Advertisement

Megalin (gp330) Is an Endocytic Receptor for Thyroglobulin on Cultured Fisher Rat Thyroid Cells*

  • Michele Marinò
    Footnotes
    Affiliations
    From the Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
    Search for articles by this author
  • Gang Zheng
    Affiliations
    From the Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
    Search for articles by this author
  • Robert T. McCluskey
    Correspondence
    To whom correspondence should be addressed: Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-5690; Fax: 617-726-5684;
    Affiliations
    From the Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129
    Search for articles by this author
  • Author Footnotes
    * This work was supported by NIDDK, National Institutes of Health Grant 46301.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Scholar of The Department of Endocrinology, University of Pisa, Italy.
Open AccessPublished:April 30, 1999DOI:https://doi.org/10.1074/jbc.274.18.12898
      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)
      The 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 saline
      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 saline
      is synthesized in thyrocytes and released into the follicle lumen, where it is stored as the major component of the colloid (
      • Dunn A.
      ,
      • Rousset B.
      • Mornex R.
      ). 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 (
      • Dunn A.
      ). 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 (
      • Dunn A.
      ,
      • Rousset B.
      • Mornex R.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Rousset B.
      ). 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 (
      • Dunn A.
      ). 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 (
      • Dunn A.
      ,
      • Rousset B.
      • Mornex R.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Rousset B.
      ,
      • Consiglio E.
      • Salvatore G.
      • Rall J.E.
      • Khon L.D.
      ,
      • Consiglio E.
      • Shifrin S.
      • Yavin Z.
      • Ambesi-Impiombato F.S.
      • Rall J.E.
      • Salvatore G.
      • Khon L.D.
      ,
      • Roitt I.M.
      • Pujol-Borrel R.
      • Hanafusa T.
      • Delves P.J.
      • Bottazzo G.F.
      • Khon L.D.
      ,
      • Miquelis R.
      • Alquier C.
      • Monsigny M.
      ,
      • Kostrouch Z.
      • Bernier-Valentin F.
      • Munari-Silem Y.
      • Rajas F.
      • Rabilloud R.
      • Rousset B.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Munari-Silem Y.
      • Rousset B.
      ,
      • Giraud A.
      • Siffroi S.
      • Lanet J.
      • Franc J.L.
      ,
      • Lemansky P.
      • Herzog V.
      ,
      • Mziaut H.
      • Bastiani P.
      • Balivet T.
      • Papandreou M.J.
      • Fert V.
      • Erregragui K.
      • Blanck O.
      • Miquelis R.
      ).
      We have previously obtained evidence suggesting that megalin (gp330) may function as a receptor for Tg (
      • Zheng G.
      • Marinò M.
      • Zhao J.
      • McCluskey R.T.
      ). Megalin (gp330) is a member of the LDL receptor family (
      • Raychowdhury R.
      • Niles J.L.
      • McCluskey R.T.
      • Smith J.A.
      ,
      • Saito A.
      • Pietromonaco S.
      • Loo A.K.C.
      • Farquhar M.G.
      ) 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 (
      • Willnow T.E.
      • Goldstein J.L.
      • Orth K.
      • Brown M.S.
      • Herz J.
      ,
      • Moestrup S.K.
      • Nielsen S.
      • Andreasen P.
      • Jørgensen K.E.
      • Nykjær A.
      • Røigaard H.
      • Gliemann J.
      • Christensen E.I.
      ,
      • Stefansson S.
      • Kounnas M.Z.
      • Henkin J.
      • Mallampalli R.K.
      • Chappell D.A.
      • Strickland D.K.
      • Argraves W.S.
      ). 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 (
      • Zheng G.
      • Bachinsky D.R.
      • Stamenkovic I.
      • Strickland D.K.
      • Brown D.
      • Andres G.
      • McCluskey R.T.
      ,
      • Lundgren S.
      • Carling T.
      • Hjalm G.
      • Juhlin C.
      • Rastad J.
      • Pihlgren U.
      • Rask L.
      • Akerstrom G.
      • Hellman P.
      ). 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 (
      • Zheng G.
      • Bachinsky D.
      • Abbate M.
      • Andres G.
