Epidermal growth factor (EGF) receptor is the prototype of the ErbB (HER) family receptor tyrosine kinases (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGF activates its receptor by inducing dimerization of the 621 amino acid EGF receptor extracellular region. We describe the 2.8 A resolution crystal structure of this entire extracellular region (sEGFR) in an unactivated state. The structure reveals an autoinhibited configuration, where the dimerization interface recently identified in activated sEGFR structures is completely occluded by intramolecular interactions. To activate the receptor, EGF binding must promote a large domain rearrangement that exposes this dimerization interface. This contrasts starkly with other RTK activation mechanisms and suggests new approaches for designing ErbB receptor antagonists.

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Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) ␣-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM ␣-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM Ϸ ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.The ErbB (or HER) family of growth factor receptor tyrosine kinases has four members: the epidermal growth factor (EGF) 1 receptor (ErbB1), ErbB2 (also known as HER2 or the Neu oncogene product), ErbB3 (HER3), and ErbB4 (HER4) (1). Each ErbB receptor has a large extracellular (EC) domain of 600 -630 amino acids, a single membrane-spanning ␣-helix, and an intracellular domain of ϳ500 amino acids that contains the tyrosine kinase domain plus regulatory sequences (2). It is now quite well established that activation of ErbB receptors involves their ligand-induced (or ligand-stabilized) oligomerization, which in turn leads to receptor trans-phosphorylation and activation within dimers or higher order oligomers (reviewed in Ref.3). In the case of ErbB1, we and others have demonstrated that the isolated EC domain of the receptor dimerizes completely upon EGF binding (4 -10). Similarly, the isolated EC domain of ErbB4 oligomerizes strongly when it binds to its growth factor ligand neuregulin 1-␤ 1 (4). These findings have led to the argument that ErbB receptor activation results directly and solely from ligand-induced oligomerization of EC domains (3,11,12). In this view, the transmembrane (TM) and intracellular domains of receptor molecules need not contribute directly to receptor oligomerization, but are instead driven together by EC domain interaction...

Structural studies have shown that ligand-induced epidermal growth factor receptor (EGFR) dimerization involves major domain rearrangements that expose a critical dimerization arm. However, simply exposing this arm is not sufficient for receptor dimerization, suggesting that additional ligand-induced dimer contacts are required. To map these contributions to the dimer interface, we individually mutated each contact suggested by crystallographic studies and analyzed the effects on receptor dimerization, activation, and ligand binding. We find that domain II contributes >90% of the driving energy for dimerization of the extracellular region, with domain IV adding little. Within domain II, the dimerization arm forms much of the dimer interface, as expected. However, a loop from the sixth disulfide-bonded module (immediately C-terminal to the dimerization arm) also makes a critical contribution. Specific ligand-induced conformational changes in domain II are required for this loop to contribute to receptor dimerization, and we identify a set of ligand-induced intramolecular interactions that appear to be important in driving these changes, effectively "buttressing" the dimer interface. Our data also suggest that similar conformational changes may determine the specificity of ErbB receptor homo-versus heterodimerization.The epidermal growth factor (EGF) receptor (also designated ErbB) family of receptor tyrosine kinases contains four members: EGFR, ErbB2/HER2, ErbB3/HER3, and ErbB4/ HER4 (32). Each mature ErbB receptor contains an extracellular ligand-binding region, a single transmembrane (TM) domain and an intracellular tyrosine kinase domain that is flanked by regulatory regions (30). ErbB receptors are controlled by at least 12 different growth factors including EGF, transforming growth factor ␣ (TGF-␣), and the neuregulins. Growth factor binding induces homo-and/or heterodimerization of the receptor, leading to trans-autophosphorylation and subsequent activation of SH2 domain-dependent downstream signaling pathways (27).ErbB receptor signaling is normally very tightly controlled, and its deregulation is linked to several epithelial cancers. Gene amplification, overexpression, and activating mutations of EGFR are seen in glioblastoma, prostate, breast, colorectal, and squamous carcinomas (18,20,21,26). Overexpression of ErbB2 (29) and other ErbB receptors occurs in mammary carcinomas and other human cancers (5). Several anticancer therapies that target ErbB receptors are now being used or tested in the clinic (1), including tyrosine kinase inhibitors (10, 15) and antibodies against ErbB receptor extracellular regions (25).X-ray crystal structures of ErbB extracellular regions in different activation states have led to a significant advance in our understanding of how receptor dimerization and activation is promoted by growth factor binding (6). Dimerization is driven entirely by receptor-receptor interactions, with a critical "dimerization arm" in the cysteine-rich domain II providing the majority of contacts acr...