Megalin (gp330) is an endocytic receptor for thyroglobulin on cultured Fisher rat thyroid cells

We recently reported that megalin (gp330), an endocytic receptor found on t he apical surface of thyroid cells, binds thyroglobulin (Tg) with high affi nity in solid phase assays. Megalin-bound Tg was releasable by heparin, Her e we show that Fisher rat thyroid (FRTL-5) cells, a differentiated rat thyr oid cell line, can bind and endocytose Tg via megalin, We first demonstrate d that FRTL-5 cells express megalin in a thyroid-stimulating hormone-depend ent manner. Evidence of Tg binding to megalin on FRTL-5 cells and on an imm ortalized rat renal proximal tubule cell line (IRPT cells), was obtained by incubating the cells with I-125-Tg, followed by chemical cross-linking and immunoprecipitation of I-125-Tg with antibodies against megalin, To invest igate cell binding further, we developed an assay in which cells were incub ated with unlabeled Tg at 4 degrees C, followed by incubation with heparin, which released almost all of the cell-bound Tg into the medium. In solid p hase experiments designed to illuminate the mechanism of heparin release, w e demonstrated that Tg is a heparin-binding protein, as are several megalin ligands, The amount of Tg released by heparin from FRTL-5 and IRPT cells, measured by enzyme-linked immunosorbent assay (ELISA), was markedly reduced by two megalin competitors, receptor-associated protein (RAP) and 1H2 (mon oclonal antibody against megalin), indicating that much of the Tg released by heparin had been bound to megalin (similar to 60-80%). The amount inhibi ted by RAP was considered to represent specific binding to megalin, which w as saturable and of high affinity (K(d)similar to 11.2 nM), Tg endocytosis by FRTL-5 and IRPT cells was demonstrated in experiments in which cells wer e incubated with unlabeled Tg at 37 degrees C, followed by heparin to remov e cell-bound Tg, The amount of Tg internalized (measured by ELISA in the ce ll lysates) was reduced by RAP and 1H2, indicating that Tg endocytosis is p artially mediated by megalin.