      • Brown D.
      • Stamenkovic I.
      • Niles J.L.
      • McCluskey R.T.
      ), 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 (
      • Zheng G.
      • Marinò M.
      • Zhao J.
      • McCluskey R.T.
      ). 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 (
      • Zheng G.
      • Marinò M.
      • Zhao J.
      • McCluskey R.T.
      ). 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 (
      • Ambesi-Impiombato F.S.
      • Parks L.A.M.
      • Coon H.G.
      ,
      • Bidey S.P.
      • Chiovato L.
      • Day A.
      • Turmaine M.
      • Gould R.P.
      • Ekins R.P.
      • Marshall N.J.
      ). Here we show that FRTL-5 cells do express megalin and that they can bind and endocytose Tg via megalin.

      DISCUSSION

      In the present study we show that megalin is a receptor for Tg endocytosis on cultured thyroid cells. We first demonstrated that FRTL-5 cells, a well studied established rat thyroid cell line (
      • Ambesi-Impiombato F.S.
      • Parks L.A.M.
      • Coon H.G.
      ,
      • Bidey S.P.
      • Chiovato L.
      • Day A.
      • Turmaine M.
      • Gould R.P.
      • Ekins R.P.
      • Marshall N.J.
      ), express megalin when cultured in standard medium containing the thyrotropic hormone (TSH). As shown by FACS analysis and immunoperoxidase staining, megalin was present on the cell surface. FRTL-5 cells are known to maintain several specific thyroid functions, most of which are TSH-dependent (
      • Ambesi-Impiombato F.S.
      • Parks L.A.M.
      • Coon H.G.
      ,
      • Bidey S.P.
      • Chiovato L.
      • Day A.
      • Turmaine M.
      • Gould R.P.
      • Ekins R.P.
      • Marshall N.J.
      ). Therefore, the demonstration that megalin expression by FRTL-5 cells is TSH-dependent indirectly supports a thyroid-related function of megalin. In particular, because TSH provides a signal for Tg internalization (
      • Dunn A.
      ,
      • Rousset B.
      • Mornex R.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Rousset B.
      ), the finding suggests a role of megalin as an endocytic receptor.
      Direct evidence of binding of Tg to megalin on FRTL-5 cells as well as on IRPT cells, a rat renal proximal tubule cell line that expresses abundant megalin (
      • Jung F.F.
      • Bachinsky D.R.
      • Tang S.S.
      • Zheng G.
      • Diamant D.
      • Haveran L.
      • McCluskey R.T.
      • Ingelfinger J.R.
      ,
      • Tang S.S.
      • Jung F.
      • Diamant D.
      • Ingelfinger J.
      ), was provided by experiments in which cells were incubated with 125I-labeled Tg followed by cross-linking and incubation of the cell extracts with antibodies against megalin, which resulted in co-immunoprecipitation of125I-Tg. In other experiments, binding of Tg to megalin on FRTL-5 and IRPT cells was demonstrated by incubation of cells with unlabeled Tg at 4 °C, followed by treatment with heparin, which released Tg into the medium, as detected by ELISA. Because almost all (∼97%) of the total cell-bound Tg was released by heparin, the amount of heparin-releasable Tg can be considered as a measure of cell surface-bound Tg.
      We previously showed in solid phase experiments that heparin dissociates Tg from purified megalin (
      • Zheng G.
      • Marinò M.
      • Zhao J.
      • McCluskey R.T.
      ). Goldstein and colleagues (
      • Goldstein L.J.
      • Basu S.K.
      • Brunschede G.Y.
      • Brown M.S.
      ) had shown earlier that heparin releases the LDL from its receptor on cultured fibroblasts and used this finding to measure the amount of binding of LDL to the LDL receptor. However, it is known that heparin can release molecules not only from members of the LDL receptor family but also from heparan sulfate proteoglycans (
      • Saxena U.
      • Klein M.G.
      • Goldberg I.G.
      ), which are expressed in many cell types, including FRTL-5 cells (
      • Emoto N.
      • Isozaki O.
      • Ohmura E.
      • Tsushima T.
      • Shizume K.
      • Demura H.
