Jennifer L. Macdonald-Obermann

The Intracellular Juxtamembrane Domain of the Epidermal Growth Factor (EGF) Receptor Is Responsible for the Allosteric Regulation of EGF Binding

We have previously shown that the binding of epidermal growth factor (EGF) to its receptor can best be described by a model that involves negative cooperativity in an aggregating system (Macdonald, J. L., and Pike, L. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 112–117). However, despite the fact that biochemical analyses indicate that EGF induces dimerization of its receptor, the binding data provided no evidence for positive linkage between EGF binding and dimer assembly. By analyzing the binding of EGF to a number of receptor mutants, we now report that in naive, unphosphorylated EGF receptors, ligand binding is positively linked to receptor dimerization but the linkage is abolished upon autophosphorylation of the receptor. Both phosphorylated and unphosphorylated EGF receptors exhibit negative cooperativity, indicating that mechanistically, cooperativity is distinct from the phenomenon of linkage. Nonetheless, both the positive linkage and the negative cooperativity observed in EGF binding require the presence of the intracellular juxtamembrane domain. This indicates the existence of inside-out signaling in the EGF receptor system. The intracellular juxtamembrane domain has previously been shown to be required for the activation of the EGF receptor tyrosine kinase (Thiel, K. W., and Carpenter, G. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 19238–19243). Our experiments expand the role of this domain to include the allosteric control of ligand binding by the extracellular domain.

Mechanics of EGF Receptor/ErbB2 kinase activation revealed by luciferase fragment complementation imaging

Binding of EGF to its receptor induces dimerization of the normally monomeric receptor. Activation of its intracellular tyrosine kinase then occurs through the formation of an asymmetric kinase dimer in which one subunit, termed the “receiver” kinase, is activated by interaction with the other subunit, termed the “activator” kinase [Zhang, et al. (2006) Cell 125: 1137–1149]. Although there is significant experimental support for this model, the relationship between ligand binding and the mechanics of kinase activation are not known. Here we use luciferase fragment complementation in EGF receptor (EGFR)/ErbB2 heterodimers to probe the mechanics of ErbB kinase activation. Our data support a model in which ligand binding causes the cis-kinase (the EGFR) to adopt the receiver position in the asymmetric dimer and to be activated first. If the EGF receptor is kinase active, this results in the phosphorylation of the trans-kinase (ErbB2). However, if the EGF receptor kinase is kinase dead, the ErbB2 kinase is never activated. Thus, activation of the kinases in the EGFR/ErbB2 asymmetric dimer occurs in a specific sequence and depends on the kinase activity of the EGF receptor.

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation

Background: The EGF receptor has multiple ligands that can induce different biological effects. Results: EGF and TGFα, but not BTC or AREG, bias the EGF receptor toward heterodimer formation with ErbB2. AREG induces dimerization with different kinetics than the other ligands. Conclusion: EGFR ligands differentially induce receptor dimerization. Significance: This contributes to the biological differences elicited by different ligands. The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers.

Dynamic analysis of the epidermal growth factor (EGF) receptor-ErbB2-ErbB3 protein network by luciferase fragment complementation imaging.

Background: The EGF receptor, ErbB2, and ErbB3 can interact to form dimers. Results: Analyses of these interactions show that ErbB3 interacts strongly with ErbB2 but weakly with the EGF receptor. All ErbB receptor interactions are enhanced by erlotinib and lapatinib. Conclusion: ErbB3 binds and is functionally affected by tyrosine kinase inhibitors. Significance: Tyrosine kinase inhibitors restructure the network of ErbB interactions. ErbB3 is a member of the ErbB family of receptor tyrosine kinases. It is unique because it is the only member of the family whose kinase domain is defective. As a result, it is obliged to form heterodimers with other ErbB receptors to signal. In this study, we characterized the interaction of ErbB3 with the EGF receptor and ErbB2 and assessed the effects of Food and Drug Administration-approved therapeutic agents on these interactions. Our findings support the concept that ErbB3 exists in preformed clusters that can be dissociated by NRG-1β and that it interacts with other ErbB receptors in a distinctly hierarchical fashion. Our study also shows that all pairings of the EGF receptor, ErbB2, and ErbB3 form ligand-independent dimers/oligomers. The small-molecule tyrosine kinase inhibitors erlotinib and lapatinib differentially enhance the dimerization of the various ErbB receptor pairings, with the EGFR/ErbB3 heterodimer being particularly sensitive to the effects of erlotinib. The data suggest that the physiological effects of these drugs may involve not only inhibition of tyrosine kinase activity but also a dynamic restructuring of the entire network of receptors.

