbB3 also was by in to ligand to a 4 results were observed in cells with ErbB3 and ErbB2 mutant not These results demonstrated that the ErbB2 kinase was of the sequence blocked homomeric association and activation effects on the activation of heteromeric we have that a sequence in ErbB3 is for transactivation by ErbB2 (29Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Based on the that mutation of blocked ligand-independent ErbB2 activation by receptor we the in ligand-dependent phosphorylation may be to in the ability of the mutant to form was on cells ErbB3 and either ErbB2 or of of ErbB2-ErbB3 complexes that mutation of the sequence of ErbB2 not the formation of The ability of to form complexes with ErbB3 is to ErbB2, complexes are These results were by not the tyrosine phosphorylation of ErbB3 in these complexes with the formation These results suggest that the region the may a role in ligand-dependent ErbB2 activation in ligand-independent ErbB2 activation. Ligand-independent ErbB2 activation occurs in a manner when the protein is at This activation is by its self-association at the plasma membrane T. A. R. Greene M.I. Oncogene. 2000; 19: PubMed Scopus Google Scholar). Although of the ligand-independent activation are a number of the molecular are The that receptor subdomains in this activation have been with ErbB2 to dimerization T. J. T. N. D. S. Ratzkin B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). In constitutive homodimerization and activation are observed with ErbB2 mutant that the O. King C.R. Pierce J.H. Di Fiore P.P. Aaronson S.A. Mol. Cell. Biol. PubMed Scopus Google Scholar) or with the M.A. J. 1996; PubMed Google Scholar). These data suggest that within the region of ErbB receptors are for constitutive activation. it is that the kinase not the kinase are important for A. J. Biol. Chem. 1995; 270: 3068-3073Abstract Full Text Full Text PDF PubMed Google Scholar). is also that the sites may transformation T. S. Y. T. K. T. Mol. Cell. Biol. PubMed Scopus Google Scholar) and that phosphorylation of these sites may this negative X. H. Greene M.I. Oncogene. 1996; Google Scholar). these suggest that within the intracellular domain contain sequences for and that this motif is distinct from the or tyrosine phosphorylation Our present to this and that of this motif may amino acid residues that the kinase domain and the phosphorylation In with ErbB3, and we a region in the ICD that was required for transactivation of ErbB2 (29Schaefer G. Akita R.W. Sliwkowski M.X. J. Biol. Chem. 1999; 274: 859-866Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Here we these and that a region in ErbB2, of amino acids is for homomeric association. the hydrophobic nature of this short sequence (VVI), it is that these residues are and form the region in a we that the segment is within a hydrophobic of the domain and that of these residues with results in a global conformational or change in protein we have the for the EGFR kinase domain. M. X. and C. in with this the in is in a hydrophobic with at the It is that of this region may that the may indirectly to binding of that these or may in other of the receptor. The of this region for homomeric association is by results the This well characterized mutant greater constitutive activation and to ErbB2 of an alteration within its domain that stabilization of formation (22Bargmann C.I. Hung M.C. Weinberg R.A. Cell. 1986; 45: 649-657Abstract Full Text PDF PubMed Scopus (877) Google Scholar, J. J.A. Greene M.I. Nature. 1989; PubMed Scopus Google Scholar). A for these is that this mutation M.J. W.J. Nature. 1989; PubMed Scopus Google Scholar). J.M. King M.C. M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) dimerization the these that the mutant not propose that the greater of dimerization in the mutant of ErbB2 is by in the of the dimerization The domain the Our results this that mutation of the sequence in the context of the mutant constitutive within the ICD of ErbB2 are for homomeric association and are by mutation of the In to homomeric ligand-dependent ErbB2 heteromeric association is likely by the X. Zhang Greene M.I. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Ligand binding may a conformational change that drives heteromeric association or it may release a negative that to occur receptor and other domains as well K.M. M.J. M.A. EMBO J. 2000; 19: PubMed Scopus Google Scholar). In either case, within the ICD contribute to the overall of the complex. the mutant can ErbB3 in a ligand-dependent manner, at Our results with those of that receptor association may from the effects of X. Zhang Greene M.I. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, K.M. M.J. M.A. EMBO J. 2000; 19: PubMed Scopus Google Scholar, X. Greene M.I. Oncogene. 1995; 10: Google Scholar). suggest that the of these may be for ErbB2 homomeric heteromeric association with other ErbB receptors. that ErbB2 self-association is by in the ICD and that within the region this association. The effects of these can be by a number of the well characterized mutation or by the of C. Stern D. Mol. Cell. Biol. 1998; 18: 5371-7379Crossref PubMed Google Scholar, H. L. C. B.J. Stern D.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar). mutation of amino acids 966–968 in ErbB2 the effect of the that are to ErbB2 association. The observation that of ErbB receptors their are active that the is with to ligand-independent ErbB2 association and may ErbB2 homodimerization O. King C.R. Pierce J.H. Di Fiore P.P. Aaronson S.A. Mol. Cell. Biol. PubMed Scopus Google Scholar). In of suggest that ligand-dependent ErbB2 association is driven primarily by in the receptors form complexes in a ligand-dependent that are high binding sites that are characteristic of V.D. Vandlen R.L. Sliwkowski M.X. FEBS Lett. 1998; PubMed Scopus Google Scholar). This of is with the that was proposed by T. H. J. Mol. Biol. 2001; PubMed Scopus Google Scholar) for EGF binding to the EGFR. In results to the that a dimerization motif within the ICD of present the of this motif is the kinase domain and the tyrosine phosphorylation that amino acids 966–968 are within this motif and a role in a that is to receptor self-association and subsequent constitutive activation. of ICD may lead to for the of cancer contain molecular that in ErbB2 protein overexpression. for and Schaefer for