      ). Nevertheless, in our experiments with FRTL-5 cells we obtained compelling evidence that most of the Tg released by heparin had been bound to megalin. Thus, when cells were incubated with Tg plus the monoclonal anti-megalin antibody 1H2 or RAP, there was roughly a 60–80% reduction in heparin releasable Tg as well as in total cell-bound Tg, which represents the proportion of megalin-bound Tg. 1H2 is entirely specific for megalin (
      • Raychowdhury R.
      • Zheng G.
      • Brown D.
      • McCluskey R.T.
      ), and, although RAP binds to certain other members of the LDL receptor family, notably LRP and the very low density lipoprotein receptor (
      • Willnow T.E.
      • Goldstein J.L.
      • Orth K.
      • Brown M.S.
      • Herz J.
      ,
      • Zheng G.
      • Bachinsky D.R.
      • Stamenkovic I.
      • Strickland D.K.
      • Brown D.
      • Andres G.
      • McCluskey R.T.
      ,
      • Mulder M.
      • Lombardi P.
      • Jansen H.
      • van Berkel T.J.C.
      • Frants R.R.
      • Havekes L.M.
      ,
      • Argraves K.M.
      • Battey F.D.
      • MacCalman C.D.
      • McCrae K.R.
      • Gafvels M.E.
      • Kozarsky K.F.
      • Chappell D.A.
      • Strauss III, J.F.
      • Strickland D.K.
      ,
      • Battey F.D.
      • Gafvels M.E.
      • FitzGerald D.J.
      • Argraves W.S.
      • Chappell D.A.
      • Strauss III, J.F.
      • Strickland D.K.
      ), these receptors are not expressed by thyroid epithelial cells or by renal proximal tubule cells in vivo(
      • Zheng G.
      • Bachinsky D.R.
      • Stamenkovic I.
      • Strickland D.K.
      • Brown D.
      • Andres G.
      • McCluskey R.T.
      ,
      • Weaver A.M.
      • Lysiak J.J.
      • Gonias S.L.
      ,
      • Takahashi S.
      • Kawarabayasi Y.
      • Nakai T.
      • Sakai J.
      • Yamamoto T.
      ). Furthermore, LRP is not expressed on FRTL-5 cells as shown here, nor as previously reported on IRPT cells (
      • Jung F.F.
      • Bachinsky D.R.
      • Tang S.S.
      • Zheng G.
      • Diamant D.
      • Haveran L.
      • McCluskey R.T.
      • Ingelfinger J.R.
      ). In addition, RAP does not bind appreciably to heparan sulfate proteoglycans nor inhibit ligand binding to heparan sulfate proteoglycans (
      • Mahley R.W.
      • Ji Z.S.
      • Brecht W.J.
      • Miranda R.D.
      • He D.
      ). Thus, in dealing with FRTL-5 or IRPT cells, inhibitory effects of RAP can be considered specific for megalin. Based on this, we showed that Tg binding to megalin on FRTL-5 cells is saturable and of high affinity (K d = ∼11.2 nm). However, the finding that binding not related to megalin (noninhibitable by RAP) also showed some degree of saturation supports the existence, in addition to megalin, of other Tg receptors on thyroid cells, as suggested in other studies (
      • Dunn A.
      ,
      • Rousset B.
      • Mornex R.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Rousset B.
      ,
      • Consiglio E.
      • Salvatore G.
      • Rall J.E.
      • Khon L.D.
      ,
      • Consiglio E.
      • Shifrin S.
      • Yavin Z.
      • Ambesi-Impiombato F.S.
      • Rall J.E.
      • Salvatore G.
      • Khon L.D.
      ,
      • Roitt I.M.
      • Pujol-Borrel R.
      • Hanafusa T.
      • Delves P.J.
      • Bottazzo G.F.
      • Khon L.D.
      ,
      • Miquelis R.
      • Alquier C.
      • Monsigny M.
      ,
      • Kostrouch Z.
      • Bernier-Valentin F.
      • Munari-Silem Y.
      • Rajas F.
      • Rabilloud R.
      • Rousset B.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Munari-Silem Y.
      • Rousset B.
      ,
      • Giraud A.
