Megalin (gp330) Is an Endocytic Receptor for Thyroglobulin on Cultured Fisher Rat Thyroid Cells
1999; Elsevier BV; Volume: 274; Issue: 18 Linguagem: Inglês
10.1074/jbc.274.18.12898
ISSN1083-351X
AutoresMichele Marinò, Gang Zheng, Robert T. McCluskey,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoWe 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 with125I-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 °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 (∼60–80%). The amount inhibited by RAP was considered to represent specific binding to megalin, which was saturable and of high affinity (K d∼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 °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. 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 with125I-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 °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 (∼60–80%). The amount inhibited by RAP was considered to represent specific binding to megalin, which was saturable and of high affinity (K d∼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 °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. Thyroglobulin (Tg) 1The abbreviations used are: Tg, thyroglobulin; LDL, low density lipoprotein; TSH, thyroid-stimulating hormone; RAP, receptor-associated protein; GST, glutathione S-transferase; FRTL-5 cells, Fisher rat thyroid cells; CHO cells, Chinese hamster ovary cells; IRPT cells, immortalized rat proximal tubule cells; OVA, ovalbumin; LRP, low density lipoprotein receptor-related protein; ELISA, enzyme-linked immunosorbent assay; ALP, alkaline phosphatase; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter; TBS, Tris-buffered saline1The abbreviations used are: Tg, thyroglobulin; LDL, low density lipoprotein; TSH, thyroid-stimulating hormone; RAP, receptor-associated protein; GST, glutathione S-transferase; FRTL-5 cells, Fisher rat thyroid cells; CHO cells, Chinese hamster ovary cells; IRPT cells, immortalized rat proximal tubule cells; OVA, ovalbumin; LRP, low density lipoprotein receptor-related protein; ELISA, enzyme-linked immunosorbent assay; ALP, alkaline phosphatase; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter; TBS, Tris-buffered salineis synthesized in thyrocytes and released into the follicle lumen, where it is stored as the major component of the colloid (1Dunn A. Braverman L.E. Utiger R.D. Werner and Ingebar's The Thyroid, A Fundamental And Clinical Text. 7th Ed. Lippincott-Raven, Philadelphia1996: 81-95Google Scholar, 2Rousset B. Mornex R. Mol. Cell. Endocrinol. 1991; 78: 89-93Crossref PubMed Scopus (37) Google Scholar). Post-transitional modifications of Tg that occur mainly at the cell-colloid interface lead to forms that are iodine-rich and that contain the thyroid hormones T4 and T3 (mature Tg). Hormone secretion requires uptake of Tg by thyrocytes, with transport to lysosomes, where proteolytic cleavage leads to release of the hormones from mature Tg molecules (1Dunn A. Braverman L.E. Utiger R.D. Werner and Ingebar's The Thyroid, A Fundamental And Clinical Text. 7th Ed. Lippincott-Raven, Philadelphia1996: 81-95Google Scholar). Internalization of Tg may result from pseudopod ingestion under certain circumstances, such as intense, acute stimulation by the thyrotropic hormone (TSH), but micropinocytosis (vesicular internalization) is thought to be the usual route (1Dunn A. Braverman L.E. Utiger R.D. Werner and Ingebar's The Thyroid, A Fundamental And Clinical Text. 7th Ed. Lippincott-Raven, Philadelphia1996: 81-95Google Scholar, 2Rousset B. Mornex R. Mol. Cell. Endocrinol. 1991; 78: 89-93Crossref PubMed Scopus (37) Google Scholar, 3Bernier-Valentin F. Kostrouch Z. Rabilloud R. Rousset B. Endocrinology. 