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 the apical surface of thyroid cells, binds thyroglobulin (Tg) with high affinity in solid phase assays. Megalin-bound Tg was releasable by heparin. Here we show that Fisher rat thyroid (FRTL-5) cells, a differentiated rat thyroid cell line, can bind and endocytose Tg via megalin. We first demonstrated that FRTL-5 cells express megalin in a thyroid-stimulating hormone-dependent manner. Evidence of Tg binding to megalin on FRTL-5 cells and on an immortalized rat renal proximal tubule cell line (IRPT cells), was obtained by incubating the cells with 125I-Tg, followed by chemical cross-linking and immunoprecipitation of 125I-Tg with antibodies against megalin. To investigate cell binding further, we developed an assay in which cells were incubated 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 phase experiments designed to illuminate the mechanism of heparin release, we 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 (monoclonal antibody against megalin), indicating that much of the Tg released by heparin had been bound to megalin ( approximately 60-80%). The amount inhibited by RAP was considered to represent specific binding to megalin, which was saturable and of high affinity (Kd approximately 11.2 nM). Tg endocytosis by FRTL-5 and IRPT cells was demonstrated in experiments in which cells were incubated with unlabeled Tg at 37 degrees C, followed by heparin to remove cell-bound Tg. The amount of Tg internalized (measured by ELISA in the cell lysates) was reduced by RAP and 1H2, indicating that Tg endocytosis is partially mediated by megalin.
Marinò
M
M
Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.
Zheng
G
G
McCluskey
R T
RT
D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis; D006493:Heparin / Q000378:metabolism; D026541:Heymann Nephritis Antigenic Complex; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011916:Rats, Inbred F344; D013954:Thyroglobulin / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism*; D013972:Thyrotropin / Q000378:metabolism
D026541:Heymann Nephritis Antigenic Complex; D008562:Membrane Glycoproteins; D013972:Thyrotropin; D006493:Heparin; D013954:Thyroglobulin