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Epidermal growth factor receptor: mechanisms of activation and signalling

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Abstract

The epidermal growth factor (EGF) receptor (EGFR) is one of four homologous transmembrane proteins that mediate the actions of a family of growth factors including EGF, transforming growth factor-α, and the neuregulins. We review the structure and function of the EGFR, from ligand binding to the initiation of intracellular signalling pathways that lead to changes in the biochemical state of the cell. The recent crystal structures of different domains from several members of the EGFR family have challenged our concepts of these processes.

Introduction

The epidermal growth factor receptor (EGFR) regulates the intracellular effects of ligands such as EGF and transforming growth factor-α (TGFα) [1], [2], [3]. For many years it has been known that upon ligand binding to the EGFR extracellular domains (collectively called the ectodomain), there is an increase in the proportion of dimerized receptor and the enzymatic activity of its intracellular tyrosine kinase domain increases greatly [4], [5], [6]. The EGFR kinase catalyses the transfer of the γ-phosphate of bound ATP to the tyrosine residues of exogenous substrates and the C-terminal domains of the EGFR, the latter in a trans manner [7], [8]. After the induction of tyrosine phosphorylation, some signalling pathways appear to start with the recognition of the C-terminal phosphotyrosines by appropriate adaptor or signalling molecules [9], [10]. The binding of ligand and activation of the EGFR kinase also induces the migration of EGFR from the caveolae/raft component of the cell membrane to the bulk membrane component [11] and the clustering of EGFR complexes into clathrin-coated pits that are subsequently internalized ([12], [13], [14]; see also Wiley et al., this issue) in a kinase-dependent manner [15], [16]. Abnormal expression and/or mutation of the EGFR has been implicated in the progression of some classes of solid tumours (see Hynes article in this issue).

The EGFR also interacts with its three known homologues, ErbB2 (also called Neu or HER2), ErbB3 (HER3), and ErbB4 (HER4), in a ligand-dependent fashion to form heterodimers [3], [17]. Differences in the C-terminal domains of these proteins results in changes to the repertoire of signalling molecules that interact with the heterodimers, thus leading to an expansion in the number of possible signalling pathways stimulated by a single ligand.

The strength and duration of intracellular signalling from the EGFR are also controlled by internalization and recycling of the receptor, which can be modulated by heterodimerization at the cell surface and by association with intracellular signalling molecules; these aspects of EGFR behavior, relating to its trafficking in cells, are reviewed elsewhere in this issue (Wiley et al.).

The mechanism of the activation of the EGFR has been studied for many years; however, much remains to be determined. Significant progress has recently occurred; the crystal structures of extracellular portions of two ErbB family members and of the EGFR kinase domain have been reported [18], [19], [20], [21]. Some of these structures reveal the modes of ligand binding, ectodomain dimerization, and the conformation of the apo-kinase domain. Full elucidation of the mechanisms of behaviour of both wild-type and oncogenic mutants of the EGFR should help with the design of new molecules to antagonize the action of the mutant or overexpressed receptor in cancer.

Section snippets

Architecture of the EGFR

The EGFR is synthesized from a 1210-residue polypeptide precursor; after cleavage of the N-terminal sequence, an 1186-residue protein is inserted into the cell membrane [22]. Over 20% of the receptor’s 170-kDa mass is N-linked glycosylation and this is required for translocation of the EGFR to the cell surface and subsequent acquisition of function [23]; overexpression of the EGFR or altered glycosylation can reveal peptide epitopes suitable for antibody therapies [24]. The sequence can be

Ligand binding to the EGFR

The three-dimensional structures of the EGF- and TGFα-bound EGFR ectodomain fragments show that EGF and TGFα bind to the EGFR in the same mode [18], [19]. Each bound ligand interacts with the L1 and L2 domains of a given EGFR molecule (Fig. 2). The conserved EGF residue Arg 41 (Arg 42 in TGFα) makes bidentate hydrogen bonds with Asp 355. Arg 41 is surrounded by Tyr 13 and Leu 15 (Phe 15 and Phe 17, respectively, in TGFα), orienting the arginine residue and shielding the salt bridge interaction

Ligand-induced EGFR oligomerization

The 2:2 ligand-EGFR complex forms on the cell surface [72]. Ligated EGFR ectodomain fragments undergo a novel mode of receptor dimerization [18], [19]; a loop from the back of the CR1 domain from one receptor molecule interacts with a pocket at the base of the CR1 loop in the partner EGFR (Fig. 3). There are also some minor contacts between the CR1 loop and the L1 and L2 domains of the partner receptor. This interface participates in the formation of the physiological active dimer on the cell