      • Siffroi S.
      • Lanet J.
      • Franc J.L.
      ,
      • Lemansky P.
      • Herzog V.
      ,
      • Mziaut H.
      • Bastiani P.
      • Balivet T.
      • Papandreou M.J.
      • Fert V.
      • Erregragui K.
      • Blanck O.
      • Miquelis R.
      ). This possibility is also supported by the finding that in co-immunoprecipitation experiments a higher proportion of Tg was precipitated by the anti-Tg antibody from FRTL-5 cells than from IRPT cells.
      Based on the finding that heparin dissociates Tg from megalin, we postulated that Tg is a heparin-binding protein, because megalin itself does not bind to heparin (
      • Kounnas M.Z.
      • Stefansson S.
      • Loukinova E.
      • Argraves K.M.
      • Strickland D.K.
      • Argraves W.S.
      ,
      • Farquhar M.G.
      • Kerjaschki D.
      • Lundstrom M.
      • Orlando R.A.
      ) and because several megalin ligands are heparin-binding proteins (
      • Kounnas M.Z.
      • Stefansson S.
      • Loukinova E.
      • Argraves K.M.
      • Strickland D.K.
      • Argraves W.S.
      ). Indeed, this prediction was confirmed by solid phase assays, which showed specific binding of Tg to heparin. This observation suggests that regions rich in positively charged amino acid residues (arginine and lysine) in the Tg molecule may contribute to its binding to megalin, as has been demonstrated for binding of certain other megalin ligands, including aprotinin and polybasic drugs (
      • Moestrup S.K.
      • Cui S.
      • Vorum H.
      • Bregengard C.
      • Bjørn C.E.
      • Norris K.
      • Gliemann J.
      • Christensen E.I.
      ).
      Experiments in which FRTL-5 and IRPT cells were incubated with unlabeled Tg at 37 °C, followed by heparin treatment to remove cell-bound Tg, showed that megalin can mediate Tg endocytosis. The detection of Tg in FRTL-5 cell lysates clearly showed that exogenous Tg had been internalized, because Tg of endogenous origin was considerably lower, as found in lysates from cells incubated in medium lacking Tg. Furthermore, the demonstration that almost all of the cell surface-bound Tg was released by heparin provides evidence that the amount of Tg found in the cell lysates represented only Tg that had been internalized. Moreover, because Tg was measured by ELISA, the amount of Tg internalized may have been underestimated, because Tg degradation during the course of the incubation should cause some loss of immunoreactivity.
      Inhibition experiments provided evidence that a certain amount of Tg uptake is mediated by megalin. Thus, internalization of Tg by FRTL-5 and IRPT cells was appreciably reduced when cells were co-incubated with exogenous Tg plus RAP or 1H2. Furthermore, we obtained evidence against the possibility that reduction of Tg uptake in FRTL-5 and IRPT cells by megalin competitors resulted merely from lowering the amount of Tg bound to the cells. Thus, the ratio of internalized Tg to cell surface bound Tg was reduced by RAP and 1H2.
      The inhibition of Tg uptake produced by megalin competitors in FRTL-5 cells was not complete (∼50%), suggesting that, in addition to megalin, other mechanisms are responsible for Tg endocytosis, as suggested by previous studies (
      • Dunn A.
      ,
      • Rousset B.
      • Mornex R.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Rousset B.
      ,
      • Consiglio E.
      • Salvatore G.
      • Rall J.E.
      • Khon L.D.
      ,
      • Consiglio E.
      • Shifrin S.
      • Yavin Z.
      • Ambesi-Impiombato F.S.
      • Rall J.E.
      • Salvatore G.
      • Khon L.D.
      ,
      • Roitt I.M.
      • Pujol-Borrel R.
      • Hanafusa T.
      • Delves P.J.
      • Bottazzo G.F.
      • Khon L.D.
      ,
      • Miquelis R.
      • Alquier C.
      • Monsigny M.
      ,
      • Kostrouch Z.
      • Bernier-Valentin F.
      • Munari-Silem Y.
      • Rajas F.
      • Rabilloud R.
      • Rousset B.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Munari-Silem Y.
      • Rousset B.
