HER2/Neu: Mechanisms of Dimerization/Oligomerization

Patrick J Brennan1, Toru Kumogai1, Alan Berezov1, Ramachandran Murali1 and Mark I Greene*,1

1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

Oncogene (2000) 19, 6093 ± 6101. principles involved remain to be de®ned. Key studies which have led to our current understanding of the Keywords: erbB; kinase; growth factor; signaling; mechanism and function of the oligomerization process tumor in erbB receptors will be outlined below, as will the outstanding questions raised by these studies.

Introduction Mechanisms of dimerization/oligomerization Within a multi-celled organism, regulation and organ- Though dimerization/oligomerization has been recog- ization require that biological signals be transmitted nized as an integral component of signaling by erbB from one cell to another, across cell membranes. Such family receptors since shortly after the discovery of is the case with growth factors, which originate from neu, the mechanism by which these receptors aggregate one site in an organism, yet need to be distributed is still not fully understood. Extracellular, transmem- throughout the organism to many cell types in order to brane, and intracellular domains of the protein have all exert their pleiotropic eects. Systems have evolved to been implicated in dimerization/oligomerization; the allow a soluble signal, a growth factor for example, to potential contribution of each of these domains to be conveyed from the extracellular space to the receptor aggregation will be discussed below. cytoplasm and nucleus of a cell, thus directing protein synthesis, cellular growth, and cellular proliferation. To solve this problem of transmitting signals across Insights from the transmembrane domain membranes, growth factors appear to induce aggregation of their receptors, leading to activation of these A role for neu in transformation and tumorigenesis has receptors and signal propagation. Unregulated, how- been suspected since its discovery as a dierentially ever, these growth signals have the potential to expressed antigen in a chemically induced rat neuroglio- promote inappropriate proliferation; receptor aggrega- blastoma (Padhy et al., 1982; Schechter et al., 1984; Shih tion in the absence of signal represents a means of et al., 1979, 1981), hence the name `neu'. Upon unregulated growth factor receptor activity which is comparison of the normal, protooncogenic form of the postulated to occur in human neoplasia. gene with oncogenic neu in the rat model, a single The neu gene encodes a 185-kDa transmembrane causative point mutation in the putative transmembrane glycoprotein, referred to as p185neu, HER2, or erbB-2, region of the protein revealed itself as the culprit lesion possessing intrinsic protein tyrosine kinase activity. The (Bargmann et al., 1986a,b). Mutation occurred at receptor consists of an extracellular domain, with four position 664 of the protein sequence, encoding a change subdomains including two cysteine rich domains, a from the hydrophobic amino acid valine to the transmembrane domain, and an intracellular domain, negatively charged glutamate. Further studies suggested consisting of a juxtamembrane region, a tyrosine kinase that this mutation garnered its transforming eect by domain, and a carboxyl tail harboring autophosphor- increasing the propensity of the receptor to form ylation sites (Figure 1). HER2 is homologous to, but aggregates (Weiner et al., 1989b). Although the mutation distinct from, other members of the erbB family, which is not observed in human tumors, the information gained includes the epidermal growth factor receptor (EGFR through the study of this mutation has provided or erbB-1), erbB-3, and erbB-4. The binding of cognate invaluable clues to the mechanism of activation and growth factors to these receptors regulates cell growth, transformation utilized by HER2/neu in human disease. proliferation, and dierentiation through the activation Early studies identi®ed that the eects of the point of receptor tyrosine kinases, triggering an incompletely mutation seen in rat neu were mediated by dimeriza- de®ned signal transduction cascade. Signal transduction. Using nondenaturing gel electrophoresis with cell tion by these receptors is believed to involve dimeriza- lysates prepared from cells overexpressing normal or tion and oligomerization, both in the form of homo- oncogenic (containing the transmembrane point muta- oligomers and hetero-oligomers in various erbB tion) forms of p185, Weiner et al. (1989a) found that receptor combinations. Though this oligomerization the majority of oncogenic p185 existed in a dimeric has been recognized for more than 20 years, and form, while normal p185 was predominantly in a though much progress has been made in de®ning the monomeric state. Co-transfection of short neu trans- critical aspects of this process, many of the mechanistic membrane sequences lacking both intracellular and extracellular domains was shown to diminish the transforming potential of full-length p185neu protein, *Correspondence: MI Greene presumably by preventing the formation of produc- HER2/Neu: mechanisms of dimerization/oligomerization PJ Brennan et al 6094 interacts with the backbone of the adjacent receptor (Smith et al., 1996; Sternberg and Gullick, 1989). Recent eorts in molecular modeling support these ®rst two models, but have not provided a means to distinguish between the two (Sajot and Genest, 2000). Structural studies using NMR and IR have suggested