Identification of a Heparin-binding Region of Rat Thyroglobulin Involved in Megalin Binding
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.30377
ISSN1083-351X
AutoresMichele Marinò, Joel A. Friedlander, Robert T. McCluskey, David Andrews,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoWe recently showed that thyroglobulin (Tg) is a heparin-binding protein and that heparin inhibits binding of Tg to its endocytic receptor megalin (gp330). Here we have identified a heparin-binding region in the carboxyl-terminal portion of rat Tg and have studied its involvement in megalin binding. Rat thyroid extracts, obtained by ammonium sulfate precipitation, were separated by column fractionation into four Tg polypeptides, with apparent masses of 660, 330, 210, and 50 kDa. As assessed by enzyme-linked immunoadsorbent assays and ligand blot binding assays, megalin bound to intact Tg (660 and 330 kDa) and, to a even greater extent, to the 210-kDa Tg polypeptide. Furthermore, the 210-kDa Tg polypeptide inhibited megalin binding to intact Tg by ∼70%. Solid phase assays showed binding of biotin-labeled heparin to intact Tg and to the 210-kDa Tg polypeptide. We characterized the 210-kDa Tg polypeptide by matrix-assisted laser desorption/ionization mass spectrometry analysis and found that it corresponds to the carboxyl-terminal portion of rat Tg. We developed a synthetic peptide corresponding to a 15-amino acid sequence in the carboxyl-terminal portion of rat Tg (Arg689–Lys703), containing a heparin-binding consensus sequence (SRRLKRP) and demonstrated heparin binding to this peptide. A rabbit antibody raised against the peptide recognized intact Tg in its native conformation and under denaturing conditions. This antibody markedly reduced heparin-binding to intact Tg, indicating that the region of native Tg corresponding to the peptide is involved in heparin binding. Furthermore, the anti-Tg peptide antibody almost completely inhibited binding of megalin to Tg, suggesting that the Tg region containing the peptide sequence is required for megalin binding. Physiologically, Tg binding to megalin on thyroid cells may be facilitated by Tg interaction with heparin-like molecules (heparan sulfate proteoglycans) via adjacent binding sites. We recently showed that thyroglobulin (Tg) is a heparin-binding protein and that heparin inhibits binding of Tg to its endocytic receptor megalin (gp330). Here we have identified a heparin-binding region in the carboxyl-terminal portion of rat Tg and have studied its involvement in megalin binding. Rat thyroid extracts, obtained by ammonium sulfate precipitation, were separated by column fractionation into four Tg polypeptides, with apparent masses of 660, 330, 210, and 50 kDa. As assessed by enzyme-linked immunoadsorbent assays and ligand blot binding assays, megalin bound to intact Tg (660 and 330 kDa) and, to a even greater extent, to the 210-kDa Tg polypeptide. Furthermore, the 210-kDa Tg polypeptide inhibited megalin binding to intact Tg by ∼70%. Solid phase assays showed binding of biotin-labeled heparin to intact Tg and to the 210-kDa Tg polypeptide. We characterized the 210-kDa Tg polypeptide by matrix-assisted laser desorption/ionization mass spectrometry analysis and found that it corresponds to the carboxyl-terminal portion of rat Tg. We developed a synthetic peptide corresponding to a 15-amino acid sequence in the carboxyl-terminal portion of rat Tg (Arg689–Lys703), containing a heparin-binding consensus sequence (SRRLKRP) and demonstrated heparin binding to this peptide. A rabbit antibody raised against the peptide recognized intact Tg in its native conformation and under denaturing conditions. This antibody markedly reduced heparin-binding to intact Tg, indicating that the region of native Tg corresponding to the peptide is involved in heparin binding. Furthermore, the anti-Tg peptide antibody almost completely inhibited binding of megalin to Tg, suggesting that the Tg region containing the peptide sequence is required for megalin binding. Physiologically, Tg binding to megalin on thyroid cells may be facilitated by Tg interaction with heparin-like molecules (heparan sulfate proteoglycans) via adjacent binding sites. OVA: ovalbumin enzyme-linked immunoadsorbent assay alkaline phosphatase Tris-buffered saline phosphate-buffered saline Megalin (gp330) is a member of the low density lipoprotein receptor family (1Raychowdhury R. Niles J.L. McCluskey R.T. Smith J.A. Science. 