      ,
      • Giraud A.
      • Siffroi S.
      • Lanet J.
      • Franc J.L.
      ,
      • Lemansky P.
      • Herzog V.
      ,
      • Mziaut H.
      • Bastiani P.
      • Balivet T.
      • Papandreou M.J.
      • Fert V.
      • Erregragui K.
      • Blanck O.
      • Miquelis R.
      ,
      • Van Den Hove M.F.
      • Couvrer M.
      • De Visscher M.
      • Salvatore G.
      ). The finding that less inhibition of uptake was produced by megalin competitors in FRTL-5 cells than in IRPT cells (∼70%) suggests that the contribution of megalin to Tg uptake is greater in IRPT cells.
      As noted earlier, there is evidence that micropinocytosis of Tg by thyrocytes occurs both through fluid phase uptake and receptor-mediated endocytosis (
      • Dunn A.
      ,
      • Rousset B.
      • Mornex R.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Rousset B.
      ,
      • Consiglio E.
      • Salvatore G.
      • Rall J.E.
      • Khon L.D.
      ,
      • Consiglio E.
      • Shifrin S.
      • Yavin Z.
      • Ambesi-Impiombato F.S.
      • Rall J.E.
      • Salvatore G.
      • Khon L.D.
      ,
      • Roitt I.M.
      • Pujol-Borrel R.
      • Hanafusa T.
      • Delves P.J.
      • Bottazzo G.F.
      • Khon L.D.
      ,
      • Miquelis R.
      • Alquier C.
      • Monsigny M.
      ,
      • Kostrouch Z.
      • Bernier-Valentin F.
      • Munari-Silem Y.
      • Rajas F.
      • Rabilloud R.
      • Rousset B.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Munari-Silem Y.
      • Rousset B.
      ,
      • Giraud A.
      • Siffroi S.
      • Lanet J.
      • Franc J.L.
      ,
      • Lemansky P.
      • Herzog V.
      ,
      • Mziaut H.
      • Bastiani P.
      • Balivet T.
      • Papandreou M.J.
      • Fert V.
      • Erregragui K.
      • Blanck O.
      • Miquelis R.
      ). However, despite extensive investigations, a specific receptor shown to have a major role in Tg uptake by thyrocytes has not previously been characterized. Consiglio et al. (
      • Consiglio E.
      • Salvatore G.
      • Rall J.E.
      • Khon L.D.
      ,
      • Consiglio E.
      • Shifrin S.
      • Yavin Z.
      • Ambesi-Impiombato F.S.
      • Rall J.E.
      • Salvatore G.
      • Khon L.D.
      ) showed the existence of a specific binding site for asialogalacto-Tg in thyroid membrane preparations as well as in cultured thyrocytes, and this was confirmed by others (
      • Roitt I.M.
      • Pujol-Borrel R.
      • Hanafusa T.
      • Delves P.J.
      • Bottazzo G.F.
      • Khon L.D.
      ). The receptor was identified in porcine thyroid membranes as a 45-kDa protein, which was suggested to be involved mainly in Tg recycling (
      • Miquelis R.
      • Alquier C.
      • Monsigny M.
      ). More recently, Lemansky and Herzog (
      • Lemansky P.
      • Herzog V.
      ) performed a study using porcine thyroid follicles, designed to investigate the role of mannose-6-phosphate receptors in Tg endocytosis through interaction with mannose-6 recognition markers on Tg N-linked glycans. Although they failed to show that mannose-6-phosphate receptors are responsible for Tg endocytosis, they did obtain evidence of specific low affinity binding sites on the apical surface of thyrocytes involved in Tg endocytosis. However, the responsible receptor was not identified. In another study, Giraudet al. (
      • Giraud A.
      • Siffroi S.
      • Lanet J.
      • Franc J.L.
      ) obtained evidence of selective, moderately high affinity binding of Tg to thyrocytes in cultured “inside-out” porcine follicles, as well as to cultured CHO and Madin-Darby canine kidney cells from other tissues. The receptor was not isolated, but its pH dependence, its presence on CHO and Madin-Darby canine kidney cells, and its apparent recognition of anionic charges indicate that it is not megalin. One reason why megalin has not been found in thyroid cells in previous studies may be related to the fact that in most of those studies primary cultures of thyroid cells or tissues were used. In this regard, we have observed that primary cultures of rat renal proximal tubule cells cease to express megalin after several days (
      • Jung F.F.