that the 664E mutation is involved in a hydrogen bonding type of interaction with its partner transmembrane domain, supporting the ®rst model (Smith et al., 1996). Mutational studies show that only Glu and Gln residues at position 664 lead to transformation, Asp and Tyr having weak eects, results consistent with a primary role for hydrogen bonding (Bargmann and Weinberg, 1988). The third model proposes that the V664E mutation leads to a global conformational change within the transmembrane domain allowing for a more stable dimeric structure. Initially, molecular modeling and other structural analyses of the protooncogenic and oncogenic forms of the neu transmembrane domain suggested that the protooncogenic form of the receptor contained a kinked transmembrane domain, while the oncogenic form of the receptor was primarily helical (Brandt-Rauf et al., 1989, 1990, 1995). This model, however, has not been supported by later moleuclar dynamics simulations or other structural studies (Sajot and Genest, 2000; Smith et al., 1996). Because the transmembrane mutation seen in neu seems sucient to cause constitutive dimerization, a similar mutation in EGFR might be expected to lead to dimerization, unregulated signaling, and transformation. There exist con¯icting data on this point, however, bringing the autonomy of the transmembrane mutation into question. Simply mutating the equivalent Val in EGFR to Glu led to a receptor complex with increased, yet still ligand-dependent, transforming Figure 1 Schematic diagram of structural organization of p185 potential (Kashles et al., 1988). A more recent study, receptors. Epidermal growth factor receptor (EGFR), Her2/neu/ however, found con¯icting results, suggesting that a erbB2, erbB3 and erbB4 are members of the super family. The numbers indicate the sequence homology among members point mutation in the EGFR transmembrane domain compared to EGF receptor. They share a high degree of primary could lead to a ligand-independent transforming and tertiary structural homology in the tyrosine kinase domain receptor complex (Beguinot et al., 1995) and a moderate degree of homology in ectodomain. C-terminus is Production of a functional neu complex requires the most variable region among the receptors more than stable dimer formation, as a steric constraint on this dimerization also appears to be crucial. Experimentally relocating the position of the tively arranged oligomers (Lofts et al., 1993). Further point mutation within the transmembrane domain only supporting a role for oligomerization within the erbB leads to a transforming receptor complex if the family, EGFR dimerization had been demonstrated mutation is placed in certain positions. Bargmann using chemical crosslinking (Fanger et al., 1989), and Weinberg (1988) noted that mutation of either sucrose gradients (Boni-Schnetzler and Pilch, 1987), residues 663 or 665, the residues immediately adjacent and ¯orescent energy transfer (Carraway et al., 1989). to the mutation observer in rat neu, to Glu did not Though much eort has been expended toward lead to transformation. Other groups have pursued this de®ning the mechanism by which the observed observation, investigating the importance of residues transmembrane mutation leads to transformation, a surrounding the point mutation and more closely clear answer has remained elusive. That this point examining the location constraints for a transforming mutation leads to increased dimerization, increased mutation (Burke et al., 1997; Cao et al., 1992). By receptor tyrosine kinase activity, and increased trans- generating a battery of both random and rationally formation potential is well accepted; how this mutation designed potential transmembrane sequences, Chen et leads to these eects remains a matter for debate. al. (1997) found a critical limitation for spacing Three models have been proposed to explain the between residues with the potential for hydrogen mechanism by which the transmembrane point muta- bonding, suggesting a possible rotational constraint. tion leads to oligomerization. The ®rst and second A transgenic mouse overexpressing protooncogenic model, which dier only slighlty, favor stabilization of erbB-2 develops tumors predominantly after sponta- interreceptor transmembrane association via additional neous mutation of juxtamembrane residues, leading to hydrogen bonds provided by the mutation; the ®rst disul®de-mediated constitutive dimerization (Siegel and model proposes that adjacent 664E residues interact, Muller, 1996). In addition to providing proof that while the second proposes that the 664E residue dimerization leads to transformation, this observation

Oncogene HER2/Neu: mechanisms of dimerization/oligomerization PJ Brennan et al 6095 provided for the use of juxtamembrane mutations as a readily controlled arti®cial experimental system for the induction of dimerization (Sorokin et al., 1994). Surprisingly, it was found that, depending on the location of the cysteine mutations introduced, stable dimers could be induced without concomitant trans- foration (Sorokin et al., 1994), a result supported by a later study utilizing a transplanted dimerization-indu- cing transmembrane domain (Burke et al., 1997). Upon further investigation, receptors with mutations that led to transforming receptor complexes all formed dimers involving the same predicted helical interface, strongly suggesting a steric constraint for p185neu activation (Burke and Stern, 1998). Figure 2 Schematic diagram of putative dimerization of EGF