Structure and Function of the Epidermal Growth Factor (EGF⧸ErbB) Family of Receptors

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This chapter reviews the family of receptors as ErbB receptors and the individual receptors, such as EGFR (epidermal growth factor receptor), HER2 (human epidermal growth factor receptor 2), HER3, or HER4. The soluble extracellular regions of these receptors are referred to as sEGFR, sHER2, sHER3, and sHER4. Both EGF and EGFR are archetypes of protein families that have undergone duplication and diversification throughout animal evolution. EGF-related ligands include EGF, transforming growth factor-α (TGFα), heparin-binding EGF-like growth factor (HB-EGF), amphiregulin, betacellulin, and several isoforms of heregulin/neuregulin. The association of ErbB receptors with human disease is also discussed. HER2 presents a particularly instructive example of growth factor receptor involvement in cancer. All structures of ErbB extracellular domains confirm the expected structural homology of the ‘L’ domains (domains I and III) to one another and to corresponding domains in type I insulin-like growth factor receptor (IGF1R). The structure of entire ErbB receptor ectodomains is provided. The structure of a complex of the EGFR extracellular region and EGF determined at low pH provides a snapshot of a likely mechanism for release of bound ligand in the low pH environment of the endosome. HER2 is unique among ErbB receptors in that no high-affinity HER2 ligand has been found, it functions as a co-receptor with each of the other ErbB receptors and transforms when overexpressed. The active-like structure of HER2 provides a structural basis for its role as the preferred heterodimerization partner among ErbB receptors.

Introduction

Epidermal Growth Factor (EGF) was among the first growth factors discovered, and study of EGF and its receptors has established many paradigms for growth factor–mediated signaling (Carpenter 1987, Cohen 1986, Cohen 1987, Schlessinger 2000, Yarden 2001). Initially isolated from the mouse submaxillary gland based on its ability to stimulate premature eye opening and tooth eruption in neonatal mice, EGF is a 53 amino-acid polypeptide derived by proteolysis from a larger precursor (Carpenter 1979, Cohen 1986). The ability of EGF to stimulate growth and differentiation of epidermal and mesodermal tissues led both to its name and to keen interest in its mode of action.

Following its isolation, EGF was shown to bind with high affinity to a specific receptor in the cell membrane and stimulate rapid activation of a protein kinase activity (Carpenter 1975, Carpenter 1978, Carpenter 1979, Das 1977, Hock 1979, Wrann 1979). Purification and characterization of the EGF receptor (EGFR) showed it to be a ∼170 kD molecular weight integral membrane glycoprotein (Cohen et al., 1982). The ligand-inducible kinase activity co-purified with EGFR, suggesting a physical linkage between the ligand binding and kinase activities, which was later verified by molecular cloning (Cohen 1980, Ullrich 1984). Early on, the EGFR kinase activity was shown to result in phosphorylation of tyrosine residues, the first such demonstration for any receptor (Ushiro and Cohen, 1980).

Molecular cloning of EGFR revealed it to be a 1186 amino acid protein with a 621 amino acid extracellular region followed by a single membrane-spanning region and a cytoplasmic tyrosine kinase (Ullrich et al., 1984). Despite nonhomologous ligand binding regions, this overall architecture is shared by many other receptors, including those for insulin, PDGF, FGF, and VEGF, and these receptors are now collectively known as receptor tyrosine kinases (RTKs) (Schlessinger, 2000). The extracellular ligand-binding region of EGFR is made up of four subdomains arranged as a tandem repeat of two types of domains (Fig. 1). The first and third domains are homologous to one another and have been designated domains I and III or L1 and L2, respectively; the second and fourth domains are also homologous to one another and have been designated domains II and IV or CR1 and CR2, respectively (Bajaj 1987, Lax 1988, Ward 1995). The CR in this case is short for “cysteine-rich” and reflects the fact that nearly 50 conserved cysteines are found in these two domains. For simplicity, this review will employ the I, II, III, and IV domain nomenclature. Studies with mutant and chimeric EGF receptors and receptor fragments demonstrated that ligand binding is mediated primarily by domain III with some contribution from regions on domain I (Kohda 1993, Lax 1989). Curiously, the presence of domain IV was shown to be slightly inhibitory to ligand binding (Elleman et al., 2001).

Once the topology and functional organization of EGFR became apparent, questions about receptor activation focused on how extracellular ligand binding activates the intracellular kinase. Early studies with fluorescent-labeled EGF showed aggregation of EGFR on the cell surface in response to ligand binding (Schechter et al., 1979), implicating receptor cross-linking as an activation mechanism. EGFR is endocytosed following ligand binding, however, and it was difficult to distinguish between aggregation as the trigger of signaling as opposed to a downstream response to receptor activation (Haigler et al., 1979). A key piece of the puzzle emerged with the observation of ligand-induced dimers of EGFR (Yarden and Schlessinger, 1987), which was the first indication that dimerization might play a role in signaling for any receptor (Heldin, 1995). Ligand-induced dimerization—more broadly induction of a specific oligomeric conformation by ligand binding—is now accepted as the signaling trigger for all RTKs (Heldin 1995, Schlessinger 2000), as memorably illustrated by the crystal structure of the complex of human growth hormone with two of its receptors (de Vos et al., 1992).

