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Abstract

  1. Top of page
  2. Abstract
  3. Evolutionary Aspects
  4. Structure–Function Relationships
  5. Perspectives
  6. Acknowledgements
  7. Literature Cited

The epidermal growth factor receptor (EGFR) regulates key processes of cell biology, including proliferation, survival, and differentiation during development, tissue homeostasis, and tumorigenesis. Canonical EGFR activation involves the binding of seven peptide growth factors. These ligands are synthesized as transmembrane proteins comprising an N-terminal extension, the EGF module, a short juxtamembrane stalk, a hydrophobic transmembrane domain, and a carboxy-terminal fragment. The central structural and functional feature is the EGF module, a sequence containing six cysteines in a conserved spacement which is responsible for binding to the EGFR. While the membrane-anchored peptide can be biologically active by juxtacrine signaling, in most cases the EGF module is proteolytically cleaved (a process termed ectodomain shedding) to release the soluble growth factor, which may act in an endocrine, paracrine, or autocrine fashion. This review summarizes the structural and functional properties of these fascinating molecules and presents selected examples to illustrate their roles in development, physiology, and pathology. J. Cell. Physiol. 218: 460–466, 2009. © 2008 Wiley-Liss, Inc.

The tyrosine-kinase epidermal growth factor receptor (EGFR, ERBB1), one of the most versatile signaling units in biology, regulates key processes of cell biology, such as proliferation, survival, and differentiation during development, tissue homeostasis, and tumorigenesis. Upon ligand binding, homodimers or heterodimers (with the related receptors ERBB2, ERBB3, and ERBB4) are formed, and kinase activation initiates multiple intracellular signaling pathways. In mammals, canonical EGFR activation involves the binding of seven peptide growth factors: EGF, transforming growth factor-α (TGFA), heparin-binding EGF-like growth factor (HBEGF), amphiregulin (AREG), betacellulin (BTC), epiregulin (EREG), and epigen (EPGN) (Harris et al., 2003). Curiously, HBEGF serves as the diphtheria toxin receptor, permitting the toxin to enter the cell via binding and subsequent endocytosis. A distinct group of growth factors called neuregulins binds ERBB3 and ERBB4 (Falls, 2003). ERBB2 has no known ligand (Klapper et al., 1999; Kochupurakkal et al., 2005) and ERBB3 has a defective kinase domain due to substitutions of essential residues (Guy et al., 1994). Therefore, both receptors act primarily as subunits of the other ERBBs. Interestingly, ERBB2/ERBB3 heterodimers are able to generate potent cellular signals (Citri et al., 2003).

The events that take place after ligand binding (including receptor homo- or heterodimerization, autophosphorylation, signal transduction, and sorting of the ligand–receptor complex to degradation or recycling) have been summarized in excellent reviews (Waterman and Yarden, 2001; Holbro and Hynes, 2004; Citri and Yarden, 2006; Yarden and Shilo, 2007). However, information sources that concisely integrate the cell biology and functional aspects of the EGFR ligands are rather scarce. The aim of this article is to provide the “basic facts” about the evolution, the structure–function relationships, and exemplify some of the multiple roles of the seven EGFR ligands in development and physiology.

Evolutionary Aspects

  1. Top of page
  2. Abstract
  3. Evolutionary Aspects
  4. Structure–Function Relationships
  5. Perspectives
  6. Acknowledgements
  7. Literature Cited

In contrast to the whole gamut of molecules in mammals, only a single receptor type that is equivalent to ERBB has been described in Caenorhabditis elegans and Drosophila melanogaster, and the number of ligands is restricted to a single peptide in the worm and to a group of four peptides in the fly. Further details concerning the phylogenetic relationships between today's EGFR ligands and its relation to gene duplication events or receptor binding affinities can be found elsewhere (Stein and Staros, 2000, 2006). The progressive phylogenetic increase in the complexity of the EGFR system seems to parallel the development of more sophisticated networks that are necessary for controlling and coordinating the cell metabolism, growth and development of vertebrates, which underlines the relevance of this growth factor system.