receptors. Dimerization of EGFR viewed down from the top. The scheme was conceived based on our molecular modeling of the Extracellular domain EGF receptor. The proposed dimerization model assumes a 2 : 2 association of EGFR : EGF ErbB receptor extracellular domains are responsible ligand binding and seem to mediate dimerization/ oligomerization in the majority of biological settings. in a cleft formed by subdomains I, II and III (Jorissen Natural variants and experimental mutations of these et al., 2000). Studies investigating the binding of EGF extracellular domains have provided clues to the nature to EGFR show a high and a low anity binding site, of ligand binding and interaction of these domains. and kinetic studies show that receptor activity is second While other members in the receptor family have order with respect to ligand (Canals, 1992). In a recent natural ligand, erbB2 has no known natural ligand to study of EGF-sEGFR interactions, a complex binding date. Despite an exhaustive search eort to ®nd a mechanism has been observed, which could not be natural ligand, erbB-2 remains an orphan receptor, and adequately described by a simple Langmuirian inter- it may very well be that this receptor functions mainly action (Domagala et al., 2000). Two sEGFR popula- in complex with other family members. HER2/neu tions have been detected in solution: one with a high shows both ligand-independent and ligand-dependent anity (2 ± 20 nM) and the other with a low anity activity, and investigation of each of these activities has (400 ± 550 nM) for EGF (Domagala et al., 2000). The provided valuable information regarding the mechan- results of these studies are complicated by the fact that isms of transformation utilized by this receptor. some groups have used soluble receptor fragments, while others have used full-length receptor present on the surface of intact cells; the anity of EGF for a Ligand-dependent dimerization soluble extracellular EGFR fragment has been shown Though erbB-2 is now thought to be an orphan to be markedly lower than for the full-length receptor receptor, it plays an active role in ligand-mediated (Brown et al., 1994). Furthermore, interpretation of signaling through heterodimerization with other erbB binding studies with erbB receptors must take into family members. Dimer formation between multiple account that the receptors are in a monomer/oligomer erbB family members, in most combinations, in equilibrium which is not governed solely by ligand response to a growing list of ligands, constitutes a concentration. Using a soluble EGFR extracellular complex signaling network (Klapper et al., 2000; domain, positive cooperativity with increasing EGF Tzahar et al., 1996). The EGFR was the ®rst receptor concentration has been demonstrated (Hurwitz et al., tyrosine kinase proposed to dimerize in response to 1991; Lemmon et al., 1997), while studies of intact cells ligand binding (Yarden and Schlessinger, 1987a,b), and have demonstrated negative cooperativity (Chamberlin this system has been well studied; an understanding of and Davies, 1998). the ligand binding mechanism in EGFR should serve Lemmon et al. (1997) investigating EGF-induced as a template for receptor ± ligand and receptor ± dimerization using soluble EGFR extracellular domain, receptor interactions within the erbB family, and proposed a model in which one EGF binds an EGFR perhaps beyond. Ligand binding mechanisms utilized monomer, followed by dimerization with a similar by other receptor families may also provide valuable EGF/EGFR complex. One possibility assumes that the clues to the mechanisms used within the erbB family. A bound EGF molecule mediates subsequent dimeriza- general scheme based on current understanding of tion; alternatively, EGF may bind to EGFR causing a EGF induced oligomerization is shown in Figure 2. conformational change favoring dimerization, without Multiple studies have concluded that EGF binds a requirement for EGF at the dimer interface (Green- EGFR with a 1 : 1 stoichiometry (Brown et al., 1994; ®eld et al., 1989; Lemmon et al., 1997). While this Domagala et al., 2000; Lemmon et al., 1997; Odaka et model may accurately represent the events involved in al., 1997; Weber et al., 1984). Subdomains I and ligand-induced oligomerization in solution, it may not especially III of the extracellular portion of EGFR re¯ect what is happening at the surface of intact cells. have been identi®ed as important in ligand binding A second model which could explain the observed (Lax et al., 1998, 1989, 1991; Lee et al., 1995; high and low binding activities of EGF for EGFR Summer®eld et al., 1996), as has subdomain IV, one involves the induction of dimerization by the binding of two cysteine rich domains (Saxon and Lee, 1999). of one EGF molecule to two EGFR, a low anity Recent structural modeling proposes that EGF binds interaction, followed by the binding of a second EGF