1991; 129: 2194-2201Crossref PubMed Scopus (38) Google Scholar). There is evidence that micropinocytosis of Tg can take place both by nonselective fluid phase uptake and receptor-mediated endocytosis, but the relative importance of these two mechanisms is uncertain (1Dunn A. Braverman L.E. Utiger R.D. Werner and Ingebar's The Thyroid, A Fundamental And Clinical Text. 7th Ed. Lippincott-Raven, Philadelphia1996: 81-95Google Scholar). Although evidence has been obtained of low affinity receptors for Tg on thyroid cells, a receptor capable of mediating Tg endocytosis has not been fully characterized (1Dunn A. Braverman L.E. Utiger R.D. Werner and Ingebar's The Thyroid, A Fundamental And Clinical Text. 7th Ed. Lippincott-Raven, Philadelphia1996: 81-95Google Scholar, 2Rousset B. Mornex R. Mol. Cell. Endocrinol. 1991; 78: 89-93Crossref PubMed Scopus (37) Google Scholar, 3Bernier-Valentin F. Kostrouch Z. Rabilloud R. Rousset B. Endocrinology. 1991; 129: 2194-2201Crossref PubMed Scopus (38) Google Scholar, 4Consiglio E. Salvatore G. Rall J.E. Khon L.D. J. Biol. Chem. 1979; 254: 5065-5076Abstract Full Text PDF PubMed Google Scholar, 5Consiglio E. Shifrin S. Yavin Z. Ambesi-Impiombato F.S. Rall J.E. Salvatore G. Khon L.D. J. Biol. Chem. 1981; 256: 10592-10599Abstract Full Text PDF PubMed Google Scholar, 6Roitt I.M. Pujol-Borrel R. Hanafusa T. Delves P.J. Bottazzo G.F. Khon L.D. Clin. Exp. Immunol. 1984; 56: 129-134PubMed Google Scholar, 7Miquelis R. Alquier C. Monsigny M. J. Biol. Chem. 1987; 262: 15291-15298Abstract Full Text PDF PubMed Google Scholar, 8Kostrouch Z. Bernier-Valentin F. Munari-Silem Y. Rajas F. Rabilloud R. Rousset B. Endocrinology. 1993; 132: 2645-2653Crossref PubMed Scopus (34) Google Scholar, 9Bernier-Valentin F. Kostrouch Z. Rabilloud R. Munari-Silem Y. Rousset B. J. Biol. Chem. 1990; 265: 17373-17380Abstract Full Text PDF PubMed Google Scholar, 10Giraud A. Siffroi S. Lanet J. Franc J.L. Endocrinology. 1997; 138: 2325-2332Crossref PubMed Scopus (24) Google Scholar, 11Lemansky P. Herzog V. Eur. J. Biochem. 1992; 209: 111-119Crossref PubMed Scopus (41) Google Scholar, 12Mziaut H. Bastiani P. Balivet T. Papandreou M.J. Fert V. Erregragui K. Blanck O. Miquelis R. Endocrinology. 1996; 137: 1370-1376Crossref PubMed Scopus (13) Google Scholar). We have previously obtained evidence suggesting that megalin (gp330) may function as a receptor for Tg (13Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (69) Google Scholar). Megalin (gp330) is a member of the LDL receptor family (14Raychowdhury R. Niles J.L. McCluskey R.T. Smith J.A. Science. 1989; 244: 1163-1165Crossref PubMed Scopus (210) Google Scholar, 15Saito A. Pietromonaco S. Loo A.K.C. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9725-9729Crossref PubMed Scopus (480) Google Scholar) and has been shown to bind multiple, unrelated ligands and to mediate endocytosis of ligands via coated pits, leading to delivery of ligands to lysosomes, where degradation occurs (16Willnow T.E. Goldstein J.L. Orth K. Brown M.S. Herz J. J. Biol. Chem. 1992; 267: 26172-26180Abstract Full Text PDF PubMed Google Scholar, 17Moestrup S.K. Nielsen S. Andreasen P. Jørgensen K.E. Nykjær A. Røigaard H. Gliemann J. Christensen E.I. J. Biol. Chem. 1993; 268: 16564-16570Abstract Full Text PDF PubMed Google Scholar, 18Stefansson S. Kounnas M.Z. Henkin J. Mallampalli R.K. Chappell D.A. Strickland D.K. Argraves W.S. J. Cell Sci. 1995; 108: 2361-2368Crossref PubMed Google Scholar). In immunohistochemical studies, megalin has been found principally on the apical surface of a restricted group of absorptive epithelial cells, including renal proximal tubule cells, epididymal cells, type II pneumocytes, and thyroid epithelial cells (19Zheng G. Bachinsky D.R. Stamenkovic I. Strickland D.K. Brown D. Andres G. McCluskey R.T. J. Histochem. Cytochem. 