Abstract
TG internalized from the colloid by megalin, bypasses the lysosomal pathway and is transported across thyrocytes by transcytosis. Although most of the intracellular mechanisms responsible for targeting of ligands to transcytosis are unknown, for certain ligands a role of lysosomal pH has been established. Thus, ligands that undergo lysosomal degradation dissociate from their receptors due to the low pH of endosomes, whereas certain ligands that undergo transcytosis fail to dissociate because they bind to their receptors at acidic pH. Here we studied the role of pH in TG transcytosis. We first investigated the effect of pH on megalin binding to TG in solid phase assays and found that, although megalin bound to TG at various pH values (ranging from 4–8), optimal binding was seen at acidic pH (ranging from 4.5–6). We then studied the effect of chloroquine (CQ) and ammonium chloride (AC), which increase endosomal pH, on transcytosis of TG across Fisher rat thyroid (FRTL-5 cells). Transcytosis assays were performed using FRTL-5 cells cultured on filters in dual chambered devices, with megalin expression only on the upper surface of the layers. TG was added to the upper chamber and transcytosed TG was measured in fluids collected from the lower chamber after incubation at 37 C. Treatment of FRTL-5 cells with CQ or AC did not affect binding and uptake of TG, but it did reduce T3 release from exogenously added TG, used as a measure of TG degradation in the lysosomal pathway. Treatment with CQ or AC resulted in an increase of transcytosis of TG across FRTL-5 cells, but only to a minimal extent (15–20%). The effects of CQ or AC and those of a megalin competitor (the monoclonal antibody 1H2, which reduced transcytosis) were not additive, suggesting that CQ and AC act on the megalin-mediated pathway. In conclusion, because TG binding to megalin is greatest at acidic pH, it is possible that TG does not dissociate from megalin in the lysosomal pathway. However, the pH-dependence of TG binding to megalin does not account for much of transcytosis, which probably occurs largely because of other mechanisms of targeting.
Similar content being viewed by others
References
Marinò M., McCluskey R.T. Role of thyroglobulin endocytic pathways in the control of thyroid hormone release. Am. J. Physiol. Cell Physiol. 2000, 279: C1295–C1306.
Rousset B., Mornex R. The thyroid hormone secretory pathway-current dogmas and alternative hypotheses. Mol. Cell Endocrinol. 1991, 78: 89–93.
Montuori N., Pacifico F., Mellone S. et al. The rat asialoglycoprotein receptor binds the amino-terminal domain of thyroglobulin. Biochem. Biophys. Res. Commun. 2000, 268: 42–46.
Marinò M., Pinchera A., McCluskey R.T., Chiovato L. Megalin in thyroid physiology and Pathology. Thyroid 2001, 11: 47–56.
Marinò M., Zheng G., Chiovato L. et al. Role of megalin (gp330) in transcytosis of thyroglobulin by thyroid cells: a novel function in the control of thyroid hormone release. J. Biol. Chem. 2000, 275: 7125–7138.
Raychowdhury R., Niles J.L., McCluskey R.T., Smith. J.A. Autoimmune target in Heymann nephritis is a glycoprotein with homology to the LDL receptor. Science 1989, 244: 1163–1165.
Saito A., Pietromonaco S., Loo A.K.C., Farquhar M.G. Complete cloning and sequencing of rat gp330/“megalin,” a distinctive member of the low density lipoprotein receptor gene family. Proc. Natl. Acad. Sci. USA 1994, 91: 9725–9729.
Zheng G., Bachinsky D.R., Stamenkovic I. et al. Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/a2MR, and the receptor-associated protein (RAP). J. Histochem. Cytochem. 1994, 42: 531–542.
Lundgren S., Carling T., Hjalm G. et al. Tissue distribution of human gp330/megalin, a putative Ca2+-sensing protein. J. Histochem. Cytochem. 1997, 45: 383–392.
Marinò M., Zheng G., McCluskey R.T. Megalin (gp330) is an endocytic receptor for thyroglobulin on cultured thyroid cells (FRTL-5 cells). J. Biol. Chem. 1999, 27: 12898–12904.
Zheng G., Marinò M., Zhao J., McCluskey R.T. Megalin (gp330): a putative endocytic receptor for thyroglobulin (TG). Endocrinology 1998, 139: 1462–1465.
Marinò M., Chiovato L., Latrofa F. et al. Circulating thyroglobulin derived from transcytosis is combined with a secretory component of its endocytic receptor megalin. J. Clin. Endocrinol. Metab. 2000, 85: 3458–3467.