Oncogene HER2/Neu: mechanisms of dimerization/oligomerization PJ Brennan et al 6096 to the previously formed EGF : 2EGFR complex in increased dimerization and transforming ability (Se- which the EGFR now bind EGF with high anity. In gatto et al., 1988). Overexpression of the protoonco- such a model, high and low anity binding sites may genic form of HER2/neu, however, either with or be explained by the asymmetrical binding of a bivalent without ampli®cation, is observed in human tumors EGF in which each EGF binds with high anity to (Dougall et al., 1994; Klapper et al., 2000). It might be one member of the EGFR dimer and with low anity, expected, from data obtained with oncogenic neu, that via a distinct epitope, to a second EGFR (Summer®eld increased dimerization/oligomerization, thus leading to et al., 1996; Tzahar et al., 1997); a two binding site increased signaling, would be seen in human tumors model is consistent with a recent structural model associated with HER2/neu overexpression; indeed, this developed for the EGFR extracellular domain (Jorissen turns out to be the case. In cell culture models, et al., 2000). moderate overexpression (receptor number *105)of In support of this model, recent electron microscopic the protooncogenic form of p185, using an SV40 analysis suggests that EGFR may exist in a pre- promoter-driven expression vector, failed to cause dimerized state, and that this state seems to correspond transformation in NIH3T3 cells. However, overexpres- with observed high anity EGF binding sites (Gadella sion (receptor number 4106) of this receptor at higher and Jovin, 1995). A theoretical kinetic analysis levels, using a retroviral promoter, was transforming proposed by Chamberlin and Davies (1998) which (Di Fiore et al., 1987b; Di Marco et al., 1990). Hence, accounts for possible clustering that may occur on the it seems that once a critical threshold level of HER2/ surface of intact cells also supports a two site model in neu expression is reached, transformation is achieved, a which EGF molecules bind sequentially. Recent single- phenomenon thought to be due to spontaneous molecule imaging of EGFR on the cell surface (Sako et dimerization/oligomerization. To directly show that al., 2000) suggests that the predominant mechanism of overexpression itself can lead to spontaneous oligomer- dimerization involves the formation of a cell-surface ization, we demonstrated that increasing the expression complex of one EGF molecule and an EGFR dimer, of protooncogenic p185neu shifted the receptor equili- followed by the direct arrest of a second EGF brium from a monomeric to an aggregated state molecule. The EGFR dimers are thus preformed before (Samanta et al., 1994). It appears that overexpression, the binding of the second EGF molecule. much like the rat neu point mutation discussed Ligand-mediated binding involving erbB-2-contain- previously, leads to unregulated oligomerization and ing heterodimers has not been as extensively studied as unregulated signaling. EGF/EGFR binding. Given the high degree of Extracellular domain of p185 is sucient to induce homology between erbB family members, models dimers. We have shown that mutant forms of p185neu developed through the study of EGF/EGFR binding lacking the intracellular portion of the receptor, both should prove a useful frame for understanding with and without the oncogenic transmembrane point interactions within the erbB family. With respect to mutation, are sucient to oligomerize with full-length erbB-2 ligand-dependent dimerization, the data that EGFR and p185neu receptors (Qian et al., 1994a,b, exist suggest a model in which heterodimerization is 1996). This binding is ligand-independent, and that mediated by ligand bivalence. ErbB-2 has been shown truncated forms both with and without the oncogenic to be the preferred dimerization partner of the other transmembrane point mutation eciently oligomerize erbB family members (Tzahar et al., 1996), and suggests that this binding is mediated by the extra- demonstrates synergistic transforming eect when cellular domain. In the reverse experiment, an expressed with EGFR (Kokai et al., 1989). Using intracellular truncation mutant of EGFR also hetero- soluble chimeric receptors, Jones et al. (1999) recently dimerizes with p185neu (Kashles et al., 1991), suggest- showed that heterodimerization with erbB2 enhances ing again that the extracellular domains of erbB ligand binding for all other erbB family members to receptors are sucient for dimerization. their respective ligands. Tzahar and colleagues (Tzaher Analysis of extracellular domain mutants derived et al., 1997), in studies of erbB-2/erbB-3 heterodimer- from erbB-1 and erbB-2 have provided clues as to ization driven by NDF/neuregulin, propose that NDF which subdomains are important for ligand binding has a high anity interaction with erbB-3 and a low and oligomerization. A truncated EGFR seen in anity interaction with erbB-2, and that NDF human glioblastoma, for example, can dimerize and simultaneously binds both receptors. Furthermore, this transform mammalian cells in a ligand-independent low anity binding site, while possessing relatively fashion (Moscatello et al., 1996) Homologous to broad speci®city, may favor erbB-2 binding, providing mammalian erbB proteins, avian viral v-erbB trunca- a model which could explain the observed preference tion mutants display ligand-independent eects, though for heterodimerization and possibly even erbB-2 the signi®cance of the dimerization status of these oncogenicity. proteins is unclear (Adelsman et al., 1996). Herstatin, a recently discovered erbB-2 alternate transcript, consisting of 340 amino acids of the erbB-2 extracellular Ligand-independent dimerization domain and 79 amino acids of novel sequence, binds As discussed previously, the transmembrane mutation full-length erbB-2, inhibiting its function (Doherty et seen in rat neu confers the ability for ligand- al., 1999). Studies with the deletion mutants suggest independent signaling. Transmembrane mutation ob- that the extracellular domains of erbB receptor that served in rat is not seen in human tumors, probably small structural components in a subdomain may because such a mutation would require a two function not only to mediate dimerization in response nucleotide mutation in the human gene (Dougall et to ligand, but also to prevent surreptitious dimeriza- al., 1994). Nevertheless if arti®cially introduced into tion. Recently we have modeled the three dimensional human HER2/neu, an equivalent mutation leads to structure of the erbB-2 extracellular domain using the