1994; 42: 531-542Crossref PubMed Scopus (280) Google Scholar, 20Lundgren S. Carling T. Hjalm G. Juhlin C. Rastad J. Pihlgren U. Rask L. Akerstrom G. Hellman P. J. Histochem. Cytochem. 1997; 45: 383-392Crossref PubMed Scopus (172) Google Scholar). Based on the assumption that physiological ligands of megalin may be identified by consideration of the composition of fluids to which it is exposed in various organs (21Zheng G. Bachinsky D. Abbate M. Andres G. Brown D. Stamenkovic I. Niles J.L. McCluskey R.T. Ann. N. Y. Acad. Sci. 1994; 737: 154-162Crossref PubMed Scopus (15) Google Scholar), we postulated that megalin on thyrocytes serves as a receptor for Tg. In support of this possibility we demonstrated in solid phase assays that purified rat megalin binds to rat Tg with high affinity (13Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (69) Google Scholar). Binding was inhibited by several known competitors of megalin, including the receptor-associated protein (RAP), antibodies to megalin, and heparin, which in addition dissociated Tg bound to megalin (13Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (69) Google Scholar). In the present study we have investigated FRTL-5 cells to determine whether they express megalin and if they are capable of binding and internalizing Tg via megalin. FRTL-5 cells, an established rat thyroid cell line, exhibit a number of thyroid-specific functions in a TSH-dependent manner (22Ambesi-Impiombato F.S. Parks L.A.M. Coon H.G. Proc Natl. Acad. Sci. U. S. A. 1980; 77: 3455Crossref PubMed Scopus (972) Google Scholar,23Bidey S.P. Chiovato L. Day A. Turmaine M. Gould R.P. Ekins R.P. Marshall N.J. J. Endocrinol. 1984; 101: 269-276Crossref PubMed Scopus (43) Google Scholar). Here we show that FRTL-5 cells do express megalin and that they can bind and endocytose Tg via megalin. Tg was purified from rat thyroids by ammonium sulfate precipitation and column fractionation, as described previously (24Esquivel P.S. Rose N.R. Kong Y.M. J. Exp. Med. 1977; 145: 1250-1263Crossref PubMed Scopus (144) Google Scholar). The Tg preparations were analyzed by Western blotting, using a rabbit anti-human Tg antibody cross-reactive with Tg from other species (Axle-Westbury, NY). Two bands were seen at about 330 and 660 kDa, corresponding to the monomeric and the dimeric forms of Tg. The RAP was used in the form of a glutathione S-transferase (GST) fusion protein. DH5α bacteria harboring the pGEX-RAP expression construct were kindly provided by Dr. Joachim Herz (University of Texas Southern Medical Center, Dallas, TX). The production of RAP-GST and GST was performed as described (25Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar). Heparin (Sigma) was used because it inhibits Tg binding to megalin in solid phase assays (13Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (69) Google Scholar), as occurs for binding of other megalin ligands (26Kounnas M.Z. Stefansson S. Loukinova E. Argraves K.M. Strickland D.K. Argraves W.S. Ann. N. Y. Acad. Sci. 1994; 737: 114-123Crossref PubMed Scopus (44) Google Scholar). Furthermore, heparin dissociates Tg bound to megalin (13Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (69) Google Scholar) and releases ligands that are bound to members of the LDL receptor family (27Goldstein L.J. Basu S.K. Brunschede G.Y. Brown M.S. Cell. 1976; 7: 85-95Abstract Full Text PDF PubMed Scopus (356) Google Scholar). A rabbit antibody, designated A55, against immunoaffinity purified megalin was described previously (28Gutmann E.J. Niles J.L. McCluskey R.T. Brown D. Am. J. Physiol. 