1989; 244: 1163-1165Crossref PubMed Scopus (213) Google Scholar, 2Saito A. Pietromonaco S. Loo A.K.C. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9725-9729Crossref PubMed Scopus (492) Google Scholar) expressed on the apical surface of certain absorptive epithelial cells, including thyroid cells (3Zheng 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 (283) Google Scholar, 4Lundgren 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 (174) Google Scholar). Based on the assumption that physiologically megalin binds to ligands to which it is exposed in different organs (5Zheng 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 thyroid cells is a receptor for thyroglobulin (Tg).1 Tg is synthesized in thyrocytes and released into the follicle lumen, where it is stored as the major component of colloid (6Dunn A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 81-84Google Scholar, 7Dunn J. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 85-95Google Scholar). Hormone secretion requires uptake of Tg by thyrocytes, with transport to lysosomes, where proteolytic cleavage leads to release of hormones from mature Tg molecules (6Dunn A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 81-84Google Scholar). Internalization of Tg may result from pseudopod ingestion, but under most conditions uptake occurs by micropinocytosis (vesicular internalization), which can take place both by nonselective fluid phase uptake and receptor-mediated endocytosis (6Dunn A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 81-84Google Scholar, 7Dunn J. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 85-95Google Scholar, 8Rousset B. Mornex R. Mol. Cell. Endocrinol. 1991; 78: 89-93Crossref PubMed Scopus (37) Google Scholar, 9Consiglio E. Salvatore G. Rall J.E. Khon L.D. J. Biol. Chem. 1979; 254: 5065-5076Abstract Full Text PDF PubMed Google Scholar, 10Consiglio 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, 11Miquelis R. Alquier C. Monsigny M. J. Biol. Chem. 1987; 262: 15291-15298Abstract Full Text PDF PubMed Google Scholar, 12Bernier-Valentin F. Kostrouch Z. Rabilloud R. Munari-Silem Y. Rousset B. J. Biol. Chem. 1990; 265: 17373-17380Abstract Full Text PDF PubMed Google Scholar, 13Bernier-Valentin F. Kostrouch Z. Rabilloud R. Rousset B. Endocrinology. 1991; 129: 2194-2201Crossref PubMed Scopus (39) Google Scholar, 14Lemansky P. Herzog V. Eur. J. Biochem. 1992; 209: 111-119Crossref PubMed Scopus (42) Google Scholar, 15Kostrouch Z. Bernier-Valentin F. Munari-Silem Y. Rajas F. Rabilloud R. Rousset B. Endocrinology. 1993; 132: 2645-2653Crossref PubMed Scopus (35) Google Scholar, 16Mziaut 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, 17Giraud A. Siffroi S. Lanet J. Franc J.L. Endocrinology. 1997; 138: 2325-2332Crossref PubMed Scopus (24) Google Scholar). In previous studies (18Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (72) Google Scholar, 19Marinò M. Zheng G. McCluskey R.T. J. Biol. Chem. 1999; 274: 12898-12904Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) we showed that megalin is a high affinity receptor for Tg and that it can mediate Tg endocytosis by cultured thyroid cells. We also demonstrated that heparin almost completely inhibits binding of megalin to Tg (18Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (72) Google Scholar, 19Marinò M. Zheng G. McCluskey R.T. J. Biol. Chem. 1999; 274: 12898-12904Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and, in studies designed to investigate the mechanism, we showed that Tg is a heparin-binding protein, as are certain other megalin ligands (19Marinò M. Zheng G. McCluskey R.T. J. Biol. Chem. 1999; 274: 12898-12904Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In the present study we show that a 15-amino acid sequence in the carboxyl-terminal portion of rat Tg, rich in positively charged residues, is involved in binding of Tg to heparin and to megalin.DISCUSSIONIn the present study we have identified a heparin-binding region in the carboxyl-terminal portion of rat Tg and have obtained evidence that it is closely related to a megalin-binding region. To characterize Tg-binding regions we used rat thyroid extracts separated by column size fractionation and, in confirmation of previous studies (6Dunn A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 81-84Google Scholar), found that they contain four major Tg polypeptides, with estimated molecular masses of 660, 330, 210, and 50 kDa. In an extension of our earlier studies (18Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (72) Google Scholar, 