Oncogene HER2/Neu: mechanisms of dimerization/oligomerization PJ Brennan et al 6097 insulin-like growth factor-1 receptor (Garrett et al., Aggregation of erbB kinase domains seem to be 1998) as a template and found that the fourth domain intrinsically linked to their activity. Activation of may be critical for dimer formation. Based on this kinase activity was observed in response to forced model, we have developed a small peptide with cystine- aggregation of a soluble EGFR intracellular domain by knot topology which shows varying degree of anity various methods, including cross-linking antibodies, to dierent members of erbB receptors. This small polycation, or chemical reduction of protein solubility peptide is also able to block the dimer formation (Mohammadi et al., 1993). Using a soluble version of (Berezov, Murali and Greene, unpublished data). These the p185neu intracellular domain, we have shown that results suggests that a relatively small fragment of the aggregation leads to an increase in the Vmax of the p185 extracellular domain has the ability to homo- receptor (LeVea et al., 1993). It is possible, then, that dimerize. the binding activity inherent in the kinase domains of erbB and other growth factor receptors may serve to mediate interactions of these domains after extracel- Intracellular domain lular domain-mediated juxtaposition, the end result being the same as with forced aggregation. Although the extracellular domain appears to be responsible for the majority of the interactions Functional consequences of oligomerization governing erbB receptor dimerization/oligomerization, evidence suggests that the intracellular domain plays It is clear that both homodimerization and hetero- more than a passive role in dimerization. For example, dimerization occur within the erbB receptor tyrosine if both full-length EGFR and an intracellular domain kinase family. To de®ne the function and the essential truncated EGFR are coexpressed, full-length receptor features of this phenomenon, a variety of receptor favors binding available full-length receptor; this mutants have been generated, and their function suggests that the intracellular domain may stabilize subsequently evaluated both alone and in combination dimer formation (Kashles et al., 1991). with other erbB receptors. The picture that has To directly address the role of the intracellular emerged suggests that oligomerization may allow domain in oligomerization, Chantry coexpressed trun- kinase domains to be positioned next to one another, cated EGFR forms lacking the extracellular domain thereby allowing cross-phosphorylation, activation, along with full-length receptor containing the intracel- and substrate phosphorylation. Several of the key lular domains of EGFR or HER2/neu, measuring the experiment which have led to these conclusions are resultant dimerization, kinase activity, and autopho- outlined below. sphorylation (Chantry, 1995); it was found that the intracellular domain, if membrane associated, was Homodimerization sucient for dimerization and kinase activation. Analysis of a panel of deletion mutants, created to Though the transforming activity of p185neu was ascertain which portions of the intracellular domain identi®ed along with the receptor itself, and though were involved in the observed dimerization, suggests receptor oligomerization was also quickly identi®ed as that the kinase domain is critical. Interestingly, a feature of this receptor, the link between dimerization intracellular domain-mediated interactions were also and transformation was not immediately accepted. The observed between EGFR and PDGF receptor as well functional consequences of homodimerization of as HER2/neu and PDGF receptor, suggesting that EGFR have also been studied, and this dimerization, dimerization may not be speci®c, but may represent which can be regulated by the presence or absence of interactions between `dimerization motifs' present ligand, has provided evidence in support of a role for within the highly conserved kinase domains of these homodimerization of erbB-2. Two possible mechanisms receptors (Chantry, 1995). The reverse experiment, for the activation of EGFR signaling by EGF have however, in which the intracellular domain of p185 been proposed; the ®rst suggests that EGF signal was expressed along with full-length EGFR, yielded transmission occurs through a single EGFR monomer, con¯icting results, as an interaction was not seen (Qian possibly through an induced conformational change et al., 1999). Modeling from our laboratory never- (Koland and Cerione, 1988). The second, initially theless supports kinase domain interaction. Dimeriza- alternative hypothesis, supports a functional role for tion and tetramerization models of EGFR and p185 dimerization in ligand-responsive signal transduction kinase domains suggest that these domains can (Yarden and Schlessinger, 1987b). The kinetics of both associate as energetically stable structures (Murali et dimerization and activation of full-length, solubilized