Comprehensive Model for Epidermal Growth Factor Receptor Ligand Binding Involving Conformational States of the Extracellular and the Kinase Domains

Abstract

The epidermal growth factor (EGF) receptor (EGFR) undergoes ligand-dependent dimerization to initiate transmembrane signaling. Although crystallographic structures of the extracellular and kinase domains are available, ligand binding has not been quantitatively analyzed taking the influence of both domains into account. Here, we developed a model explicitly accounting for conformational changes of the kinase and extracellular domains, their dimerizations and ligand binding to monomeric and dimeric receptor species. The model was fitted to ligand binding data of suspended cells expressing receptors with active or inactive kinase conformations. Receptor dimers with inactive, symmetric configuration of the kinase domains exhibit positive cooperativity and very weak binding affinity for the first ligand, whereas dimers with active, asymmetric kinase dimers are characterized by negative cooperativity and subnanomolar binding affinity for the first ligand. The homodimerization propensity of EGFR monomers with active kinase domains is ∼100-times higher than that of dimers with inactive kinase domains. Despite this fact, constitutive, ligand-independent dimers are mainly generated from monomers with inactive kinase domains due to the excess of such monomers in the membrane. The experimental finding of increased positive cooperativity at high expression levels of EGFR was recapitulated by the model. Quantitative prediction of ligand binding to different receptor species revealed that EGF binds to receptor monomers and dimers in an expression-level dependent manner without significant recruitment of monomers to dimers upon EGF stimulation below the phase transition temperature of the membrane. Results of the fitting offer unique insight into the workings of the EGFR.

Different conformations of EGF-induced receptor dimers involved in signaling and internalization

“…Previously, agonist-induced conformational models of the EGFR 38, 39 and the role of agonist binding kinetics in ligand bias have been described 40, 41 . For TK inhibitors, such studies are less well known, whereas it could improve understanding of their mode of action.…”

It Takes More than Two to Tango: Complex, Hierarchal, and Membrane-Modulated Interactions in the Regulation of Receptor Tyrosine Kinases

“…The two major classes of these small molecule inhibitors differ in whether they recognize the active or inactive conformation of the KD [ 52 ]. Several studies found that type I inhibitors, stabilizing the active conformation of the kinase, increase the formation of EGFR dimers harboring inhibited KDs, while type II inhibitors, stabilizing the inactive kinase structure, inhibit homodimerization altogether [ 53 , 54 , 55 ]. However, lapatinib, a type II inhibitor, has been shown to latch ErbB2 into a conformation that predisposes it to form head-to-head KD dimers with ErbB3, which explains the unexpected synergy between this kinase inhibitor and heregulin in promoting cell proliferation [ 56 ].…”

“…Several studies reported negative cooperative EGF binding curves [ 63 , 64 ], and these studies have been rationalized by linking negative cooperativity to the apparent heterogeneity of EGF binding sites [ 65 ], and by the observation of a squeezed, restrained ligand-binding site in the unliganded receptor in a singly liganded dimer in the Drosophila EGFR [ 66 ]. However, several findings put the issue into a different perspective, including the significant structural differences between how human and Drosophila EGFRs are activated [ 36 ], the loss of negative cooperative binding in studies of isolated, human EGFR ECDs [ 67 ], and the repeated observation of positive, cooperative ligand binding to EGFR [ 53 , 68 , 69 , 70 ]. It has been proposed that the inactive and active conformations of the KD are coupled to the tethered and untethered structures of the ECD, respectively, assumed to display different cooperativities [ 70 ].…”

“…It has been proposed that the inactive and active conformations of the KD are coupled to the tethered and untethered structures of the ECD, respectively, assumed to display different cooperativities [ 70 ]. A comprehensive, quantitative model [ 53 ] invoked the formation of chains of preformed, unliganded dimers harboring symmetric kinase dimers [ 71 ] and their escape from these superclusters to explain positive cooperativity. This model also accounts for the expression level dependence of apparent cooperativity and for the existence of preformed dimers [ 53 ].…”

“…A comprehensive, quantitative model [ 53 ] invoked the formation of chains of preformed, unliganded dimers harboring symmetric kinase dimers [ 71 ] and their escape from these superclusters to explain positive cooperativity. This model also accounts for the expression level dependence of apparent cooperativity and for the existence of preformed dimers [ 53 ].…”