Intracellular retention of thyroglobulin in the absence of the low-density lipoprotein receptor-associated protein (RAP) is likely due to premature binding to megalin in the biosynthetic pathway

Objective: The low-density lipoprotein receptor associated protein (RAP) is expressed by thyroid epithelial cells (TEC) in a TSH-dependent manner. In the thyroid RAP functions as a molecular chaperone for the thyroglobulin (Tg) endocytic receptor megalin/LRP2, which is retained intracellularly in RAP KO mice rather than being expressed on the apical membrane of TEC, its usual location. RAP binds also to Tg, which is also retained intracellularly in RAP KO mice, thereby suggesting a role of RAP in Tg secretion. Here we investigated whether Tg intracellular retention in the absence of RAP is due to premature Tg-megalin interactions during the biosynthetic pathway or to a direct action of RAP on Tg secretion. Methods: We performed immunoprecipitation experiments in thyroid extracts from RAP KO and WT mice. In addition, we investigated Tg secretion in COS-7 cells co-transfected with human RAP (hRAP) and mouse Tg (mTg). Results: An anti-megalin megalin precipitated greater amounts of Tg in thyroid extracts from RAP KO than from WT mice, suggesting increased intracellular interactions between megalin and Tg in the absence of RAP. COS-7 cells transiently transfected with hRAP, mTg or both, expressed the two proteins accordingly. RAP was found almost exclusively in cell extracts, whereas Tg was found both in extracts and media, as expected from the knowledge that RAP is ER-resident and that Tg is secreted. Regardless of whether cells were transfected with mTg alone or were co-transfected with hRAP, similar proportions of the total Tg synthesized were detected in cell extracts and media. Conclusions: The intracellular retention of Tg in the absence of RAP is likely due to its premature interaction with megalin, whereas RAP does not seem to affect Tg secretion directly.