al., 1996). In addition, the models suggest that the EGFR were found to be second-order, with an heterodimer interaction is more favorable than that of identical rate constant, a correlation which strongly either homodimer, an observation which could con- suggests that the two processes are linked (Canals, tribute to observed synergistic signaling (Kokai et al., 1992). EGFR has been shown to transform NIH3T3 1989), and even to the favored binding partner status cells when overexpressed, though this transformation is enjoyed by p185 (Graus-Porta et al., 1997; Tzahar et ligand-dependent (Di Fiore et al., 1987a). An en- al., 1996). The model also proposed dimerization of gineered EGFR mutant which no longer binds EGF erbB receptors may be in¯uenced by electrostatic with high anity, yet is constitutively dimerized, is charge distribution near membrane proximal regions transforming when expressed in NIH3T3 cells, suggest- (Murali et al., 1996). This hypothesis suggests that ing that dimerization may be the critical function of proper orientation of receptors is critical for dimeriza- ligand binding (Sorokin, 1995). tion. This feature has been observed in the EPO Further evidence that the function of EGFR is receptor complex (Livnah et al., 1998). dependent on dimerization/oligomerization has come

Oncogene HER2/Neu: mechanisms of dimerization/oligomerization PJ Brennan et al 6098 from the use of antibodies. Cross-linking antibodies are dependent on the ratio of truncated and full-length capable of activating EGFR, an observation which has receptor (Qian et al., 1996). Stable dimers consisting of been documented by a number of groups using T691stop and p185neu are observed, and likely antibodies which recognize distinct epitopes of the preclude the interaction of p185neu kinase domains, EGFR extracellular domain (Schreiber et al., 1981; preventing transphosphorylation. The end result of this Spaargaren et al., 1991; Yarden and Schlessinger, situation is the formation of dimers which do not 1987b); Non-crosslinking Fab fragments of these contain phosphorylation-dependent docking sites, antibodies, however, do not activate the receptor much the same as in cells expressing only kinase- (Spaargaren et al., 1991). In contradiction with the inactive neu forms. above results, another antibody, capable of reducing EGFR dimerization, had little eect on EGFR Heterodimerization of HER2/neu and EGFR autophosphorylation (Carraway and Cerione, 1993). As with the EGFR receptor, overexpression of erbB- Early observations suggested that protooncogenic p185 2 at sucient levels transforms NIH3T3 cells (Di Fiore activation is EGF dependent, despite that EGF does et al., 1987b); unlike the EGFR, however, this not bind p185, implying that EGFR may be involved transformation is ligand-independent. As discussed in p185 signal transduction (Dougall et al., 1994). previously, overexpression leads to spontaneous recep- Transformation by either protooncogenic p185 or tor oligomerization, and as with the EGFR receptor, EGFR alone requires high receptor expression; how- oligomerization is thought to be necessary for signaling ever, expression of both receptors at moderate levels is and subsequent transformation. In order to establish a sucient to cause transformation (Kokai et al., 1989), role for oligomerization, and to de®ne the mechanism which is dependent on continuous cell surface expres- of activation and signaling utilized by erbB receptors, sion of both receptors (Wada et al., 1990). The we and others have generated a variety of mutant apparent synergy in transformation observed with forms of these proteins. A full-length kinase-inactive erbB-2 and EGFR may be particularly signi®cant, as mutant of oncogenic neu, T757, when expressed in a these two receptors have been shown to be upregulated background de®cient of both EGFR and erbB-2 concomitantly in breast as well as other tumors proteins, retains the ability to oligomerize, yet is not (Dougall et al., 1994; Klapper et al., 2000). transforming, indicating the critical importance of Investigating the link between p185 and EGFR kinase activity to transformation (Qian et al., 1995). signaling, our laboratory identi®ed a protein ± protein Such a kinase-inactive mutant has been shown to have interaction between the two receptors using cross- the ability to competitively inhibit the function of wild- linking reagents (Wada et al., 1990). The observed type p185neu when co-expressed (Akiyama et al., 1991; interaction was found to be stimulated by EGF (Qian Messerle et al., 1994), likely by preventing the et al., 1992; Spivak-Kroizman et al., 1992; Wada et al., formation of productive dimers. TAPstop, a mutant 1990), though heterodimers were also detected in the in which the carboxy terminus has been truncated, absence of EGF (Qian et al., 1994a). These results were including potential autophosphorylation sites, retains con®rmed by non-denaturing gel electrophoresis, and transforming ability, although at a reduced level the interactions found to be non-covalent in nature compared to full-length p185neu. TD5, an ectodo- (Qian et al., 1992). Heterodimerization of soluble main-deleted form of neu, also retains transforming extracellular domains of HER2/neu and EGFR has ability, though like TAPstop, a lower level than also been demonstrated (Spivak-Kroizman et al., 1992). p185neu.. Interestingly if either TAPstop or TD5is In addition to the two EGF anities observed for cells coexpressed with the kinase-inactive mutant, T757, an expressing