The unliganded epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase, exists as an inactive transmembrane monomer; biochemical data indicate that ligand binding stimulates receptor dimerization and tyrosine autophosphorylation. Binding studies, however, have failed to show positive linkage between ligand binding and dimerization, as one would expect if ligand binding promoted dimer assembly. Macdonald-Obermann and Pike used a series of EGFR mutants stably expressed in Chinese hamster ovary (CHO) cells, which do not have endogenous EGFRs, to explore the basis for these apparently contradictory findings. Consistent with previous studies, they found that 125I-EGF binding to wild-type receptors failed to show positive linkage: The affinity of ligand binding to the unoccupied dimer was similar to that for the monomer. In experiments with a catalytically inactive mutant, however, or with a truncated mutant that could not undergo autophosophorylation in the C-terminal tail, EGF bound to the unoccupied dimer with much greater affinity than it did to the monomer. In all three cases, ligand binding showed negative cooperativity, so that EGF bound with lower affinity to the second site on the dimer than to the first. Analyses of two additional truncation mutants indicated that an intracellular juxtamembrane domain previously implicated in kinase activation was crucial for positive linkage and negative cooperativity of EGF binding, as did analysis of a mutant in which the juxtamembrane domain can undergo palmitoylation. The authors thus conclude that the unphosphorylated receptor does, in fact, show positive linkage between ligand binding and dimerization but that receptor activation and autophosphorylation affect binding affinity, masking this early linkage.
J. L. Macdonald-Obermann, L. J. Pike, The intracellular juxtamembrane domain of the epidermal growth factor (EGF) receptor is responsible for the allosteric regulation of EGF binding. J. Biol. Chem. 284, 13570–13576 (2009). [Abstract] [Full Text]