Structure–Function Relationships

  1. Top of page
  2. Abstract
  3. Evolutionary Aspects
  4. Structure–Function Relationships
  5. Perspectives
  6. Acknowledgements
  7. Literature Cited

The EGFR ligands are synthesized as type I transmembrane proteins that comprise an N-terminal extension (pro-region), the EGF module, a short juxtamembrane stalk, a hydrophobic transmembrane domain, and a carboxy-terminal fragment, also known as the cytoplasmic tail (CT; Fig. 1A). EGF is unique in having a total of nine EGF motifs, although only the one adjacent to the cell membrane is functional as an EGFR-binding domain. Via proteolytic processing, a “soluble” growth factor containing the EGF module can be released into the extracellular milieu (see below). Overall protein sequence identity between EGFR ligands is low (∼25%) and these proteins also differ concerning the distribution of glycosylation sites, the presence of a heparin-binding domain, and other biochemical characteristics (Harris et al., 2003).

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Figure 1. Structural aspects of the mammalian EGFR ligands. A: Schematic representation of the membrane-anchored precursor form of the seven mammalian EGFR ligands: epidermal growth factor (EGF), transforming growth factor-α (TGFA), heparin-binding EGF-like growth factor (HBEGF), amphiregulin (AREG), betacellulin (BTC), epiregulin (EREG), and epigen (EPGN). B: The secondary structure of the EGF module of human EGF showing the interaction between the six conserved cysteines and the resulting loops.

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The EGF module

The central structural and functional feature (i.e., the sequence responsible for the interaction with the EGFR) is the EGF module (also known as the EGF domain or motif), a roughly 40 amino acids long sequence containing six cysteines in a conserved spacement (Fig. 1B). The cysteines are arranged as three disulfide bridges between C1 and C3, C2 and C4, and C5 and C6, forming the A, B, and C loops, respectively. The single amino acid between the B and C loops is known as the “hinge residue” because it is believed to function as a hinge along which the two segments of the protein can move (van Zoelen et al., 2000).

EGF motifs are not confined to EGFR ligands but are present in single or multiple copies in dozens of structurally and functionally unrelated proteins, including extracellular matrix (ECM) and cell adhesion proteins, blood coagulation factors and proteins related to the immune response. Several poxviruses also encode EGF-like motifs, which, although not essential for viral replication, enhance virulence and stimulate cell proliferation at the primary infection site. The wide distribution and putative lack of specific function of this motif has puzzled researchers for decades (Engel, 1989; Davis, 1990). Although EGF-like motifs may predominantly function only as structural spacers, strong activation of the EGFR has been demonstrated for some poxviruses (Tzahar et al., 1998) and the matrix component tenascin-c (Swindle et al., 2001). In spite of very low receptor-binding affinity, these proteins are able to exert mitogenic activity that is equivalent to or even more potent than “classical” ligands by evading the main signaling attenuation mechanisms (receptor and ligand internalization and degradation), resulting in sustained signal transduction. For tenascin-c, embedded in the ECM, the mechanism may be a constant re-presentation of the ligand following dissociation from the receptor (Swindle et al., 2001). Poxviruses achieve strong mitogenic effects by attenuating receptor degradation and ubiquitination (Tzahar et al., 1998).

Structural and molecular evolutionary analyses (Stein and Staros, 2000, 2006) have identified several structural features that distinguish legitimate ERBB ligands from non-ERBB ligands: (i) the spacing of EGF-motif cysteines can be formulated in a considerably more restrictive fashion. For instance, the spacement CX7 CX4–5 CX10 CX CX8 C, in which X can be any amino acid, would include only the EGFR ligands, neuregulin-1, and the tomoregulins; (ii) the EGF motif is encoded by two exons with a splice site between the fourth and fifth cysteine; (iii) the functional EGF module is located within approximately 25 residues of the transmembrane domain. The presence and spacing of additional specific residues further distinguish the EGFR ligands from the neuregulins and other EGF module-containing proteins at the structural level and define high affinity binding to the EGFR (Groenen et al., 1994; van Zoelen et al., 2000; van der Woning et al., 2006).