1989; 257: C397-C407Crossref PubMed Google Scholar, 29Raychowdhury R. Zheng G. Brown D. McCluskey R.T. Am. J. Pathol. 1996; 148: 1613-1623PubMed Google Scholar). A previously described mouse anti-megalin monoclonal antibody, designated 1H2, has been shown to react with ectodomain epitopes in the second cluster of ligand binding repeats (29Raychowdhury R. Zheng G. Brown D. McCluskey R.T. Am. J. Pathol. 1996; 148: 1613-1623PubMed Google Scholar). A rabbit antibody against RAP-GST and a mouse antibody against the low density lipoprotein receptor-related protein (LRP) were previously described (19Zheng G. Bachinsky D.R. Stamenkovic I. Strickland D.K. Brown D. Andres G. McCluskey R.T. J. Histochem. Cytochem. 1994; 42: 531-542Crossref PubMed Scopus (280) Google Scholar). The above antibodies were used as purified IgG preparations unless otherwise specified. A goat antibody against GST was purchased from Amersham Pharmacia Biotech. Alkaline phosphatase (ALP)-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG were obtained from Bio-Rad. ALP-conjugated mouse anti-goat IgG was obtained from Axle. Horseradish peroxidase- and ALP-conjugated goat anti-mouse IgG were obtained from Sigma. Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG and goat anti-rabbit IgG were obtained from Cappel (Durham, NC). Biotin-labeled goat anti-mouse IgG was obtained from Vector (Burlingame, CA). 200 μg of rat Tg were radiolabeled with 125I-Na (NEN Life Science Products) using IODO beads (Pierce), according to the manufacturer's instructions. The specific activity of the preparations ranged from 1,500 to 7,000 cpm/ng. Fisher rat thyroid cells (FRTL-5) (American Type Culture Collection, Manassas, VA) were cultured as described previously (22Ambesi-Impiombato F.S. Parks L.A.M. Coon H.G. Proc Natl. Acad. Sci. U. S. A. 1980; 77: 3455Crossref PubMed Scopus (972) Google Scholar, 23Bidey S.P. Chiovato L. Day A. Turmaine M. Gould R.P. Ekins R.P. Marshall N.J. J. Endocrinol. 1984; 101: 269-276Crossref PubMed Scopus (43) Google Scholar) in Coon's F12 medium, supplemented with 5% fetal calf serum and with a mixture of six hormones, including TSH (10 milliunits/ml). In some experiments the medium was replaced with fresh medium lacking TSH for 24 h, after which TSH was sometimes readded. An immortalized rat renal proximal tubule cell line (IRPT), which expresses megalin but not LRP, was established as described previously (30Jung F.F. Bachinsky D.R. Tang S.S. Zheng G. Diamant D. Haveran L. McCluskey R.T. Ingelfinger J.R. Kid. Int. 1998; 53: 358-366Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 31Tang S.S. Jung F. Diamant D. Ingelfinger J. Exp. Nephrol. 1994; 2: 127PubMed Google Scholar). IRPT cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. CHO cells, a Chinese hamster ovary cell line that lacks megalin and expresses LRP (32Ji Z.S. Brecht W.J. Miranda R.D. Hussain M.H. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1993; 268: 10160-10167Abstract Full Text PDF PubMed Google Scholar), were cultured in Ham's medium supplemented with 10% fetal bovine serum. Cells were detached from the plates, washed with Tris buffered-saline (TBS), and incubated in plastic tubes for 1 h at 4 °C with the mouse monoclonal anti-megalin antibody (1H2), with the rabbit polyclonal anti-megalin antibody (A55), with the anti-LRP antibody (tissue culture supernatant), or, as controls, with purified mouse or rabbit IgG. Anti-megalin antibodies and control IgGs were used at a concentration of 20 μg/ml in binding buffer (TBS, 5 mm CaCl2, 0.5 mm MgCl2, 5% fetal bovine serum). After washing with TBS, FITC-conjugated rabbit anti-mouse IgG (1:250) or goat anti-rabbit IgG (1:1500) secondary antibodies were added for 1 h at 4 °C in binding buffer. Cells were then washed and analyzed