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 ( The EGF 2 receptor is a tyrosine kinase composed of an ϳ620-amino-acid extracellular domain that recognizes and binds EGF, a single pass ␣-helical transmembrane domain, and an intracellular tyrosine kinase domain, encompassing roughly residues 685-950 (1). In addition, the receptor contains an ϳ230-amino-acid-long C-terminal tail that contains the bulk of the sites of receptor autophosphorylation (2-4). An intracellular juxtamembrane domain of about 40 residues connects the transmembrane domain to the kinase domain and has been shown to be crucial in the allosteric activation of the EGF receptor kinase (5, 6).In the membrane, the EGF receptor exists as a monomer, but a wealth of data indicate that the binding of EGF induces the formation of EGF receptor dimers (7-10). Dimerization appears to be mediated in large part by the extracellular domain of the receptor, which is comprised of four subdomains, designated I through IV. X-ray crystallography data suggest that in the absence of ligand, the extracellular domain is held in a closed configuration through the interaction of loops or arms that extend from the backs of subdomains II and IV (11). Upon binding of EGF, this intramolecular tether is released, allowing the receptor to adopt an open conformation in which EGF is tightly bound between subdomains I and III. In this configuration, the "dimerization arm" that was previously involved in tethering the receptor closed mediates the formation of a backto-back EGF receptor dimer (12, 13).Analyses of the binding of 125 I-EGF to its receptor have invariably resulted in concave up Scatchard plots that have been interpreted as indicating the presence of two classes of EGF binding sites. However, we have recently used global analysis of the binding of 125 I-EGF to cells expressing increasing levels of EGF receptors to show that EGF binding is best described by a model involving negative cooperativity in an aggregating system (14) (see Fig. 6). Ligand binding is negatively cooperative if the binding of ligand to the first site on a dimer reduces the affinity of the ligand for binding to the second site on the dimer.The concept of cooperativity only applies to existing dimers. It does not relate to the effect of ligand on the assembly or disassembly of those dimers. The effect of ligand on the formation of receptor dimers is captured in the concept of linkage (15,16). If ligand binding is positively linked to dimer formation, then ligand promotes the assembly of receptor dimers. In a monomer-dimer equilibrium, positive linkage arises when a ligand binds with higher affinity to the first site on the dimer than to the monomer. Under these circumstances, the ligand will preferentially bind to the dimer, shifting the equilibrium in favor of the dimeric species. In the case of the EGF receptor, biochemical data suggest that EGF induces recept...

Background:The EGF receptor has multiple ligands that can induce different biological effects. Results: EGF and TGF␣, 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.

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.T he EGF receptor is a member of the ErbB family of receptor tyrosine kinases that also includes ErbB2, ErbB3, and ErbB4 (1, 2). All ErbB receptors contain an extracellular ligand-binding domain, a single pass transmembrane domain, an intracellular tyrosine kinase, and a C-terminal tail (3). The EGF receptor, ErbB3, and ErbB4 are activated through the binding of a family of homologous ligands (4). Unique among the ErbB receptors, ErbB2 has no known ligand (5, 6).Binding of an ErbB ligand to its receptor induces dimerization of the receptor through interaction of the extracellular domains. Essentially all combinations of ErbB receptors are possible. However, the ligandless ErbB2 appears to be the preferred heterodimerization partner among ErbB receptors (7,8).Ligand-induced dimerization of the extracellular domains leads to the activation of the intracellular tyrosine kinase through the formation of an asymmetric kinase dimer (9-11). In the asymmetric dimer, the C lobe of the activator kinase interacts with the N lobe of the receiver kinase in a manner similar to that in which cyclin A interacts with cyclin-dependent kinase (12). This interaction leads to the activation of the receiver kinase, which then phosphorylates the C-terminal tail of the activator kinase.Although a substantial body of evidence supports this model for EGF receptor kinase activation (9-11, 13), it is not clear how the binding of ligand to the extracellular domain directs the assembly of the intracellular asymmetric kinase dimer. In particular, if ligand binds to one subunit in an ErbB dimer, which kinase domain adopts the activator and which adopts the receiver position in the asymmetric dimer, or is the choice made randomly? Once formed, does the asymmetric kinase dimer readily shift from one configuration to the reciprocal one, activating each kinase in turn, or is this a con...

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, TGF␣, 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.

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.