      • Bachinsky D.R.
      • Tang S.S.
      • Zheng G.
      • Diamant D.
      • Haveran L.
      • McCluskey R.T.
      • Ingelfinger J.R.
      ). Furthermore, we have found that primary cultures of porcine thyroid cells or follicles cease to express megalin after a few days, although megalin is normally expressed on pig thyroid cells.
      M. Marinò, G. Zheng, and R. T. McCluskey, unpublished observations.
      In summary, evidence obtained in our previous (
      • Zheng G.
      • Marinò M.
      • Zhao J.
      • McCluskey R.T.
      ) and present studies supports the conclusion that megalin can function as a receptor on thyrocytes capable of mediating binding and uptake of Tg. Receptors other than megalin probably contribute to this process and fluid phase pinocytosis may play a major role (
      • Dunn A.
      ,
      • Rousset B.
      • Mornex R.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Rousset B.
      ,
      • Consiglio E.
      • Salvatore G.
      • Rall J.E.
      • Khon L.D.
      ,
      • Consiglio E.
      • Shifrin S.
      • Yavin Z.
      • Ambesi-Impiombato F.S.
      • Rall J.E.
      • Salvatore G.
      • Khon L.D.
      ,
      • Roitt I.M.
      • Pujol-Borrel R.
      • Hanafusa T.
      • Delves P.J.
      • Bottazzo G.F.
      • Khon L.D.
      ,
      • Miquelis R.
      • Alquier C.
      • Monsigny M.
      ,
      • Kostrouch Z.
      • Bernier-Valentin F.
      • Munari-Silem Y.
      • Rajas F.
      • Rabilloud R.
      • Rousset B.
      ,
      • Bernier-Valentin F.
      • Kostrouch Z.
      • Rabilloud R.
      • Munari-Silem Y.
      • Rousset B.
      ,
      • Giraud A.
      • Siffroi S.
      • Lanet J.
      • Franc J.L.
      ,
      • Lemansky P.
      • Herzog V.
      ,
      • Mziaut H.
      • Bastiani P.
      • Balivet T.
      • Papandreou M.J.
      • Fert V.
      • Erregragui K.
      • Blanck O.
      • Miquelis R.
      ,
      • Van Den Hove M.F.
      • Couvrer M.
      • De Visscher M.
      • Salvatore G.
      ). In any case, the ability of megalin to bind Tg with high affinity raises interesting questions about its function in vivo. High affinity receptors serve to mediate endocytosis of ligands that are generally present in low concentrations in extracellular fluids and thus serve to compete effectively with fluid phase uptake. However, Tg in the colloid is very highly concentrated (100–200 mg/ml), which is consistent with the notion that nonspecific fluid phase uptake is the major mechanism for Tg endocytosis and hormone release (
      • Dunn A.
      ). The function of a high affinity receptor for Tg on thyroid cells may therefore be to regulate the extent of endocytosis only under special circumstances. High affinity receptor binding should lead to an increase of Tg endocytosis, which would be expected to result in delivery to lysosomes, with hormone release. However, it is also possible that the receptor could divert Tg from the usual endocytic pathway, as through recycling or transcytosis. Further studies are needed to define the role of megalin in thyroid hormone release.

      Acknowledgments

      We are indebted to Dr. Ivan Stamenkovic and Dr. David Andrews for their critical reading of the manuscript and for helpful discussions.

      REFERENCES

        • Dunn A.
        Braverman L.E. Utiger R.D. Werner and Ingebar's The Thyroid, A Fundamental And Clinical Text. 7th Ed. Lippincott-Raven, Philadelphia1996: 81-95
        • Rousset B.
        • Mornex R.
        Mol. Cell. Endocrinol. 1991; 78: 89-93
        • Bernier-Valentin F.
        • Kostrouch Z.
        • Rabilloud R.
        • Rousset B.
