Effect of brefeldin A and lysosomotropic reagents on intracellular trafficking of epidermal growth factor and transferrin in Madin-Darby canine kidney epithelial cells

Abstract

To regulate intracellular sorting of epidermal growth factor (EGF) or transferrin (Tf), the effect of brefeldin A (BFA) or lysosomotropic reagents was investigated. To examine the effect of them on the net transcellular transport of 125I-EGF or 125I-Tf, their transcytosis was investigated in the presence or absence of reagents. For the investigation of their fate after internalization, radiolabeled ligands were internalized at 37°C, followed by extensive washing and subsequent incubation at 37°C in the ligand-free medium (pulse-chase study). BFA enhanced transcytosis of 125I-Tf, but had no effect on 125I-EGF. Kinetic analysis in the pulse-chase study showed that BFA does not affect cell-surface binding or intracellular sorting of EGF, while it only increases the transcytosis rate constant of Tf. From the lysosomotropic reagents study, both ammonium chloride and monensin suppressed transcytosis and recycling as well as the degradation of EGF, while both chloroquine and bafilomycin A selectively suppressed the degradation process with only a minimal effect on transcytosis, resulting in an increase in the amount transcytosed. It is concluded the that enhancement effect of BFA on transcytosis depends upon the type of receptor targeted. Lysosomotropic reagents can be divided into two types as far as the specificity of the effect on the net amount of EGF transcytosed in Madin-Darby canine kidney (MDCK) cells is concerned.