Targeting of thyroglobulin to transcytosis following megalin-mediated endocytosis: evidence for a preferential ph-independent pathway

Abstract TG internalized from the colloid by megalin, bypasses the lysosomal pathway and is transported across thyrocytes by transcytosis. Although most of the intracellular mechanisms responsible for targeting of ligands to transcytosis are unknown, for certain ligands a role of lysosomal pH has been established. Thus, ligands that undergo lysosomal degradation dissociate from their receptors due to the low pH of endosomes, whereas certain ligands that undergo transcytosis fail to dissociate because they bind to their receptors at acidic pH. Here we studied the role of pH in TG transcytosis. We first investigated the effect of pH on megalin binding to TG in solid phase assays and found that, although megalin bound to TG at various pH values (ranging from 4-8), optimal binding was seen at acidic pH (ranging from 4.5-6). We then studied the effect of chloroquine (CQ) and ammonium chloride (AC), which increase endosomal pH, on transcytosis of TG across Fisher rat thyroid (FRTL-5 cells). Transcytosis assays were performed using FRTL-5 cells cultured on filters in dual chambered devices, with megalin expression only on the upper surface of the layers. TG was added to the upper chamber and transcytosed TG was measured in fluids collected from the lower chamber after incubation at 37 C. Treatment of FRTL-5 cells with CQ or AC did not affect binding and uptake of TG, but it did reduce T3 release from exogenously added TG, used as a measure of TG degradation in the lysosomal pathway. Treatment with CQ or AC resulted in an increase of transcytosis of TG across FRTL-5 cells, but only to a minimal extent (15-20%). The effects of CQ or AC and those of a megalin competitor (the monoclonal antibody 1H2, which reduced transcytosis) were not additive, suggesting that CQ and AC act on the megalin-mediated pathway. In conclusion, because TG binding to megalin is greatest at acidic pH, it is possible that TG does not dissociate from megalin in the lysosomal pathway. However, the pH-dependence of TG binding to megalin does not account for much of transcytosis, which probably occurs largely because of other mechanisms of targeting.