        Endocrinology. 1991; 129: 2194-2201
        • Consiglio E.
        • Salvatore G.
        • Rall J.E.
        • Khon L.D.
        J. Biol. Chem. 1979; 254: 5065-5076
        • Consiglio E.
        • Shifrin S.
        • Yavin Z.
        • Ambesi-Impiombato F.S.
        • Rall J.E.
        • Salvatore G.
        • Khon L.D.
        J. Biol. Chem. 1981; 256: 10592-10599
        • Roitt I.M.
        • Pujol-Borrel R.
        • Hanafusa T.
        • Delves P.J.
        • Bottazzo G.F.
        • Khon L.D.
        Clin. Exp. Immunol. 1984; 56: 129-134
        • Miquelis R.
        • Alquier C.
        • Monsigny M.
        J. Biol. Chem. 1987; 262: 15291-15298
        • Kostrouch Z.
        • Bernier-Valentin F.
        • Munari-Silem Y.
        • Rajas F.
        • Rabilloud R.
        • Rousset B.
        Endocrinology. 1993; 132: 2645-2653
        • Bernier-Valentin F.
        • Kostrouch Z.
        • Rabilloud R.
        • Munari-Silem Y.
        • Rousset B.
        J. Biol. Chem. 1990; 265: 17373-17380
        • Giraud A.
        • Siffroi S.
        • Lanet J.
        • Franc J.L.
        Endocrinology. 1997; 138: 2325-2332
        • Lemansky P.
        • Herzog V.
        Eur. J. Biochem. 1992; 209: 111-119
        • Mziaut H.
        • Bastiani P.
        • Balivet T.
        • Papandreou M.J.
        • Fert V.
        • Erregragui K.
        • Blanck O.
        • Miquelis R.
        Endocrinology. 1996; 137: 1370-1376
        • Zheng G.
        • Marinò M.
        • Zhao J.
        • McCluskey R.T.
        Endocrinology. 1998; 139: 1462-1465
        • Raychowdhury R.
        • Niles J.L.
        • McCluskey R.T.
        • Smith J.A.
        Science. 1989; 244: 1163-1165
        • Saito A.
        • Pietromonaco S.
        • Loo A.K.C.
        • Farquhar M.G.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9725-9729
        • Willnow T.E.
        • Goldstein J.L.
        • Orth K.
        • Brown M.S.
        • Herz J.
        J. Biol. Chem. 1992; 267: 26172-26180
        • Moestrup S.K.
        • Nielsen S.
        • Andreasen P.
        • Jørgensen K.E.
        • Nykjær A.
        • Røigaard H.
        • Gliemann J.
        • Christensen E.I.
        J. Biol. Chem. 1993; 268: 16564-16570
        • Stefansson S.
        • Kounnas M.Z.
        • Henkin J.
        • Mallampalli R.K.
        • Chappell D.A.
        • Strickland D.K.
        • Argraves W.S.
        J. Cell Sci. 1995; 108: 2361-2368
        • Zheng G.
        • Bachinsky D.R.
        • Stamenkovic I.
        • Strickland D.K.
        • Brown D.
        • Andres G.
        • McCluskey R.T.
        J. Histochem. Cytochem. 1994; 42: 531-542
        • Lundgren S.
        • Carling T.
        • Hjalm G.
        • Juhlin C.
        • Rastad J.
        • Pihlgren U.
        • Rask L.
        • Akerstrom G.
        • Hellman P.
        J. Histochem. Cytochem. 1997; 45: 383-392
        • Zheng G.
        • Bachinsky D.
        • Abbate M.
        • Andres G.
        • Brown D.
        • Stamenkovic I.
        • Niles J.L.
        • McCluskey R.T.
        Ann. N. Y. Acad. Sci. 1994; 737: 154-162
        • Ambesi-Impiombato F.S.
        • Parks L.A.M.
        • Coon H.G.
        Proc Natl. Acad. Sci. U. S. A. 1980; 77: 3455
        • Bidey S.P.
        • Chiovato L.
        • Day A.
        • Turmaine M.
        • Gould R.P.
        • Ekins R.P.
        • Marshall N.J.
        J. Endocrinol. 1984; 101: 269-276
        • Esquivel P.S.
