Structural Evidence for Loose Linkage between Ligand Binding and Kinase Activation in the Epidermal Growth Factor Receptor

Abstract

The mechanisms by which signals are transmitted across the plasma membrane to regulate signaling are largely unknown for receptors with single-pass transmembrane domains such as the epidermal growth factor receptor (EGFR). A crystal structure of the extracellular domain of EGFR dimerized by epidermal growth factor (EGF) reveals the extended, rod-like domain IV and a small, hydrophobic domain IV interface compatible with flexibility. The crystal structure and disulfide cross-linking suggest that the 7-residue linker between the extracellular and transmembrane domains is flexible. Disulfide cross-linking of the transmembrane domain shows that EGF stimulates only moderate association in the first two ␣-helical turns, in contrast to association throughout the membrane over five ␣-helical turns in glycophorin A and integrin. Furthermore, systematic mutagenesis to leucine and phenylalanine suggests that no specific transmembrane interfaces are required for EGFR kinase activation. These results suggest that linkage between ligand-induced dimerization and tyrosine kinase activation is much looser than was previously envisioned.Fundamental to cellular physiology is the ability to transmit extracellular signals across the cell membrane to trigger intracellular responses. Although the extracellular and intracellular portions of cell surface receptors are responsible for detecting ligands and initiating signal cascades, respectively, transmembrane (TM) domains are thought to play critical roles by specifically associating and propagating signals across the phospholipid bilayer. However, the mechanisms by which single-pass TM domains associate and conduct signals are poorly understood.The epidermal growth factor receptor (EGFR) is the prototypical type I TM receptor tyrosine kinase. EGFR and related members of the ErbB family-ErbB2, ErbB3, and ErbB4-contain a glycosylated extracellular ligand binding domain; a singlepass TM domain; and intracellular juxtamembrane, tyrosine kinase, and autophosphorylation domains. The extracellular domain of EGFR binds polypeptide growth factor ligands, such as epidermal growth factor (EGF), to stimulate an array of intracellular signaling cascades that regulate normal and oncogenic cellular growth and proliferation (3,17,36). In one model of growth factor-dependent EGFR activation, ligand binding promotes receptor dimerization and activation of intracellular protein tyrosine kinase activity (35); other models suggest that receptors are predimerized on the cell surface and ligand binding alters the equilibrium between inactive and active dimeric (or higher-order oligomeric) configurations (9, 29).Structural mechanisms of growth factor-mediated receptor dimerization and allosteric kinase domain activation have been proposed from recent crystal structures of isolated extracellular ligand binding domains (7) and intracellular tyrosine kinase domains (37). The orientation between the four extracellular domains is dramatically altered upon ligand binding, which frees interfaces that are masked in ...

A single ligand is sufficient to activate EGFR dimers

“…1C). This observation suggests that unliganded EGFRs can serve as both a donor and an acceptor kinase and that extracellular asymmetry is not absolutely coupled to intracellular asymmetry, consistent with studies suggesting a loose linkage between ligand binding and kinase activation (13). A recent report using a luciferase fragment complementation assay showed that normal activation of EGFR/ErbB2 heterodimers required the EGFR kinase to be active, suggesting that the liganded partner (EGFR) could initially only function as an acceptor kinase and that extraand intracellular asymmetry are coupled (14).…”

“…2 and SI Appendix, Fig. S4, and Table S3) (11,13,15,16). The C-terminal juxtamembrane regions of domain IV are closely apposed when well ordered, suggesting interactions between transmembrane regions in active receptor dimers, but regions of domain IV homologous to those involved in direct dimer contacts in EGFR:EGF complexes (13) are mostly disordered and few specific intersubunit domain IV contacts are resolvable.…”

“…3). Dimers of sErbB4:Nrg1β and sEGFR:EGF (13,16) are similar to one another but differ from TGFα−bound dimers of a truncated form of EGFR (tEGFR) comprising the N-terminal 3 extracellular domains (15) (Fig. 3).…”

“…Superposing ErbB2 extracellular regions on either domain II or domain IV contact regions of EGFR/ErbB dimers demonstrates that ErbB2 is not capable of simultaneously forming favorable domain II dimer contacts and bringing the juxtamembrane regions of domain IV into close proximity (Fig. 5), which appears to be a feature of ErbB signaling (4,13,24). Unlike Drosophila EGFR, ligand binding is not required for ErbB2 to participate in active signaling complexes owing to the presence of ligand-binding homologs, which allowed ErbB2 to evolve a fixed straight domain II conformation and specialize as a heterodimerization partner.…”

Conformational flexibility of the ErbB2 ectodomain and trastuzumab antibody complex as revealed by molecular dynamics and principal component analysis

“…All EGFR receptors contain three different regions: an extracellular (ectodomain, ECD) ligand-binding region, a single membrane-spanning domain and a cytoplasmatic tyrosine kinase domain. The extracellular EGFR domains have been crystallographically elucidated by several research groups [3][4][5][6][7][8][9][10]. The x-ray structure-based models show the appearance of two large homologous domains (L) and two cysteinerich domains (CR), in the order L1-CR1-L2-CR2 which is simply known as I-II-III-IV domains, (see Scheme 1).…”