Intracellular Trafficking of Epidermal Growth Factor Family Ligands Is Directly Influenced by the pH Sensitivity of the Receptor/Ligand Interaction

Abstract

Using members of the epidermal growth factor (EGF) family as well as site-directed recombinant human EGF mutants, we investigated how ligand binding properties influence endosomal sorting. Mouse EGF (mEGF), human EGF (hEGF), and transforming growth factor alpha (TGF alpha) bind to the human EGF receptor (EGFR) with similar affinities at pH 7.4. However, the binding properties of these ligands have substantially different pH sensitivities resulting in varying degrees of dissociation from the receptors at lower pH levels characteristic of endosomes. We employed a steady-state sorting assay to determine the fraction of ligand sorted to recycling versus degradation as a function of the number of intracellular ligand molecules in mouse B82 fibroblasts. mEGF, hEGF, and TGF alpha display significantly different steady-state endosomal sorting patterns which correspond to the extent of their dissociation at endosomal pH. Moreover, several recombinant hEGF mutants with differing affinities exhibit altered endosomal sorting compared to hEGF, demonstrating a similar direct relationship between ligand binding properties and endosomal sorting outcomes. Intracellular trafficking of the EGF ligands was also monitored by measuring the observed degradation rate constants. These likewise show marked differences that correlate with the differing pH sensitivities of the ligands' binding properties.