Ectodomain shedding

Under physiological conditions, EGFR activity is largely dependent on ligand availability (spatiotemporal expression) and their post-translational processing. Without doubt, the most important post-translational modification is the proteolytic release of an extracellular fragment containing the EGF module (Fig. 2). A recent study provides evidence that both the specific sequence of the cleavage site (which is poorly conserved) and the overall length of the juxtamembrane domain are the major determinants of cleavage efficiency (Hinkle et al., 2004). Ectodomain shedding occurs in response to diverse physiological and pharmacological agonists. ADAM (a disintegrin and metalloprotease) proteins, membrane-anchored metalloproteases, have been implicated in the shedding of several EGFR ligands (Sahin et al., 2004). Remarkably, mice lacking ADAM17 recapitulate several phenotypes of animals deficient in EGFR ligands (see below), such as defects in epithelial maturation, branching morphogenesis of the lung, and malformed heart valves (Peschon et al., 1998; Zhao et al., 2001; Shi et al., 2003). This provides strong evidence for the in vivo relevance of this enzyme in EGFR processing, and for ectodomain shedding as a whole.

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Figure 2. Ectodomain shedding of EGFR ligands and its consequences for signaling. Membrane-bound molecules can activate the EGFR of neighbor cells (juxtacrine mechanism). Following proteolytic release, the soluble EGF module may activate the EGFR of neighbor cells (paracrine), distant cells (endocrine), or at the own cell membrane (autocrine). Ectodomain shedding also results in a free cytoplasmic tail, which can interact with other cytoplasmic proteins to modulate gene expression. See the main text for further explanations.

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The “soluble” growth factor may bind and activate receptors on distant cells, neighbor cells, or on the cell of its origin, mechanisms termed endocrine, paracrine, or autocrine, respectively (Fig. 2). Depending on the cellular context, growth factor shedding may be indispensable. For instance, blocking the release of EGFR ligands inhibited growth and migration in several EGFR-dependent cell lines (Dong et al., 1999) and greatly retarded wound re-epithelialization due to impaired keratinocyte migration (Tokumaru et al., 2000). In the late 1980s, different laboratories showed that membrane-anchored TGFA can activate EGFRs present on membranes of adjacent cells (Brachmann et al., 1989; Wong et al., 1989) and the term “juxtacrine” was proposed to describe this mode of intercellular interaction (Anklesaria et al., 1990). Since then, juxtacrine activity has been reported for several other EGFR ligands and growing evidence suggests that this mode of action may induce a different biological outcome as compared to the effects elicited by soluble growth factors (Singh and Harris, 2005). The importance of a tight regulation of ectodomain shedding has been compellingly demonstrated for HBEGF by gene replacement experiments. Mice expressing uncleavable HBEGF develop severe heart failure and enlarged heart valves (resembling the phenotype of full HBEGF knockout), while mice expressing a constitutively soluble HBEGF exhibit severe hyperplasia in skin and heart (Yamazaki et al., 2003).

Numerous investigations during the last decade have revealed that G-protein-coupled receptors (GPCRs), the largest group of cell surface receptors, are able to utilize the EGFR as a downstream signaling partner for generating potent mitogenic signals. Initially described by Daub et al. (1996), EGFR transactivation by GPCRs was later shown to be a metalloprotease-dependent process: stimulation of GPCRs leads to the stimulation of a metalloprotease, resulting in EGFR ligand shedding and in the activation of EGFR-downstream cascades (Prenzel et al., 1999). Since then, this phenomenon (also known as triple membrane-passing signal) has been linked to the modulation of cell proliferation, apoptosis, and migration in a variety of cancer types and other diseases and became a paradigm for inter-receptor cross-talk (reviewed in Fischer et al., 2003).

The cytoplasmic tail: a new frontier?