19Marinò M. Zheng G. McCluskey R.T. J. Biol. Chem. 1999; 274: 12898-12904Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), we showed that megalin bound not only to intact Tg but also to the 210-kDa Tg polypeptide, and to an even greater extent than to intact Tg. In addition, the 210-kDa Tg polypeptide inhibited binding of intact Tg to megalin by ∼70%, indicating that it includes a major Tg-binding site for megalin. Furthermore, heparin bound to the 210-kDa Tg polypeptide, and co-incubation with the 210-kDa polypeptide substantially reduced (by ∼60%) heparin binding to Tg, indicating that the 210-kDa Tg polypeptide contains heparin-binding sites. MALDI-MS analysis provided evidence that the 210-kDa Tg polypeptide represents the carboxyl-terminal two-thirds of rat Tg, and in support of this, a rabbit antibody raised against a synthetic peptide deduced to be in the carboxyl-terminal portion of rat Tg recognized the 210-kDa Tg polypeptide as well as intact Tg.We postulated that even though intact Tg is anionic (pI∼4.6), it interacts with heparin through regions containing clusters of positively charged amino acids (arginine and lysine), as previously shown for the majority of heparin-binding proteins (29Cardin A.D. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar, 30Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (595) Google Scholar, 31Moestrup S.K. Cui S. Vorum H. Bregengard C. Bjørn C.E. Norris K. Gliemann J. Christensen E.I. J. Clin. Invest. 1995; 96: 1404-1413Crossref PubMed Scopus (294) Google Scholar, 32Conrad H.E. Conrad H.E. Heparin-binding Proteins. Academic Press, San Diego1998: 183-202Crossref Google Scholar). By analysis of the published partial sequences of rat Tg (27Di Lauro R. Obici S. Condliffe D. Ursini V.M. Musti A. Moscatelli C. Avvedimento V.E. Eur. J. Biochem. 1985; 148: 7-11Crossref PubMed Scopus (73) Google Scholar, 28Graves P.N. Davies T.F. Mol. Endocrinol. 1990; 4: 155-161Crossref PubMed Scopus (24) Google Scholar), we identified a 7-amino acid sequence (SRRLKRP) in the carboxyl-terminal portion, corresponding to Ser693–Pro699, with characteristics of a "consensus sequence" for heparin binding, as defined by Cardin and Weintraub (29Cardin A.D. Weintraub H.J.R. Atherosclerosis. 1989; 9: 21-32Google Scholar, 30Wilson C. Wardell M.R. Weisgraber K.H. Mahley R.W. Agard D.A. Science. 1991; 252: 1817-1822Crossref PubMed Scopus (595) Google Scholar, 31Moestrup S.K. Cui S. Vorum H. Bregengard C. Bjørn C.E. Norris K. Gliemann J. Christensen E.I. J. Clin. Invest. 1995; 96: 1404-1413Crossref PubMed Scopus (294) Google Scholar, 32Conrad H.E. Conrad H.E. Heparin-binding Proteins. Academic Press, San Diego1998: 183-202Crossref Google Scholar), namelyXBBXBBX, where B is a basic amino acid and X is any nonbasic/nonacidic amino acid. We obtained a synthetic peptide (Tg peptide 1) corresponding to a 15-amino acid sequence in the carboxyl-terminal portion of rat Tg (Arg689–Lys703: RELPSRRLKRPLPVK) that contains the heparin-binding consensus sequence and showed that biotin-labeled heparin binds to this peptide but not to a 15-amino acid control peptide in which the four inner arginine and lysine residues were replaced by glycine.The heparin binding affinity of Tg peptide 1 was lower than that of intact Tg, as shown by calculations of the K d in solid phase assays (Tg peptide 1, 12.5 μm; intact Tg, 47 nm) and by the finding that Tg peptide 1 bound to a heparin-agarose column to a lower extent than intact Tg. Furthermore, the extent of inhibition of heparin binding to Tg produced by Tg peptide 1 was lower than that produced by several known heparin-binding proteins or polypeptides (lipoprotein lipase, lactoferrin, protamine, and polylysine), and Tg peptide 1 was a weaker inhibitor than intact Tg of heparin binding to the above heparin-binding peptides/proteins. The apparently anomalous ability of Tg peptide 1 to reduce binding of heparin to intact Tg to a greater extent than the 210-kDa Tg polypeptide in our assays may be explained by the much smaller molar amounts of the peptide in the 210-kDa polypeptide. Thus, the 210-kDa polypeptide was used at a concentration of 0.476 μm (100 μg/ml), with a concentration of the sequence corresponding to Tg peptide 1 of 0.0047 μm, as compared with concentrations of Tg peptide 1 (used alone) up to 250 μm.The greater heparin binding affinity of intact Tg as compared with Tg peptide 1 suggests that other heparin-binding regions are present in the Tg molecule and/or that the native conformation of Tg is important in heparin binding. To pursue this, we further analyzed the two known partial sequences (27Di Lauro R. Obici S. Condliffe D. Ursini V.M. Musti A. Moscatelli C. Avvedimento V.E. Eur. J. Biochem. 