by flow cytometry using a FACSCAN from Becton Dickinson (Mountain View, CA). Cell extracts were prepared with 1% Triton X-100, 1% deoxycholate (both from Fisher) and were briefly sonicated. The extracts were then subjected to SDS-polyacrylamide gel electrophoresis under nonreducing conditions and blotted onto nitrocellulose membranes, which were incubated with tissue culture supernatant containing the mouse monoclonal anti-megalin antibody (1H2) or with the rabbit polyclonal anti-megalin antibody (A55), followed by horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibodies. Bands were detected using a chemiluminescent substrate kit (Kirkegard & Perry Laboratories, Gaithersburg, MD). Cells were cultured on glass coverslips and fixed with 2% paraformaldehyde-l-lysine-sodium periodate. The preparations were blocked with avidin D (0.1 mg/ml in PBS) and incubated with tissue culture supernatant containing the mouse monoclonal anti-megalin antibody (1H2) followed by biotin-labeled goat anti-mouse IgG secondary antibody (1:200 in PBS) or with the secondary antibody alone. Staining was detected using the peroxidase labeled biotin-avidin complex (Vector). 3,3′-Diaminobenzidine (Sigma) was used for the color reaction. Cells were incubated with serum-free Dulbecco's modified Eagle's medium for 1 h at 37 °C and then blocked in 0.5% ovalbumin (OVA, Sigma), 25 mm Tris, 150 mm NaCl, 5 mmCaCl2, 0.5 mm MgCl2, pH 8.0, for 1 h at 4 °C. Cells were then incubated with 10 nm of 125I-Tg for 4 h at 4 °C in blocking buffer, followed by cross-linking with the homobifunctional cross-linker 3,3′-dithiobis[sulfosuccinimidyl propionate] (Pierce) (0.5 mm in PBS) for 30 min at 4 °C. After washing with TBS, cells were lysed with 1% Triton X-100, 1% deoxycholate and briefly sonicated. Cell lysates (500 μg) were immunoprecipitated with the rabbit anti-megalin antibody (A55 anti-serum, 1:200) or with the rabbit anti-human Tg antibody (1:200), or, as control, with normal rabbit serum, using protein A beads (Pierce). The precipitates were separated by 4–16% SDS-polyacrylamide gel electrophoresis under nonreducing conditions and analyzed by autoradiography. Radioactivity in the cell lysates and in the precipitates was counted with a γ counter. Cells were cultured in 96-well tissue culture plates until 80–100% confluence was reached. Cells were incubated with unlabeled Tg or, as controls, with RAP-GST or GST in Coon's F12 medium containing 5 mmCaCl2, 0.5 mm MgCl2, and 0.5% OVA. For inhibition experiments, FRTL-5 and IRPT cells were incubated with Tg alone, or in the presence of megalin inhibitors, namely RAP-GST (200 μg/ml) or 1H2 (200 μg/ml) or, as controls, with GST (200 μg/ml) or normal mouse IgG (200 μg/ml). After 4 h of incubation at 4 °C, the cells were washed twice with ice-cold PBS to remove nonspecifically bound proteins and then incubated for 1 h at 4 °C with ice-cold heparin (10–100 units/ml in PBS), to release receptor-bound proteins from the cell surface. In certain experiments, to measure total cell-bound Tg, cell lysates were prepared by treating the cells with ice-cold H2O on ice, immediately after incubation with Tg at 4 °C. The amounts of Tg, RAP-GST or GST in the heparin wash or in the cell lysates were measured by ELISA. For this purpose, 96-well microtiter plates were coated overnight at 4 °C with the heparin wash or with the cell lysates, blocked with bovine serum albumin (Sigma) and incubated with rabbit anti-human Tg (1:500), rabbit anti-RAP (20 μg/ml), or goat anti-GST (1:1000) antibodies, followed by ALP-conjugated goat anti-rabbit IgG (1:3000) or mouse anti-goat IgG (1:2500) secondary antibodies. After incubation withp-nitrophenyl-phosphate (Sigma), absorbance at 405 nm was determined with an El–311 ELISA microplate reader. The amount of Tg, RAP-GST, or GST was calculated using a standard obtained by coating the wells with 10 μg/ml of purified Tg, RAP-GST, or GST and was normalized for the total amount of protein in the cell lysates, measured with a commercial kit (Bio-Rad). 