only the EGFR, three anity states were increase in transforming potency is observed. While in identi®ed in cells expressing heterodimers, the addi- cells expressing only T757, autophosphorylation of this tional anity state being higher than those observed mutant is not observed, double mutant cells contain with EGFR only (Wada et al., 1990); this suggests that T757 which has been phosphorylated at the carboxyl oligomers which contain erbB-2 have a higher anity tail. Furthermore, the autophosphorylated T757 is for ligand, an observation which has been con®rmed capable of binding SH2-containing signal transduction and extended to other erbB-2-containing erbB family proteins. These experiments suggest a model in which heterodimers (Jones et al., 1999). Heterodimers of oligomerization allows trans-phosphorylation of bind- EGFR and p185 are also formed preferentially over ing partners which is dependent on the intrinsic kinase either homodimeric form (Qian et al., 1994b), which activity of the erbB receptor. That the introduction of may further contribute to their transforming potential. a kinase-de®cient neu leads to an increase in Since this initial observation, a series of mutants transforming potency in the double mutant cell lines have been constructted in order to investigate the suggests that transformation may not be directly functional interaction between p185neu and EGFR. dependent on the intrinsic kinase activity of the erbB When expressed with EGFR, a kinase-inactive HER2/ receptor, but rather on the docking of other signaling neu mutant, N757, was found to be trans-phosphory- molecules at erbB autophosphorylation sites. lated in and EGF-dependent fashion, suggesting that The critical aspect of erbB oligomerization may be EGFR may be responsible for HER2/neu phosphory- juxtaposition of the kinase domains of binding lation in the wild type oligomer (Qian et al., 1994b). partners. Studies of mutant neu forms in which the An intracellular truncation of p185, N691stop, also intracellular domain has been deleted, such as underwent EGF-dependent dimerization with EGFR, T691stop from our laboratory, support the notion that though the resulting complex was not active (Qian et kinase domain interaction is critical. Coexpression of al., 1994b); this suggests that activation is a trans- T691stop along with oncogenic neu results in a loss of process, and that the absence of a binding partner transforming activity, the inhibition observed being precludes activation, auto or otherwise. These results

Oncogene HER2/Neu: mechanisms of dimerization/oligomerization PJ Brennan et al 6099 provided evidence that erbB-2 is involved in the these puri®ed receptors, higher order oligomerization reciprocal activation of EGFR, showing that it is states were again observed (Samanta et al., 1994). more than a mere substrate of the EGFR. Further Similar results have been seen using soluble EGFR studies with the N757 and N691stop mutants provide fragments, in which ligand-induced dimers and trimers evidence for the functional importance of heterodimer were demonstrated after chemical crosslinking (Hur- formation; each of these mutations abolished the witz et al., 1991). Using a structure-based analysis, transformation and tumorigenicity seen with forma- Lax et al. (1991) have also demonstrated the tion of the wild type heterodimer (Qian et al., 1994a). formation of dimers, trimers, and higher order The dominant-negative interaction of the truncated oligomers using a soluble EGFR extracellular domain N691stop, eective in both cis and trans, could fragment. Though these results using puri®ed recep- potentially be used therapeutically. T691stop, a tors are suggestive, there are obvious limitations to version of this mutant with the transmembrane such in vitro studies. mutation observed in rat neu, binds in a ligand- Additional evidence for oligomerization comes from independent fashion, and should therefore be an even an early study into the kinetics of EGF binding to more eective inhibitor of erbB receptor function. To p185/EGFR heterodimers. As mentioned previously, test the therapeutic potential of this construct in a Wada et al. (1990) described three binding anities for pathologic setting, O'Rourke et al. (1997) expressed EGF in cells expressing both EGFR and p185. T691stop in a cell line established from human Monoclonal antibody treatment, led to the removal glioblastoma; inhibition of transformation and tumor- of p185 from the surface of the cells without aecting igenicity was observed. EGFR levels, as this antibody is thought to bind only The interactions observed between p185/EGFR to p185 homodimers. After treatment with this anti- heterodimers appear to be conceptually parallel to body, only two anity states remained. A possible those observed with homodimers of either receptor. model which could explain this binding behavior would Similar to the studies outlined above, experiments have predict that the high anity binding site was provided been done utilizing EGFR mutants, and the results are by a heterooligomeric complex consisting of two consistent with those obtained using mutant erbB-2 homodimers. The situation, however, is complex and receptors. Coexpression of HER2/neu with an EGFR would require further, more directed studies in order to lacking most of the intracellular domain demonstrated con®dently draw such a conclusion. that the kinase activity of HER2/neu could be inhibited More physiologically relevant data supporting re- in a trans-negative fashion, similar