1985; 148: 7-11Crossref PubMed Scopus (73) Google Scholar, 28Graves P.N. Davies T.F. Mol. Endocrinol. 1990; 4: 155-161Crossref PubMed Scopus (24) Google Scholar) of rat Tg and identified another putative heparin-binding consensus sequence (XBBXBX), from Ala801 to Gly806 (ARRTRG). We developed a second 15-amino acid synthetic Tg peptide (Tg peptide 2), corresponding to Met796–Ile810 (MASLWARRTRGNVFI), which included this heparin-binding consensus sequence. However, we found no binding of biotin-labeled heparin to this peptide. No other heparin-binding consensus sequences could be identified in the two known partial sequences of rat Tg. To obtain information about the remaining sequences of Tg, we analyzed the complete sequence of mouse Tg (Ref. 34Caturegli P. Vidalain P.O. Vali M. Aguilera-Galaviz L.A. Rose N.R. Clin. Immunol. Immunopathol. 1997; 85: 221-226Crossref PubMed Scopus (33) Google Scholar, GenBankTM accession number 008710), which is highly homologous to rat Tg, and identified only the same two heparin-binding consensus sequences found in rat Tg, corresponding to Tg peptide 1 and Tg peptide 2. Nevertheless, a contribution of other regions of Tg to its heparin binding affinity cannot be excluded. Thus, heparin-binding segments of proteins can be different from the Cardin and Weintraub model, and separate regions rich in positively charged amino acid residues can be brought together by the folding of the protein (32Conrad H.E. Conrad H.E. Heparin-binding Proteins. Academic Press, San Diego1998: 183-202Crossref Google Scholar). The heparin binding affinity of intact Tg may thus depend in part on the conformation of the molecule. The optimal way to study how conformation affects the heparin binding ability of a protein is to analyze its three-dimensional structure, to locate regions rich in positively charged residues, and to determine whether they are brought together by the folding of the protein. However, this approach is not currently possible in the case of Tg, because its three-dimensional structure is not known.Another way to study putative heparin-binding regions of a protein is by site-directed mutagenesis studies. However, this approach is unlikely to be feasible in the case of Tg. So far only Arvan and associates (35Kim P.S. Hossain S.A. Park Y.N. Lee I. Yoo S.E. Arvan P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9909-9913Crossref PubMed Scopus (105) Google Scholar) have succeeded in transfecting a nearly full-length Tg gene in cultured cells. However, they found that point mutations in the carboxyl-terminal portion abolished secretion of the protein, because of its retention in the endoplasmic reticulum, thereby making it impossible to obtain preparations of purified mutated Tg. The above study supports the notion that certain Tg mutations described in humans, especially in the carboxyl-terminal portion, result in misfolding of the protein in the endoplasmic reticulum with impaired secretion by thyroid cells (7Dunn J. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 85-95Google Scholar, 36Medeiros-Neto G. Targovnik H.M. Vassart G. Endocr. Rev. 1993; 14: 165-183PubMed Google Scholar, 37Medeiros-Neto G. Kim P.S. Yoo S.E. Vono J. Targovnik H.M. Camargo R. Hossain S.A. Arvan P. J. Clin. Invest. 1996; 98: 2838-2844Crossref PubMed Google Scholar, 38Perlmutter D.H. Lab. Invest. 1999; 79: 623-638PubMed Google Scholar).Our approach to the identification of a heparin-binding region, namely through the use of synthetic peptides, can be criticized because such peptides may have different conformations from those of the corresponding segments in the native protein. This may be one reason why intact Tg has a greater heparin binding affinity than Tg peptide 1. Despite this reservation, evidence obtained in the present study supports the conclusion that a continuous sequence of the 15-amino acid Tg peptide 1 corresponds to an active heparin-binding region present on the surface of native, intact Tg. Thus, a rabbit antibody raised against Tg peptide 1 recognized native, intact Tg as shown by immunoprecipitation experiments, indicating that its antigenic determinants are present on the surface of the Tg molecule. Furthermore, this antibody recognized Tg by Western blotting, under denaturing conditions, where conformational epitopes are