96-well microtiter plates were coated overnight at 4 °C with rat Tg or, as control, with OVA, at a concentration of 100 μg/ml in PBS. After blocking with bovine serum albumin, plates were incubated for 3 h at room temperature with a biotin-labeled heparin-albumin complex (Sigma) or, as control, with biotin-labeled albumin (Sigma) (in PBS, 0.05% Tween 20, 0.5% bovine serum albumin), followed by ALP-conjugated streptavidin (Vector, 1:3000) andp-nitrophenyl-phosphate. Absorbance was determined at 405 nm. For inhibition experiments, biotin-labeled heparin-albumin was added to the wells alone or in the presence of unlabeled heparin (500 units/ml). Cells were seeded in 96-well tissue culture plates and cultured until 80–100% confluence was reached. Cells were then incubated at 37 °C with unlabeled rat Tg or, as controls, with unlabeled RAP-GST or GST in Coon's F12 medium containing 5 mm CaCl2, 0.5 mmMgCl2, and 0.5% OVA. For inhibition experiments, Tg (10 μg/ml) was added to the plates together with one of the following: RAP-GST (200 μg/ml), 1H2 (200 μg/ml), or, as controls, GST (200 μg/ml) or normal mouse IgG (200 μg/ml). After 6 h of incubation the cells were washed twice with PBS and incubated with ice-cold heparin, as described above, to remove cell surface bound proteins, which were measured by ELISA. The cells were then lysed to measure internalized Tg, RAP-GST, or GST, using deionized H2O on ice. The amount of cell protein was measured in an aliquot of the cell lysate. Internalized Tg, RAP-GST or GST were measured in the cell lysates by ELISA, as described above. As shown in Fig.1 A, FRTL-5 cells were found to express megalin by FACS analysis, using either the mouse monoclonal (1H2) or the rabbit polyclonal (A55) anti-megalin antibodies. In addition, immunoperoxidase staining with 1H2 demonstrated surface megalin on FRTL-5 cells (Fig. 1 B). Megalin was also detected in FRTL-5 cell extracts by Western blotting (Fig. 1 C). In agreement with previous reports (30Jung F.F. Bachinsky D.R. Tang S.S. Zheng G. Diamant D. Haveran L. McCluskey R.T. Ingelfinger J.R. Kid. Int. 1998; 53: 358-366Abstract Full Text PDF PubMed Scopus (25) Google Scholar, 31Tang S.S. Jung F. Diamant D. Ingelfinger J. Exp. Nephrol. 1994; 2: 127PubMed Google Scholar), IRPT cells were found to express megalin by FACS analysis, whereas megalin was not detected on CHO cells (not shown). Because LRP binds many of the same ligands as megalin, we studied its expression on FRTL-5 cells by FACS analysis and, as shown in Fig. 1 D, no LRP was found. In a previous study we showed that IRPT cells do not express surface LRP (30Jung F.F. Bachinsky D.R. Tang S.S. Zheng G. Diamant D. Haveran L. McCluskey R.T. Ingelfinger J.R. Kid. Int. 1998; 53: 358-366Abstract Full Text PDF PubMed Scopus (25) Google Scholar). To investigate whether megalin expression by FRTL-5 cells is regulated by TSH, cells were cultured for 24 h in medium lacking TSH, sometimes followed by 48 h of culture in fresh medium containing TSH. As shown in Fig. 2, megalin expression was reduced at 24 h after TSH deprivation, as assessed by FACS analysis. The staining for megalin ranged from 46 to 69% of the staining before TSH deprivation, depending on the antibody used. Readdition of TSH resulted in increased megalin expression, reaching 88–90% of the levels found before TSH deprivation. The results indicate that TSH is required for high levels of megalin expression