Different epidermal growth factor receptor (EGFR) agonists produce unique signatures for the recruitment of downstream signaling proteins

The EGF receptor can bind seven different agonist ligands. Although each agonist appears to stimulate the same suite of downstream signaling proteins, different agonists are capable of inducing distinct responses in the same cell. To determine the basis for these differences, we used luciferase fragment complementation imaging to monitor the recruitment of Cbl, CrkL, Gab1, Grb2, PI3K, p52 Shc, p66 Shc, and Shp2 to the EGF receptor when stimulated by the seven EGF receptor ligands. Recruitment of all eight proteins was rapid, dose-dependent, and inhibited by erlotinib and lapatinib, although to differing extents. Comparison of the time course of recruitment of the eight proteins in response to a fixed concentration of each growth factor revealed differences among the growth factors that could contribute to their differing biological effects. Principal component analysis of the resulting data set confirmed that the recruitment of these proteins differed between agonists and also between different doses of the same agonist. Ensemble clustering of the overall response to the different growth factors suggests that these EGF receptor ligands fall into two major groups as follows: (i) EGF, amphiregulin, and EPR; and (ii) betacellulin, TGFα, and epigen. Heparin-binding EGF is distantly related to both clusters. Our data identify differences in network utilization by different EGF receptor agonists and highlight the need to characterize network interactions under conditions other than high dose EGF.

The membrane-proximal intracellular domain of the epidermal growth factor receptor underlies negative cooperativity in ligand binding.

Background: The EGF receptor exhibits negative cooperativity in ligand binding. Results: Mutation of the most membrane-proximal portion of the intracellular domain abrogates negative cooperativity. Conclusion: Residues 645–665 of the EGF receptor are involved in the genesis of negative cooperativity. Significance: These data demonstrate how alterations of the intracellular domain of the EGF receptor can lead to changes in ligand binding by the extracellular domain. The binding of EGF induces dimerization of its receptor, leading to the stimulation of its intracellular tyrosine kinase activity. Kinase activation occurs within the context of an asymmetric dimer in which one kinase domain serves as the activator for the other kinase domain but is not itself activated. How ligand binding is related to the formation and dynamics of this asymmetric dimer is not known. The binding of EGF to its receptor is negatively cooperative—that is, EGF binds with lower affinity to the second site on the dimer than to the first site on the dimer. In this study, we analyzed the binding of 125I-EGF to a series of EGF receptor mutants in the intracellular juxtamembrane domain and demonstrate that the most membrane-proximal portion of this region plays a significant role in the genesis of negative cooperativity in the EGF receptor. The data are consistent with a model in which the binding of EGF to the first site on the dimer induces the formation of one asymmetric kinase dimer. The binding of EGF to the second site is required to disrupt the initial asymmetric dimer and allow the formation of the reciprocal asymmetric dimer. Thus, some of the energy of binding to the second site is used to reorient the first asymmetric dimer, leading to a lower binding affinity and the observed negative cooperativity.

Quantitation of the Effect of ErbB2 on Epidermal Growth Factor Receptor Binding and Dimerization

Background: ErbB2 is the preferred dimerization partner for the epidermal growth factor (EGF) receptor. Results: Heterodimerization with ErbB2 increases the affinity of the EGFR for EGF and increases the level of dimers maintained at any given concentration of EGF. Conclusion: ErbB2 modulates EGF receptor affinity and dimer stability. Significance: This study elucidates the molecular basis for the enhanced binding of EGF to EGFR/ErbB2 heterodimers. The epidermal growth factor (EGF) receptor is a member of the ErbB family of receptors that also includes ErbB2, ErbB3, and ErbB4. These receptors form homo- and heterodimers in response to ligand with ErbB2 being the preferred dimerization partner. Here we use 125I-EGF binding to quantitate the interaction of the EGF receptor with ErbB2. We show that the EGFR/ErbB2 heterodimer binds EGF with a 7-fold higher affinity than the EGFR homodimer. Because it cannot bind a second ligand, the EGFR/ErbB2 heterodimer is not subject to ligand-induced dissociation caused by the negatively cooperative binding of EGF to the second site on the EGFR homodimer. This increases the stability of the heterodimer relative to the homodimer and is associated with enhanced and prolonged EGF receptor autophosphorylation. These effects are independent of the kinase activity of ErbB2 but require back-to-back dimerization of the EGF receptor with ErbB2. Back-to-back dimerization is also required for phosphorylation of ErbB2. These findings provide a molecular explanation for the apparent preference of the EGF receptor for dimerizing with ErbB2 and suggest that the phosphorylation of ErbB2 occurs largely in the context of the EGFR/ErbB2 heterodimer, rather than through lateral phosphorylation of isolated ErbB2 subunits.