Several studies have identified the CT of EGFR ligands as a site of important protein interactions, able to elicit multiple functions. These include, most notably, to mediate the correct trafficking and delivery of TGFA to the cell surface (Fernandez-Larrea et al., 1999) and to carry basolateral sorting information for TGFA (Dempsey et al., 2003) and AREG (Brown et al., 2001). Recently, the CT of EGF was shown to alter the distribution and post-translational modification of the microtubular system and to increase the production of microtubule-associated proteins 1b and 2 in human thyroid carcinoma cells (Pyka et al., 2005). While the main task of the CT may be related to ligand trafficking and delivery to the cell surface, a new, exciting possibility emerged with the identification of the transcriptional repressors promyelocytic leukemia zinc finger (PLZF, Nanba et al., 2003) and B-cell leukemia 6 (Bcl6, Kinugasa et al., 2007) as binding proteins of HBEGF CT (Fig. 2). PLZF and Bcl6 are transcriptional repressors and negatively regulate the cell cycle, and interaction with HBEGF CT reversed the decreased expression of PLZF and Bcl6 target genes reviewed in Higashiyama et al. (2008). Thus, ectodomain shedding appears to initiate a bidirectional signaling: the released growth factor containing the EGF domain activates the EGFR and, concomitantly, the CT may reverse intracellular gene repression (derepression; Fig. 2). Supporting this model, it was recently shown that HBEGF and its shed remnant are targeted from the plasma membrane to the inner nuclear membrane—where they can bind to transcriptional repressors—via a retrograde membrane trafficking pathway (Hieda et al., 2008).

EGFR ligand function: roles in development and physiology

As inherent components of the EGFR signaling system, the EGFR ligands are expressed in several tissues and elicit manifold actions during development and in adult mammals. It is therefore initially surprising that HBEGF is the only ligand whose absence results in postnatal lethality—due to malformed heart valves, hypertrophic cardiomyocytes, and hypoplastic lungs (Iwamoto et al., 2003; Jackson et al., 2003)—while mice lacking AREG (Luetteke et al., 1999), BTC (Jackson et al., 2003), EGF (Luetteke et al., 1999), EREG (Mann et al., 1993; Lee et al., 2004; Shirasawa et al., 2004), TGFA (Luetteke et al., 1993), and even triple null mice deficient for AREG, EGF, and TGFA (Luetteke et al., 1999) are viable. These observations clearly point to a high degree of functional redundancy compensation within the family of EGFR ligands. Nevertheless, many of these mouse lines show specific phenotypes, and careful examination under challenge situations further disclosed homeostasis defects in apparently normal tissues. For example, while only mammary gland defects were initially reported in AREG knockout mice, further studies additionally revealed reduced tibial trabecular bone (Qin et al., 2005) and impaired liver regeneration after partial liver resection (Berasain et al., 2005). Interestingly, many phenotypes observed in mice lacking individual EGFR ligands, such as the hair follicle and eyelid closure defects in TGFA knockout mice (Luetteke et al., 1993; Mann et al., 1993), or the heart valve defects and the lung immaturity in HBEGF-deficient animals (Iwamoto et al., 2003; Jackson et al., 2003), are also observed in mice lacking the EGFR (Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et al., 1995; Chen et al., 2000).

Additional evidence for specific actions of these proteins also comes from in vitro studies and from the phenotype of transgenic animals overexpressing individual EGFR ligands (see below). Often, as for BTC (Schneider et al., 2005) and EGF (Chan and Wong, 2000; Wong et al., 2000), overexpressing the EGFR ligand results in profuse, complex phenotypes which are in sharp contrast to the inconspicuousness of mice lacking the respective ligand (Luetteke et al., 1999; Jackson et al., 2003), rendering this approach more suitable for the study of potential actions of these molecules. Transgenic mice overexpressing EGFR ligands have also been very informative concerning the role of these molecules in tumorigenesis and other diseases. The most prominent and classical example is the large array of neoplastic lesions observed in mice overexpressing TGFA (Jhappan et al., 1990; Matsui et al., 1990; Sandgren et al., 1990), including hepatocarcinogenesis, pancreatic metaplasia, dysplasia of the coagulation gland epithelium, and mammary adenocarcinoma. Since then, the EGFR ligands have been implicated in the pathogenesis of a large number of disorders. These include a role for EREG in mediating breast cancer metastasis to the lung (Minn et al., 2005), isolated recessive renal hypomagnesemia as a consequence of impaired basolateral sorting of EGF (Groenestege et al., 2007), and increased TGFA expression as the causing agent of the rare gastric disorder Ménétrier disease (Dempsey et al., 1992; Takagi et al., 1992; Coffey et al., 2007), just to mention a few cases.

