Megalin-mediated transcytosis of thyroglobulin by thyroid cells is a calmodulin-dependent process

Megalin, a multiligand receptor expressed on the apical surface of thyroid cells, mediates transepithelial transport (transcytosis) of thyroglobulin (Tg) across thyrocytes, resulting in diversion of Tg from the lysosomal pathway and reduction of the extent of thyroid hormone release from internalized Tg molecules. The calcium regulatory protein calmodulin facilitates some forms of transcytosis. Here we investigated the role of calmodulin in megalin-mediated transcytosis of Tg by thyroid cells. For this purpose, we studied the effect of calmodulin antagonists on Tg transcytosis by Fisher rat thyroid cells (FRTL-5), an established, differentiated thyroid cell line. FRTL-5 cells were cultured on permeable filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper surface. Unlabeled Tg was added to the upper chamber at 37 degrees C, and transcytosed Tg was detected by enzyme-linked immunosorbent assay (ELISA) in fluids collected 1 hour later from the lower chamber. To study the role of calmodulin in Tg transcytosis, cells were preincubated with one of two calmodulin antagonists, either trifluoperazine or W7. Both antagonists markedly reduced transcytosis of Tg by FRTL-5 cells. These inhibitory effects and those of a monoclonal antimegalin antibody were not additive, indicating that calmodulin acts on the megalin-mediated pathway. Furthermore, trifluoperazine increased the extent of triiodothyronine (T3) release from exogenously added Tg by FRTL-5 cells, indicating that Tg transported in the calmodulin-dependent, megalin-mediated pathway, bypasses the lysosomal pathway.