        • Rose N.R.
        • Kong Y.M.
        J. Exp. Med. 1977; 145: 1250-1263
        • Herz J.
        • Goldstein J.L.
        • Strickland D.K.
        • Ho Y.K.
        • Brown M.S.
        J. Biol. Chem. 1991; 266: 21232-21238
        • Kounnas M.Z.
        • Stefansson S.
        • Loukinova E.
        • Argraves K.M.
        • Strickland D.K.
        • Argraves W.S.
        Ann. N. Y. Acad. Sci. 1994; 737: 114-123
        • Goldstein L.J.
        • Basu S.K.
        • Brunschede G.Y.
        • Brown M.S.
        Cell. 1976; 7: 85-95
        • Gutmann E.J.
        • Niles J.L.
        • McCluskey R.T.
        • Brown D.
        Am. J. Physiol. 1989; 257: C397-C407
        • Raychowdhury R.
        • Zheng G.
        • Brown D.
        • McCluskey R.T.
        Am. J. Pathol. 1996; 148: 1613-1623
        • Jung F.F.
        • Bachinsky D.R.
        • Tang S.S.
        • Zheng G.
        • Diamant D.
        • Haveran L.
        • McCluskey R.T.
        • Ingelfinger J.R.
        Kid. Int. 1998; 53: 358-366
        • Tang S.S.
        • Jung F.
        • Diamant D.
        • Ingelfinger J.
        Exp. Nephrol. 1994; 2: 127
        • Ji Z.S.
        • Brecht W.J.
        • Miranda R.D.
        • Hussain M.H.
        • Innerarity T.L.
        • Mahley R.W.
        J. Biol. Chem. 1993; 268: 10160-10167
        • Limbird L.E.
        Cell Surface Receptor: A Short Course on Theory And Methods. 2nd Ed. Kluwer, Boston1996
        • Farquhar M.G.
        • Kerjaschki D.
        • Lundstrom M.
        • Orlando R.A.
        Ann. N. Y. Acad. Sci. 1994; 737: 96-113
        • Saxena U.
        • Klein M.G.
        • Goldberg I.G.
        J. Biol. Chem. 1990; 265: 12880-12886
        • Emoto N.
        • Isozaki O.
        • Ohmura E.
        • Tsushima T.
        • Shizume K.
        • Demura H.
        Endocrinol. Metab. 1994; 1: 123-130
        • Mulder M.
        • Lombardi P.
        • Jansen H.
        • van Berkel T.J.C.
        • Frants R.R.
        • Havekes L.M.
        J. Biol. Chem. 1993; 268: 9369-9375
        • Argraves K.M.
        • Battey F.D.
        • MacCalman C.D.
        • McCrae K.R.
        • Gafvels M.E.
        • Kozarsky K.F.
        • Chappell D.A.
        • Strauss III, J.F.
        • Strickland D.K.
        J. Biol. Chem. 1995; 270: 26550-26557
        • Battey F.D.
        • Gafvels M.E.
        • FitzGerald D.J.
        • Argraves W.S.
        • Chappell D.A.
        • Strauss III, J.F.
        • Strickland D.K.
        J. Biol. Chem. 1994; 269: 23268-23273
        • Weaver A.M.
        • Lysiak J.J.
        • Gonias S.L.
        J. Lipid Res. 1997; 38: 1841-1850
        • Takahashi S.
        • Kawarabayasi Y.
        • Nakai T.
        • Sakai J.
        • Yamamoto T.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9252-9256
        • Mahley R.W.
        • Ji Z.S.
        • Brecht W.J.
        • Miranda R.D.
        • He D.
        Ann. N. Y. Acad. Sci. 1994; 737: 39-52
        • Moestrup S.K.
        • Cui S.
        • Vorum H.
        • Bregengard C.
        • Bjørn C.E.
        • Norris K.
        • Gliemann J.
        • Christensen E.I.
        J. Clin. Invest. 1995; 96: 1404-1413
        • Van Den Hove M.F.
        • Couvrer M.
        • De Visscher M.
        • Salvatore G.
        Eur. J. Biochem. 1982; 122: 415-422