title: Megalin (gp330) is an endocytic receptor for thyroglobulin on cultured fisher rat thyroid cells.
abstract: 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.
authors.affiliation: Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.
authors.forename: M
authors.initials: M
authors.lastname: Marinò
authors.forename: G
authors.initials: G
authors.lastname: Zheng
authors.forename: R T
authors.initials: RT
authors.lastname: McCluskey
mesh_terms: 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
chemical_list: D026541:Heymann Nephritis Antigenic Complex; D008562:Membrane Glycoproteins; D013972:Thyrotropin; D006493:Heparin; D013954:Thyroglobulin