lost, suggesting that the epitopes with which the antibody reacts are probably not entirely conformational (39Berzofsky J.A. Berkower I.J. Paul W.E. Fundamental Immunology. Raven Press Ltd., New York1993: 235-282Google Scholar). In addition, the anti-Tg peptide 1 antibody markedly reduced heparin binding to Tg, indicating that the region recognized by the antibody is involved in heparin binding. However, the inhibition of heparin binding to intact Tg produced by the anti-Tg peptide 1 antibody was not complete (∼70%), which supports the conclusion that the Tg region corresponding to Tg peptide 1 is not entirely responsible for the heparin binding ability of intact Tg.Our experiments performed with "mutants" of Tg peptide 1 provide evidence in favor of an important role of the charge of this sequence in determining its heparin binding ability. Thus, when the sequence of Tg peptide 1 was randomly mixed, the peptide still retained ∼80% of its heparin binding capacity, whereas substitutions of any of the inner positively charged residues resulted in a striking reduction of heparin binding, down to ∼20–30% of the binding ability of the wild type sequence. The inability of Tg peptide 2 to bind heparin, even though it contains a putative heparin-binding consensus sequence, may be attributed to the lower number of positively charged residues as compared with Tg peptide 1 (three versus six positive residues). However, other factors, including conformation of the peptide and spatial relationship between positively charged residues, may also be important.The main aim of the present study was to identify heparin-binding regions that are related to megalin-binding sites. As we have shown previously (18Zheng G. Marinò M. Zhao J. McCluskey R.T. Endocrinology. 1998; 139: 1462-1465Crossref PubMed Scopus (72) Google Scholar, 19Marinò M. Zheng G. McCluskey R.T. J. Biol. Chem. 1999; 274: 12898-12904Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), heparin competes with megalin for Tg binding and can dissociate Tg from megalin. Therefore, we postulated that heparin-binding regions of Tg are closely related to megalin-binding sites. Although megalin did not bind to Tg peptide 1 and the peptide did not inhibit binding of megalin to intact Tg, the antibody raised against the synthetic Tg peptide 1 almost completely inhibited megalin binding to Tg, indicating that the Tg sequence corresponding to the peptide is involved in megalin binding. In this regard, previous studies dealing with proteins that bind to heparin and to cell surface receptors, including members of the low density lipoprotein receptor family, have provided evidence that heparin and receptor-binding sites lie adjacent to each other (40Conrad H.E. Conrad H.E. Heparin-binding Proteins. Academic Press, San Diego1998: 367-409Crossref Google Scholar). For some of these proteins efficient binding and uptake by cell surface receptors requires binding to heparin-like molecules, notably heparan sulfate proteoglycans (40Conrad H.E. Conrad H.E. Heparin-binding Proteins. Academic Press, San Diego1998: 367-409Crossref Google Scholar, 41Wong P. Hampton B. 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Strickland D.K. J. Biol. Chem. 1993; 268: 14168-14175Abstract Full Text PDF PubMed Google Scholar, 49Mulder M. Lombardi P. Jansen H. Van Berkel T.J.C. Frants R.R. Havekes L.M. J. Biol. Chem. 1993; 268: 9369-9375Abstract Full Text PDF PubMed Google Scholar). In these cases, binding is believed to occur via side-by-side binding sites for heparan sulfate proteoglycans and for the receptor. These considerations raise the possibility that Tg may interact with cell surface heparan sulfate proteoglycans. Indeed, heparan sulfate proteoglycans are expressed by thyroid cells in a TSH-dependent manner (50Emoto N. Isozaki O. Ohmura E. Tsushima T. Shizume K. Demura H. Endocrinol. Metab. 1994; 1: 123-130Google Scholar, 51Emoto N. Isozaki O. Shizume K. Tsushima T. Demura H. Thyroid. 