Below, we summarize the known roles of EGFR ligands in selected mammalian tissues and biological processes to illustrate the modus operandi of the EGFR system.

Epidermis and hair follicle

EGFR ligands are of special importance in these tissues, regulating cell proliferation, migration, adhesion, and inflammatory processes (Pastore et al., 2008; Schneider et al., 2008). Genetically modified mice lacking or overexpressing individual EGFR ligands have been widely used to study EGFR ligand actions in the skin (reviewed in Schneider et al., 2008). These studies, summarized in Figure 3A, provided important insights into the roles of these molecules in processes such as wound healing (HBEGF, Shirakata et al., 2005), psoriasis (AREG, Cook et al., 1997, 2004), and tumorigenesis (TGFA, Vassar and Fuchs, 1991; Dominey et al., 1993; Jhappan et al., 1994; Shibata et al., 1997). Furthermore, the unique dermatological phenotypes observed in these mice are particularly well suited in demonstrating their ability to elicit specific effects and, at the same time, to (at least partially) compensate for the lack of another EGFR ligand family member.

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Figure 3. Selected examples of the roles of EGFR ligands in physiological processes. A: The skin and hair follicle phenotypes of genetically modified mice lacking or overexpressing individual EGFR ligands. Mice with altered expression of EPGN are not available. B: HBEGF, TGFA, and AREG expression is induced by parathormone (PTH) in bone cells and AREG regulates osteoblast proliferation and differentiation. C: Luteinizing hormone (LH) promotes the expression of AREG, BTC, and EREG in ovarian granulosa cells. These peptides, once released into the follicular fluid, activate the EGFR of granulosa and cumulus cells, regulating follicle maturation and ovulation. D: HBEGF is expressed in the uterine epithelium surrounding the blastocyst during the onset of implantation (left part, red arrow). Right part: embryo implantation, visualized as blue bands in the uterus of control mice (+/+), is frequently delayed or deferred in HBEGF-deficient mice (−/−). Pictures in D are reproduced from Xie et al. (2007).

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Bone formation

More than 20 years ago, cell culture experiments have suggested that EGFR ligands stimulate the proliferation of preosteoblasts while inhibiting their further differentiation into osteoblasts (Kumegawa et al., 1983; Ng et al., 1983; Hata et al., 1984). In vivo studies have confirmed these actions: transgenic mice overexpressing EGF display hyperproliferation of osteoblasts (Chan and Wong, 2000), and “humanized” mice (this is, with a human EGFR cDNA replacing the endogenous mouse EGFR gene with the outcome of low EGFR activity) exhibit accelerated osteoblast differentiation (Sibilia et al., 2003). More recently, bone-forming agents such as parathormone (PTH) were shown to rapidly induce the expression of AREG, HBEGF, and TGFA mRNAs (Fig. 3B, Qin et al., 2003). AREG appears to be particularly important in this context, acting as a potent growth factor for preosteoblasts while completely inhibiting their final differentiation (Qin et al., 2005). Thus, AREG emerges as an important element for the expansion of the pool of committed preosteoblasts and therefore a regulator of final bone mass. Supporting this concept, AREG knockout mice showed significantly reduced tibial trabecular bone (Qin et al., 2005). It has also been known for many years that, besides these effects on osteoblasts, EGFR ligands such as EGF and TGFA stimulate bone resorption (Stern et al., 1985; Ibbotson et al., 1986; Lorenzo et al., 1986; Takahashi et al., 1986), implying that these growth factors regulate the formation and activity of osteoclasts. This was recently shown to be an indirect effect: EGFR ligands act on osteoblasts, decreasing osteoprotegerin expression and increasing monocyte chemoattractant protein 1 expression, consequently stimulating osteoclast formation (Zhu et al., 2007). Collectively, the effects of the EGFR system on osteoblasts and osteoclasts suggest a potential application of anti-EGFR drugs for the treatment of cancer patients with bone metastasis (Normanno et al., 2005).