Palmitoylation of the EGF Receptor Impairs Signal Transduction and Abolishes High-Affinity Ligand Binding †

The intracellular juxtamembrane domain of the EGF receptor has been shown to be involved in the stimulation of the receptors tyrosine kinase activity. To further explore the function of this portion of the EGF receptor, a consensus site for protein palmitoylation was inserted at the beginning of the juxtamembrane domain of the receptor. The altered EGF receptor incorporated [(3)H]palmitate, demonstrating that it was palmitoylated. Compared to the wild-type EGF receptor, the palmitoylated EGF receptor was significantly impaired in EGF-stimulated receptor autophosphorylation as well as ligand-induced receptor internalization. While both the wild-type and the palmitoylated EGF receptors exhibited a similar propensity to associate with lipid rafts, only the wild-type receptor exited lipid rafts in response to EGF. Binding of [(125)I]EGF to the wild-type EGF receptor showed a curvilinear Scatchard plot with both high- and low-affinity forms of the receptor. By contrast, the palmitoylated receptor exhibited only low-affinity EGF binding. These data suggest that the cytoplasmic juxtamembrane domain is involved not only in the transmission of the proliferative signal generated by ligand binding but also in facilitating the adoption of the high-affinity conformation by the extracellular ligand binding domain.

Asp-960/Glu-961 Controls the Movement of the C-terminal Tail of the Epidermal Growth Factor Receptor to Regulate Asymmetric Dimer Formation

The epidermal growth factor (EGF) receptor is a tyrosine kinase that dimerizes in response to ligand binding. Ligand-induced dimerization of the extracellular domain of the receptor promotes formation of an asymmetric dimer of the intracellular kinase domains, leading to stimulation of the tyrosine kinase activity of the receptor. We recently monitored ligand-promoted conformational changes within the EGF receptor in real time using luciferase fragment complementation imaging and showed that there was significant movement of the C-terminal tail of the EGF receptor following EGF binding (Yang, K. S., Ilagan, M. X. G., Piwnica-Worms, D., and Pike, L. J. (2009) J. Biol. Chem. 284, 7474–7482). To investigate the structural basis for this conformational change, we analyzed the effect of several mutations on the kinase activity and luciferase fragment complementation activity of the EGF receptor. Mutation of Asp-960 and Glu-961, two residues at the beginning of the C-terminal tail, to alanine resulted in a marked enhancement of EGF-stimulated kinase activity as well as enhanced downstream signaling. The side chain of Asp-960 interacts with that of Ser-787. Mutation of Ser-787 to Phe, which precludes this interaction, also leads to enhanced receptor kinase activity. Our data are consistent with the hypothesis that Asp-960/Glu-961 represents a hinge or fulcrum for the movement of the C-terminal tail of the EGF receptor. Mutation of these residues destabilizes this hinge, facilitating the formation of the activating asymmetric dimer and leading to enhanced receptor signaling.

Epidermal growth factor receptors containing a single tyrosine in their C-terminal tail bind different effector molecules and are signaling-competent

The EGF receptor is a classic receptor tyrosine kinase. It contains nine tyrosines in its C-terminal tail, many of which are phosphorylated and bind proteins containing SH2 or phosphotyrosine-binding (PTB) domains. To determine how many and which tyrosines are required to enable EGF receptor-mediated signaling, we generated a series of EGF receptors that contained only one tyrosine in their C-terminal tail. Assays of the signaling capabilities of these single-Tyr EGF receptors indicated that they can activate a range of downstream signaling pathways, including MAP kinase and Akt. The ability of the single-Tyr receptors to signal correlated with their ability to bind Gab1 (Grb2-associated binding protein 1). However, Tyr-992 appeared to be almost uniquely required to observe activation of phospholipase Cγ. These results demonstrate that multiply phosphorylated receptors are not required to support most EGF-stimulated signaling but identify Tyr-992 and its binding partners as a unique node within the network. We also studied the binding of the isolated SH2 domain of Grb2 (growth factor receptor-bound protein 2) and the isolated PTB domain of Shc (SHC adaptor protein) to the EGF receptor. Although these adapter proteins bound readily to wild-type EGF receptor, they bound poorly to the single-Tyr EGF receptors, even those that bound full-length Grb2 and Shc well. This suggests that in addition to pTyr-directed associations, secondary interactions between the tail and regions of the adapter proteins outside of the SH2/PTB domains are important for stabilizing the binding of Grb2 and Shc to the single-Tyr EGF receptors.