Document Detail


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

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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