Female reproduction

EGFR ligands have been recently identified as important molecules in mediating diverse aspects of female reproduction, including the attainment of sexual maturity, ovarian follicle maturation, and embryo implantation (reviewed in Schneider and Wolf, 2008). For instance, luteinizing hormone (LH) actions include the induction of AREG, BTC, and EREG expression in ovarian mural granulosa cells. These peptides, in turn, activate EGFRs within the mural layer or are released from the cell surface into the follicular fluid and activate the EGFRs on cumulus cells, triggering cumulus expansion and the acquisition of oocyte developmental competence and finally ovulation (Park et al., 2004; Hsieh and Conti, 2005; Conti et al., 2006). Figure 3C shows a simplified scheme illustrating these processes. Accordingly, defects in follicle maturation and ovulation were observed in mice lacking AREG or EREG (Hsieh et al., 2007), or overexpressing BTC (Gratao et al., 2008) or TGFA (Ma et al., 1994). These findings provide an explanation for the profound effects exerted by LH on cumulus cells in spite of their lack of LH receptors and their failure to respond to LH treatment in vitro. Among the EGFR ligands, HBEGF has been shown to be particularly important for the initial interaction of the embryo with the uterine luminal epithelium. HBEGF expression is induced specifically at the site of embryo apposition (Das et al., 1994) and it represents the only EGFR ligand able to induce a decidual response (Paria et al., 2001; Fig. 3D). Recently, severe implantation defects, in part compensated by enhanced expression of AREG, were reported in HBEGF-deficient mice (Xie et al., 2007; Fig. 3D).

Perspectives

  1. Top of page
  2. Abstract
  3. Evolutionary Aspects
  4. Structure–Function Relationships
  5. Perspectives
  6. Acknowledgements
  7. Literature Cited

Massague and Pandiella brilliantly stated 15 years ago that “…membrane-anchored growth factor precursors are precursors in the biochemical sense but not in the biological sense” (Massague and Pandiella, 1993). Recent data on the importance of juxtacrine signaling and the description of interaction partners and actions of the CT of EGFR ligands have confirmed the correctness of this statement and expanded its significance. Further study of this aspect is essential for understanding the complex functionality of the EGFR system. It is a thrilling question whether some known effects attributed to the soluble growth factor are in fact, even if partially, the result of CT activities. Importantly, understanding why the EGFR ligands are transmembrane peptides will help to unravel the biology of other membrane-anchored growth factors and cytokines.

While the EGFR and ERBB2 are targets of successful cancer therapies, approaches targeting the EGFR ligands for therapeutic purposes are just beginning to be explored. An attractive alternative is the inhibition of shedding agents (for instance, ADAM17), a promising strategy once selective inhibitors become available and off-target effects are reduced (Kenny and Bissell, 2007; Merchant et al., 2008).

Finally, further functional characterization of EPGN, the last identified EGFR ligand (Strachan et al., 2001), is urgently needed. EPGN is a potent mitogen whose relatively low receptor affinity is compensated by a prolonged presence in the extracellular milieu and by ineffective inactivation of the ligand–receptor complex (Kochupurakkal et al., 2005). Thus, it is certainly no exaggeration that exciting years of EGFR ligand research are coming up.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Evolutionary Aspects
  4. Structure–Function Relationships
  5. Perspectives
  6. Acknowledgements
  7. Literature Cited

We thank Dr. S.K. Dey for the pictures showing uterine implantation sites. Our studies of EGFR ligands are supported by the Deutsche Forschungsgemeinschaft (GRK 1029).

Literature Cited

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  2. Abstract
  3. Evolutionary Aspects
  4. Structure–Function Relationships
  5. Perspectives
  6. Acknowledgements
  7. Literature Cited
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