Mostov K.E., Verges M., Altschuler Y. Membrane traffic in polarized epithelial cells. Curr. Op. Cell Biol. 2000, 12: 483–490.
Casanova J.E., Wang X., Kumar R. et al. Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells. Mol. Biol. Cell. 1999, 10: 47–61.
Van IJzendoorn S.C., Tuvim M.J., Weimbs T., Dickey B.F., Mostov K.E. Direct interaction between Rab3b and the polymeric immunoglobulin receptor controls ligand-stimulated transcytosis in epithelial cells. Dev. Cell. 2002, 2: 219–228.
Marinò M., Friedlander J.A., McCluskey R.T., Andrews D. Identification of a heparin-binding region of rat thyroglobulin involved in megalin binding. J. Biol. Chem. 1999, 274: 30377–30386.
Raychowdhury R., Zheng G., Brown D., McCluskey R.T. Induction of Heymann nephritis with a gp330/megalin fusion protein. Am. J. Pathol. 1996,: 1613–1623.
Moestrup S.K., Verroust P.J. Megalin- and cubilin-mediated endocytosis of protein-bound vitamins, lipids, and hormones in polarized epithelia. Ann. Rev. Nutr. 2001, 21: 407–428.
Herz J., Goldstein J.L., Strickland D.K., Ho Y.K., Brown M.S. 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/ a2-macroglobulin receptor. J. Biol. Chem. 1991, 266: 21232–21238.
Ambesi-Impiombato F.S., Parks L.A., Coon H.G. Culture of hormone-dependent functional epithelial cells from rat thyroids. Proc. Natl. Acad. Sci. USA 1980, 77: 3455–3459.
Tasevski V., Benn D., Peters G., Luttrell B., Simpson A. The Fischer rat thyroid cell line FRTL-5 exhibits a nondiploid karyotype. Thyroid 1998, 8: 623–626.
Zimmermann-Belsing T., Rasmussen A.K, Feldt-Rasmussen U. Lack of thyroglobulin production supports the finding of the FRTL-5 cells with a nondiploid karyotype. Thyroid 1999, 9: 519–520.
Marinò M., McCluskey R.T. Megalin-mediated transcytosis of thyroglobulin by thyroid cells is a calmodulin-dependent process. Thyroid 2000, 10: 461–499.
Consiglio E., Salvatore G., Rall J.E., Khon L.D. Thyroglobulin interactions with thyroid plasma membranes. The existence of specific receptors and their potential role. J. Biol. Chem. 1979, 254: 5065–5076.
Marinò M., Chiovato L., Lisi S., Pinchera A., McCluskey R.T. Phosphoinositide 3-kinase inhibits megalin-mediated transcytosis of thyroglobulin across thyroid epithelial cells at a post-sorting level. Eur. J. Endocrinol. 2001, 145: 477–483.
Brix K., Lemansky P., Herzog V. Evidence for extracellularly acting cathepsins mediating thyroid hormone liberation in thyroid epithelial cells. Endocrinology 1996, 137: 1963–1974.
Homewood C.A., Warhurst D.C., Peters W., Baggaley V.C. Lysosomes, pH and the anti-malarial action of chloroquine. Nature 1972, 235: 50–52.
Dautry-Varsat A. Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor. Biochimie 1986, 68: 375–381.
Marinò M., Andrews D., Brown D., McCluskey R.T. Transcytosis of retinol-binding protein across renal proximal tubule cells after megalin (gp 330)-mediated endocytosis. J. Am. Soc. Nephrol. 2001, 12: 637–648.
Dehouck B., Fenart L., Dehouck M.P., Pierce A., Torpier G., Cecchelli R.A. New function for the LDL receptor: transcytosis of LDL across the blood-brain barrier. J. Cell Biol. 1997, 25: 877–889.
Fillebeen C., Descamps L., Dehouck M.P. et al. Receptormediated transcytosis of lactoferrin through the bloodbrain barrier. J. Biol. Chem. 1999, 12: 7011–7017.
Author information
Authors and Affiliations
Corresponding author
Additional information
This work was supported by the American Thyroid Association Research Grant 1999–2000 (to MM), by a Grant from MIUR (Italian Ministry of Education, University and Research, year 2001) (to AP and MM) and by a Grant from AIRC (Italian Association for Cancer Research, year 2001) (to AP and MM).
Rights and permissions
About this article
Cite this article
Marinò, M., Lisi, S., Pinchera, A. et al. Targeting of thyroglobulin to transcytosis following megalin-mediated endocytosis: Evidence for a preferential pH-independent pathway. J Endocrinol Invest 26, 222–229 (2003). https://doi.org/10.1007/BF03345161
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03345161