pH-dependent receptor/ligand dissociation as a determining factor for intracellular sorting of ligands for epidermal growth factor receptors in rat hepatocytes

Both epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) bind to EGF receptors and TGFalpha has been reported to be more potent than EGF as far as many biological effects are concerned. One possible reason for this is thought to be the difference in their dissociation from the receptors in intracellular acidic compartments, which may affect the final pathway (lysosomal degradation or recycling to cell-surface) of endocytosed ligands. This study was aimed at clarifying the relationship between intracellular dissociation from the receptors and the fate of the endocytosed ligands. First, I-125-human EGF (hEGF), mouse EGF (mEGF), or human TGFalpha (hTGFalpha) was prebound to cell-surface receptors or intracellularly preloaded, followed by further incubation at 37 degreesC in primary cultured rat hepatocytes. In these experiments, the magnitude of the dissociation rate constant (k(off)) of each ligand at pH 6.0, which is similar to that inside early endosomes, was found to be in the following order: hTGFalpha>mEGF>hEGF. The recycled portion of endocytosed ligands was also in the order: hTGFalpha>mEGF>hEGF. Digitonin treatment of preloaded cells revealed that the intracellular dissociation of hTGFalpha was more rapid than that of hEGF. Moreover, several histidine-inserted or -deleted hEGF mutants were prebound to rat liver sinusoidal membrane vesicle, followed by further incubation at 37 degreesC. The dissociation rate of histidine-deleted hEGF mutants was less rapid than that of hEGF itself. These results suggested that efficient dissociation in the earlier intracellular compartment leads the endocytosed ligands to be recycled to the cell surface whereas late dissociation results in intracellular degradation in hepatocytes. Thus, one possible strategy to improve their stability in the circulation may be a change in intracellular ligand/receptor dissociation with a minimal effect on the affinity for receptors on the cell-surface and histidine residues may partly contribute to the pH-sensitive dissociation. (C) 2002 Elsevier Science B.V. All rights reserved.