Intracellular retention of thyroglobulin in the absence of the low-density lipoprotein receptor-associated protein (RAP) is likely due to premature binding to megalin in the biosynthetic pathway.
OBJECTIVE
The low-density lipoprotein receptor associated protein (RAP) is expressed by thyroid epithelial cells (TEC) in a TSH-dependent manner. In the thyroid RAP functions as a molecular chaperone for the thyroglobulin (Tg) endocytic receptor megalin/LRP2, which is retained intracellularly in RAP KO mice rather than being expressed on the apical membrane of TEC, its usual location. RAP binds also to Tg, which is also retained intracellularly in RAP KO mice, thereby suggesting a role of RAP in Tg secretion. Here we investigated whether Tg intracellular retention in the absence of RAP is due to premature Tg-megalin interactions during the biosynthetic pathway or to a direct action of RAP on Tg secretion.


METHODS
We performed immunoprecipitation experiments in thyroid extracts from RAP KO and WT mice. In addition, we investigated Tg secretion in COS-7 cells co-transfected with human RAP (hRAP) and mouse Tg (mTg).


RESULTS
An anti-megalin megalin precipitated greater amounts of Tg in thyroid extracts from RAP KO than from WT mice, suggesting increased intracellular interactions between megalin and Tg in the absence of RAP. COS-7 cells transiently transfected with hRAP, mTg or both, expressed the two proteins accordingly. RAP was found almost exclusively in cell extracts, whereas Tg was found both in extracts and media, as expected from the knowledge that RAP is ER-resident and that Tg is secreted. Regardless of whether cells were transfected with mTg alone or were co-transfected with hRAP, similar proportions of the total Tg synthesized were detected in cell extracts and media.


CONCLUSIONS
The intracellular retention of Tg in the absence of RAP is likely due to its premature interaction with megalin, whereas RAP does not seem to affect Tg secretion directly.
Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy.|Neurobiology Laboratory of Biology, Scuola Normale Superiore, Piazza Dei Cavalieri 1, Pisa, Italy.
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S
Lisi
Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy.|DiaSorin S.p.A, Saluggia, Italy.
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R
Botta
Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy.
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G
Rotondo Dottore
Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy.
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M
Leo
Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy.
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F
Latrofa
Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy.
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P
Vitti
Department of Clinical and Experimental Medicine, Endocrinology Unit I, University of Pisa and University Hospital of Pisa, Via Paradisa 2, 56124, Pisa, Italy. michele.marino@med.unipi.it.
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M
Marinò
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D053898:Biosynthetic Pathways; D015153:Blotting, Western; D005260:Female; D006801:Humans; D026563:LDL-Receptor Related Protein-Associated Protein / Q000502:physiology*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D011485:Protein Binding; D013954:Thyroglobulin / Q000378:metabolism*
D015415:Biomarkers; D026563:LDL-Receptor Related Protein-Associated Protein; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D013954:Thyroglobulin
LDL receptor associated protein; Megalin; RAP; Thyroglobulin; Thyroid