Prior to molecular cloning, amino-acid sequence data from EGFR revealed that the ErbB oncogene of the avian erythroblastosis virus encodes a truncated form of EGFR (Downward et al., 1984). This truncated form is missing most of the extracellular region but includes a constitutively active kinase region that is responsible for unregulated growth of infected cells (Frykberg 1983, Yamamoto 1983). Demonstration that an oncogene encoded an activated form of a growth factor receptor provided exciting insight into the origins of cancer and presaged discovery of the involvement of EGFR and related receptors in the genesis and severity of many human cancers (Blume-Jensen 2001, Holbro 2003, Tang 1998). The nature of the ErbB oncogene also indicated that the extracellular region not only mediates ligand-dependent activation but also contributes to maintaining the kinase in an inactive state in the absence of ligand.

Section snippets

The EGF and EGFR Families

Both EGF and EGFR are archetypes of protein families that have undergone duplication and diversification throughout animal evolution (Muller 1996, Stein 2000). C. elegans utilizes a single homolog of both EGFR (Let-23) and EGF (Lin-3), Drosophila utilizes a single EGFR (DER) and four EGF homologs (Vein, Spitz, Gurken, and Argos), and humans utilize four EGFR and at least 11 EGF homologs (Stein and Staros, 2000). The four human EGFR homologs are known as both the HER (HER1, HER2, HER3, and HER4

Association of ErbB Receptors with Human Disease

EGFR became the first cell-surface receptor linked to cancer when Cohen and colleagues demonstrated downregulation of EGFR following transformation of cultured cells with specific oncogenic viruses (Todaro et al., 1976). The significance of this association was not immediately clear, but discovery that the ErbB oncogene encoded an activated form of the EGFR kinase established a clear link between inappropriate EGFR activity and cancer (Downward et al., 1984). In the 20 years since this

Structure of Individual ErbB Receptor Domains

Despite intense interest in ErbB receptors, high-resolution structural information has been slow in coming, owing largely to difficulties expressing and crystallizing these cysteine-rich glycoproteins. Fortunately, this situation has been remedied in the last year with publication of high-resolution crystal structures of active and inactive forms of the extracellular region of EGFR (sEGFR) (Ferguson 2003, Garrett 2002, Ogiso 2002), the extracellular region of HER2 (sHER2) both alone and

Structure of Entire ErbB Receptor Ectodomains

Although the structure of subdomains within ErbB receptor extracellular regions was anticipated from their homology to the IGF1R subdomains, their arrangement in intact receptors was not. The structures of unliganded HER3 and unactivated EGFR extracellular regions revealed a ∼15 Å beta-hairpin loop that extends from domain II to interact with a pocket at the C-terminus of domain IV (Fig. 4). This interaction, akin to a “snap” or “tether,” constrains the EGFR and HER3 extracellular regions into

HER2

HER2 is unique among ErbB receptors in that no high-affinity HER2 ligand has been found, it functions as a co-receptor with each of the other ErbB receptors, and it is transforming when overexpressed (Di Fiore 1987, Klapper 1999). Much attention has been focused on HER2 because it is activated in many cancers (Holbro 2003, Slamon 1987, Tang 1998) and a HER2-targeted therapy, the monoclonal anti-HER2 antibody Herceptin, has demonstrated efficacy in a subset of breast cancers (Slamon et al., 2001

Therapeutic Anti-HER2 Antibodies

A major development in the treatment of breast cancer has been the success of Herceptin, an anti-HER2 monoclonal antibody, in treating the 20–25% of breast cancers in which HER2 is overexpressed (Slamon et al., 2001). Although not a cure—average life expectancy is extended 4–5 months—Herceptin has demonstrated that targeting activated ErbB receptors in cancer can be beneficial and holds out the hope of improved therapies. A particular advantage of specific receptor-targeted therapies is that

Remaining Questions

Recent structural studies have greatly advanced our understanding of signaling by members of the ErbB family of receptors, but many key questions remain. Principal among these is how events on the outside of the cell are communicated across the plasma membrane and lead to activation of the kinase activity. Structural and biochemical studies clearly point to receptor dimers as key elements of signaling complexes (Garrett 2002, Ogiso 2002, Yarden 1987). Not all dimers are capable of signaling,

Acknowledgements

I thank Mark Lemmon, Mark Sliwkowski, and members of my lab for helpful discussions and HHMI and NIH for funding the work in my laboratory.

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