Ligand-induced activation of the EGFR

In the absence of ligand binding, the EGFR exists on cells as both monomers and dimers [72], [73], [86], [87]. Yet ligand binding to the EGFR kinase is required to elevate the receptor’s tyrosine kinase activity. The position-dependent effects of adding a cysteine residue in the membrane-proximal part of the EGFR’s extracellular region suggests that a ligand-associated orientation of the EGF dimer is required for activation of the tyrosine kinase domains [73]. Clearly, dimerization of the EGFR,

Molecular targets perturbed by the activation of the EGFR

The EGFR exerts its function in the cellular environment mainly, if not exclusively, via its tyrosine kinase activity. Tyrosine phosphorylation of cellular substrates is thus the first and crucial step in transducing EGFR-mediated signals. It is often difficult to determine whether a protein, phosphorylated in response to cellular stimulation with EGF, is a direct substrate of the EGFR kinase or it is phosphorylated following EGFR-dependent activation of other cellular kinases. Given the

Physical association between EGFR and signalling proteins

Phosphorylation of the EGFR’s C-terminus, be it autophosphorylation or transphosphorylation by other kinases such as Src and Jak-2 [97], [107], provides specific docking sites for the SH2 or PTB domains of intracellular signal transducers and adaptors, leading to their colocalization and to the assembly of multicomponent signalling “particles.” Signalling proteins that associate directly with the EGFR in this manner, and the EGFR tyrosines that mediate the association, are listed in Table 2.

Signalling pathways activated by the EGFR

Given the functional diversity of proteins that complex with, or are phosphorylated by, the EGFR, it is hardly surprising that EGF stimulation of a cell results in the simultaneous activation of multiple pathways. These pathways are often functionally interlinked and ideally should not be considered in isolation; however, for the sake of simplicity we will discuss them individually and in particular attempt to describe the earliest steps of their EGFR-mediated activation.

The role of the EGF family of ligands and EGFR in mammalian physiology and pathology

A vast body of knowledge has been accumulating in recent years on the role of the EGF family of ligands and receptors in embryonic development, physiology, and pathology. Thanks to the power of genetic screens, much of the progress on the developmental role of the EGF/EGFR system has come from studies on invertebrates, such as Drosophila and C. elegans. The developmental aspects of EGF/EGFR signalling, both in invertebrates and in mammals, are covered elsewhere in this issue (Shilo and

Gain-of-function: EGFR and its ligands

Apart from the in vitro data, suggesting a role of EGF/EGFR in cell proliferation, evidence has been accumulating that overexpression of the ligands and/or receptors, as well as ligand-independent receptor activation, occurs in many epithelial cancers, most notably gliomas and breast, pancreas, and liver carcinoma. What is not clear is whether this overexpression/activation is indeed causative for the formation of tumours or occurs during tumour progression. The use of transgenic animals has

Effects of the loss of EGFR function

While the gain-of-function experiments address mainly the role of the EGF/EGFR system in abnormal proliferation, loss-of-function mice have shed some light on the developmental and physiological role of the system. Since EGF is produced by the submaxillary glands, sialoadenectomy was initially used as a tool to investigate the effects of reduced EGF levels in vivo. In these studies the organs most affected were the mammary gland [218] and the epidermis [219]. In both organs there was a

Cell motility: EGF receptor–integrin cooperativity

Cell migration is a complex, coordinated process that allows cells to reach specific destinations during embryonic development, to maintain the cellular architecture of self-renewing tissues, repair wounds, and to defend against infectious agents [230], [231], [232]. Signals from several classes of receptors play critical roles in the regulation of cell movement; integrins, through their ability to signal and form adhesive contacts linking the extracellular matrix (ECM) and the actin

Concluding notes

Much progress has been made in understanding the mechanism of EGFR activation upon ligand binding. However, there are many basic questions that must be answered about the nature of the EGFR on the cell surface, i.e., the nature of the inactive, unliganded EGFR monomer and dimer: how ligand induces the conformational transition in the ectodomain; how ligand binding stimulates the activation of the kinase; and whether mechanisms such as secondary dimerization or the formation of higher order

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    1

    Current address: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA.

    2

    Current address: Stem Cell laboratory, Peter McCallum Cancer Institute St. Andrew’s Place, East Melbourne, Victoria 3002, Australia.

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