on FRTL-5 cells. In experiments designed to obtain evidence of Tg binding to cell surface megalin, FRTL-5 and IRPT cells were incubated with 125I-Tg at 4 °C, followed by cross-linking and immunoprecipitation with anti-Tg or anti-megalin antibodies. As shown in Fig.3 A, both anti-Tg and anti-megalin antibodies produced a high molecular mass band at the same size, indicating Tg bound to megalin on the cell surface. Another band at approximately 50 kDa was produced by the anti-megalin antibody and not by the anti-Tg antibody. The identity of this product is unknown. As shown in Fig. 3 B, the proportion of 125I-Tg precipitated by the rabbit anti-Tg antibody from FRTL-5 cells was 19.9% of the total amount of 125I-Tg in the cell lysates, whereas that obtained from IRPT cells was 11.39%. The results are consistent with the presence of Tg receptors on thyroid cells in addition to megalin, as suggested by previous studies (1Dunn A. Braverman L.E. Utiger R.D. Werner and Ingebar's The Thyroid, A Fundamental And Clinical Text. 7th Ed. Lippincott-Raven, Philadelphia1996: 81-95Google Scholar, 2Rousset B. Mornex R. Mol. Cell. Endocrinol. 1991; 78: 89-93Crossref PubMed Scopus (37) Google Scholar, 3Bernier-Valentin F. Kostrouch Z. Rabilloud R. Rousset B. Endocrinology. 1991; 129: 2194-2201Crossref PubMed Scopus (38) Google Scholar, 4Consiglio E. Salvatore G. Rall J.E. Khon L.D. J. Biol. Chem. 1979; 254: 5065-5076Abstract Full Text PDF PubMed Google Scholar, 5Consiglio E. Shifrin S. Yavin Z. Ambesi-Impiombato F.S. Rall J.E. Salvatore G. Khon L.D. J. Biol. Chem. 1981; 256: 10592-10599Abstract Full Text PDF PubMed Google Scholar, 6Roitt I.M. Pujol-Borrel R. Hanafusa T. Delves P.J. Bottazzo G.F. Khon L.D. Clin. Exp. Immunol. 1984; 56: 129-134PubMed Google Scholar, 7Miquelis R. Alquier C. Monsigny M. J. Biol. Chem. 1987; 262: 15291-15298Abstract Full Text PDF PubMed Google Scholar, 8Kostrouch Z. Bernier-Valentin F. Munari-Silem Y. Rajas F. Rabilloud R. Rousset B. Endocrinology. 1993; 132: 2645-2653Crossref PubMed Scopus (34) Google Scholar, 9Bernier-Valentin F. Kostrouch Z. Rabilloud R. Munari-Silem Y. Rousset B. J. Biol. Chem. 1990; 265: 17373-17380Abstract Full Text PDF PubMed Google Scholar, 10Giraud A. Siffroi S. Lanet J. Franc J.L. Endocrinology. 1997; 138: 2325-2332Crossref PubMed Scopus (24) Google Scholar, 11Lemansky P. Herzog V. Eur. J. Biochem. 1992; 209: 111-119Crossref PubMed Scopus (41) Google Scholar, 12Mziaut H. Bastiani P. Balivet T. Papandreou M.J. Fert V. Erregragui K. Blanck O. Miquelis R. Endocrinology. 1996; 137: 1370-1376Crossref PubMed Scopus (13) Google Scholar). The proportion of 125I-Tg precipitated by the anti-megalin antibody was 8.48% from FRTL-5 cells and 11.81% from IRPT cells. We previously showed that heparin dissociates Tg from megalin in solid phase assays (13Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (69) Google Scholar). Here we studied the ability of heparin to dissociate Tg bound to megalin on cells, to be used as a measure of cell bound Tg. Cells were incubated with unlabeled Tg at 4 °C, followed by incubation with heparin. In addition, some cells were incubated with RAP (used as a RAP-GST fusion protein), as a positive megalin binding control, or with GST, as a negative control, before heparin treatment. The amounts of Tg, RAP-GST, and GST in the heparin wash were measured by ELISA. As shown in Fig. 4 A, heparin was found to release Tg and RAP-GST from FRTL-5 and IRPT cells. Although FRTL-5 cells are capable of synthesizing Tg (22Ambesi-Impiombato F.S. Parks L.A.M. Coon H.G. Proc Natl. Acad. Sci. U. S. A. 1980; 77: 3455Crossref PubMed Scopus (972) Google Scholar, 23Bidey S.P. Chiovato L. 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