to the eects of the ceptor oligomerization has come from imaging studies N691stop p185 mutant. Coexpression of a kinase- of EGFR receptors, using both electron microscopy de®cient EGFR with HER2/neu led to trans-phosphor- (van Belzen et al., 1988) and time-resolved ¯uorescence ylation of the mutant EGFR (Spivak-Kroizman et al., imaging microscopy (Gadella and Jovin, 1995). Fluor- 1992). Interestingly, tyrosine-phosphorylated dimers of escence imaging microscopy suggests that EGF treat- kinase-de®cient EGFR were observed in these cells, ment quickly causes EGFR aggregation/clustering, and which is surprising, given the inability of these that a subset of EGFR receptors are pre-dimerized receptors to trans-phosphorylate. It is possible that (Gadella and Jovin, 1995). These studies raise the the receptors were phosphorylated as part of a larger possibility that erbB signaling in intact cells, while HER2/neu-containing oligomeric complex. A second requiring dimer formation, may actually be function- possibility which would explain this observation ally activated by aggregation of receptor complexes, involves secondary dimerization, which involves an complexes which need not contain only members of the activated receptor dissociating from an oligomeric erbB family. Although no microscopic studies have complex and subsequently phosphorylating a pre- directly addressed the aggregation of erbB-2 receptors, viously inactive receptor; this idea has been supported oligomerization would likely represent a general for erbB family proteins (Gamett et al., 1997). mechanism of activation within and likely beyond the erbB family, and thus its demonstration with any member of the erbB family would greatly support the Oligomerization importance of this phenomenon. As discussed above, activation of erbB-2 receptors To further test the possibility that erbB protein can clearly involves the formation of dimeric receptor undergo higher order aggregation, we used molecular complexes, the dimer serving as the minimal function- modeling to assess the energetic and structural ing unit for erbB receptor signaling. Several lines of feasibility of such a complex (Murali et al., 1996). evidence, however, suggest that higher order aggre- Modeling using the intracelluar domains of EGFR and gated structures, in addition to dimers, may function p185neu predicts that a tetrameric complex with a 2 : 2 in erbB signaling. The idea that aggregation state may EGFR : p185neu stoichiometry would be energetically function in signal regulation is not novel; for example, favorable. An interesting aspect of this model is the it has been suggested that E. coli Tar receptor, which prediction that the interactions between subunits diers exists as a dimer, forms tetramers in its highest between the dimeric and the tetrameric forms; in the activity state (Cochran and Kim, 1996). Though no tetrameric form, the interactions are predicted to de®nitive evidence has been produced demonstrating a involve the autophosphorylation tyrosines. The possi- functional role for higher-order aggregation states in bility exists, then that tetramerization occurs only after the erbB family, the evidence which exists is extremely dimerization and cross-phosphorylation, and that this provocative. LeVea et al. (1993) working with the full- transition is involved in signal regulation. Tetrameriza- length neu receptor generated in baculovirus, found tion/oligomerization could prove to be a central that the transmembrane mutation led to an increase in principle in receptor signaling, and its further investi- aggregated receptor forms. In further studies using gation should prove fascinating.

Oncogene HER2/Neu: mechanisms of dimerization/oligomerization PJ Brennan et al 6100 Conclusions and prospectus transformation suggested that any reduction in HER2/ neu signaling may lead to phenotypic reversion. Though much has been discovered regarding the Therapy directed toward this end, Herceptin, an anti- mechanisms employed by the erbB family of receptors, HER2/neu monoclonal antibody, has proven clinical many basic questions remain. An erbB crystal structure eectiveness in the treatment of a subset of breast has been elusive, and though modeling insights have cancers (Shak, 1999). Current and future research into been helpful, crystallographic data would likely greatly the mechanisms by which HER2/neu promotes tumor- advance current notions. Resolving the normal and igenesis will most likely lead to additional therapeutics. tumorigenic receptor and ligand combinations involved For example, since it appears that oligomerization is in the erbB signaling network should also be of value. necessary for erbB signal transduction, agents capable Additonally, although many candidate downstream of preventing oligomerization should also prevent signal transduction pathways have been identi®ed, the signaling. Tumors which overexpress both HER2/neu relative importance of each of these pathways to and EGFR represent another clinical target, possibly normal function and malignancy remains largely involving bispeci®c antibodies directed at heterooligo- unknown. mers of these receptors. If future trends follow those Our current understanding of HER2/neu signaling that have already passed, further elucidation of erbB has already translated into clinical success. That a signaling should prove both scienti®cally and medically threshold of HER2/neu overexpression is necessary for rewarding.

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