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HER2/Neu: Mechanisms of Dimerization/Oligomerization

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HER2/Neu: Mechanisms of Dimerization/Oligomerization Oncogene (2000) 19, 6093 ± 6101 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc 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 aggrega- tion 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 transduc- tion. 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 transmem- brane 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 proto- oncogenic and oncogenic forms of the neu transmem- brane 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 transforma- tion. 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
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