1995; 5: 455-460Crossref PubMed Scopus (11) Google Scholar), and we have obtained preliminary evidence suggesting that heparan sulfate proteoglycans can facilitate binding of Tg to cultured thyroid cells.2 Thus, the inhibition produced by the antibody against Tg peptide 1 on megalin binding to intact Tg may be explained by a steric effect. The finding that the carboxyl-terminal portion of rat Tg (210-kDa polypeptide), which bound very avidly to megalin, only partially inhibited binding of megalin to intact Tg suggests that other binding sites for megalin may be present in the amino-terminal portion of Tg. In this regard, multiple binding sites for megalin that are separate but functionally related to a heparin-binding domain have been previously demonstrated in the 39-kDa low density lipoprotein receptor-associated protein, also known as RAP, a surrogate megalin ligand (52Orlando R.A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3161-3165Crossref PubMed Scopus (61) Google Scholar).The identification of a heparin-binding region of Tg that is also implicated in binding to megalin is of potential interest in the understanding of the pathogenesis of certain thyroid diseases. For example, mutations of the Tg gene resulting in amino acid substitutions are associated with certain sporadic or familial, congenital forms of goiter, and some of these mutations are located in the carboxyl-terminal portion of Tg (7Dunn J. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 85-95Google Scholar, 35Kim P.S. Hossain S.A. Park Y.N. Lee I. Yoo S.E. Arvan P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9909-9913Crossref PubMed Scopus (105) Google Scholar, 36Medeiros-Neto G. Targovnik H.M. Vassart G. Endocr. Rev. 1993; 14: 165-183PubMed Google Scholar, 37Medeiros-Neto G. Kim P.S. Yoo S.E. Vono J. Targovnik H.M. Camargo R. Hossain S.A. Arvan P. J. Clin. Invest. 1996; 98: 2838-2844Crossref PubMed Google Scholar, 38Perlmutter D.H. Lab. Invest. 1999; 79: 623-638PubMed Google Scholar). The mechanisms by which these mutations lead to a Tg accumulation in thyroid follicles and, consequently, to goiter, have not been completely clarified, although, as noted above, a defect in intracellular trafficking of Tg because of misfolding of the molecule in the endoplasmic reticulum is thought to be important (7Dunn J. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 85-95Google Scholar, 35Kim P.S. Hossain S.A. Park Y.N. Lee I. Yoo S.E. Arvan P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9909-9913Crossref PubMed Scopus (105) Google Scholar, 36Medeiros-Neto G. Targovnik H.M. Vassart G. Endocr. Rev. 1993; 14: 165-183PubMed Google Scholar, 37Medeiros-Neto G. Kim P.S. Yoo S.E. Vono J. Targovnik H.M. Camargo R. Hossain S.A. Arvan P. J. Clin. Invest. 1996; 98: 2838-2844Crossref PubMed Google Scholar, 38Perlmutter D.H. Lab. Invest. 1999; 79: 623-638PubMed Google Scholar). We postulate that modifications of the Tg structure may also result in impaired Tg endocytosis via megalin, thereby promoting excessive retention of Tg in the colloid. Clearly, additional studies are needed to further characterize the Tg-binding site for megalin and to investigate whether mutations of the Tg molecule may be responsible for altered storage of Tg as a consequence of impaired Tg-megalin interactions. Megalin (gp330) is a member of the low density lipoprotein receptor family (1Raychowdhury R. Niles J.L. McCluskey R.T. Smith J.A. Science. 1989; 244: 1163-1165Crossref PubMed Scopus (213) Google Scholar, 2Saito A. Pietromonaco S. Loo A.K.C. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9725-9729Crossref PubMed Scopus (492) Google Scholar) expressed on the apical surface of certain absorptive epithelial cells, including thyroid cells (3Zheng 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 (283) Google Scholar, 4Lundgren 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 (174) Google Scholar). Based on the assumption that physiologically megalin binds to ligands to which it is exposed in different organs (5Zheng 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 thyroid cells is a receptor for thyroglobulin (Tg).1 Tg is synthesized in thyrocytes and released into the follicle lumen, where it is stored as the major component of colloid (6Dunn A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 81-84Google Scholar, 7Dunn J. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 85-95Google Scholar). Hormone secretion requires uptake of Tg by thyrocytes, with transport to lysosomes, where proteolytic cleavage leads to release of hormones from mature Tg molecules (6Dunn A. Braverman L.E. Utiger R.D. Werner and Ingbar's The Thyroid: A Fundamental and Clinical Test. Lippincott-Raven, Philadelphia, PA1996: 81-84Google Scholar). Internalization of Tg may result from pseudopod ingestion, but under most conditions uptake occurs by micropinocytosis (vesicular internalization), which can take place both by nonselective fluid phase uptake and receptor-mediated endocytosis (6Dunn A. Brav
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