| Megalin (gp330) is an endocytic receptor for thyroglobulin on cultured fisher rat thyroid cells. | |
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MedLine Citation:
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PMID: 10212279 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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M Marinò; G Zheng; R T McCluskey |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 274 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 1999 Apr |
Date Detail:
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Created Date: 1999-06-03 Completed Date: 1999-06-03 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 12898-904 Citation Subset: IM |
Affiliation:
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Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Cells, Cultured Endocytosis* Heparin / metabolism Heymann Nephritis Antigenic Complex Membrane Glycoproteins / metabolism* Protein Binding Rats Rats, Inbred F344 Thyroglobulin / metabolism* Thyroid Gland / cytology, metabolism* Thyrotropin / metabolism |
| Grant Support | |
ID/Acronym/Agency:
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DK 46301/DK/NIDDK NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Heymann Nephritis Antigenic Complex; 0/Membrane Glycoproteins; 9002-71-5/Thyrotropin; 9005-49-6/Heparin; 9010-34-8/Thyroglobulin |
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