Binding of the Low Density Lipoprotein Receptor-Associated Protein (RAP) to Thyroglobulin (Tg): Putative Role of RAP in the Tg Secretory Pathway

Michele Marinò, Luca Chiovato, Simonetta Lisi, Aldo Pinchera and Robert T. McCluskey

Department of Endocrinology (M.M., L.C., S.L., A.P.), University of Pisa, 56124 Pisa, Italy; and Pathology Research Laboratory (M.M., R.T.M.), Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

Address all correspondence and requests for reprints to: Michele Marinò, Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy. E-mail: m.marino{at}endoc.med.unipi.it


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The 39–44 kDa protein known as the receptor-associated protein binds to members of the low density lipoprotein receptor family and is found within cells that express these receptors. The receptor-associated protein has been shown to prevent premature binding of ligands to the receptors in the endoplasmic reticulum and to promote proper folding and transport of the receptors in the secretory pathway. In the thyroid, megalin (a low-density lipoprotein receptor family member) serves as an endocytic receptor for thyroglobulin. Here we present evidence that the receptor-associated protein can bind to thyroglobulin, which suggests a novel function of the receptor-associated protein, namely binding of certain megalin ligands possibly during the biosynthetic pathway.

In solid-phase assays thyroglobulin was shown to bind to the receptor-associated protein with moderately high affinity (mean between Kd and Ki = 39.8 nM), in a calcium-dependent and saturable manner. The receptor-associated protein also bound to a native carboxyl-terminal 230-kDa thyroglobulin polypeptide, which markedly reduced binding of intact thyroglobulin to the receptorassociated protein, indicating that the receptor-associated protein binding sites of thyroglobulin are located in the carboxyl-terminal portion of the molecule. In addition to thyroglobulin, the receptor-associated protein specifically bound to another megalin ligand, namely lipoprotein lipase. Because lipoprotein lipase markedly reduced receptor-associated protein binding to thyroglobulin, we concluded that the receptor-associated protein uses the same binding site/s to bind to thyroglobulin and lipoprotein lipase. Evidence of thyroglobulin binding to the receptor-associated protein was also obtained in vivo and in cultured thyroid cells. Thus, anti-receptor-associated protein antibodies precipitated intact thyroglobulin from extracts prepared from rat thyroids and cultured thyroid cells (FRTL-5 cells). Chase experiments after inhibition of protein synthesis in FRTL-5 cells showed that thyroglobulin interacts with the receptor-associated protein shortly after the beginning of thyroglobulin biosynthesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE LOW DENSITY lipoprotein (LDL) receptor family comprises several cysteine-rich endocytic receptors, including the LDL receptor itself, the LDL receptor-related protein (LRP), megalin (gp330), and the VLDL receptor (1, 2, 3, 4, 5). LRP and megalin, the two largest members of the family, can bind multiple, unrelated, and, to some extent, overlapping ligands. The expression of these receptors differs among various organs, and their physiological function is to internalize ligands present in fluids to which they are exposed, usually with delivery of ligands to lysosomes (1, 2, 3, 4, 5). A 39- to 44-kDa receptor-associated protein (RAP) is present in all epithelial cell types where LRP and megalin are expressed (1, 2, 3, 4, 5, 6). In vitro RAP can block binding of virtually all ligands to LRP and megalin (1, 2, 3, 4, 5, 6). In vivo RAP is an endoplasmic reticulum resident protein that bears a retention sequence (HNEL). RAP functions as a molecular chaperone for members of the LDL receptor family, by preventing premature ligand binding within the endoplasmic reticulum and by promoting proper folding and assembly of the receptors, thereby facilitating their transport to the cell surface along the secretory pathway (1, 2, 3, 4, 5).

In recent studies (7, 8, 9) we have shown that megalin is a high-affinity endocytic receptor for thyroglobulin (Tg), the precursor of thyroid hormones. Tg is synthesized by thyroid cells and released into the follicle lumen, where it is stored as the major component of colloid (10, 11, 12, 13, 14, 15). Hormone secretion requires uptake of Tg by thyrocytes, with transport to lysosomes, where proteolytic cleavage leads to the release of thyroid hormones (10, 11, 12, 13, 14, 15). Megalin is expressed on the apical surface of thyrocytes (6, 16), where it mediates endocytosis of Tg from the colloid, after which Tg is not transported to lysosomes, as are most megalin ligands, but is rather transcytosed across thyrocytes to the basolateral surface, from which Tg is released into the bloodstream (17, 18, 19). This unusual function of megalin competes with other mechanisms of Tg endocytosis, probably mainly nonselective fluid phase micropinocytosis, which lead to the delivery of Tg to lysosomes (10, 11, 12, 13, 14, 15).

Expression of RAP by thyroid cells has been demonstrated in several species, including humans and rats (6, 16). Here we show that RAP binds to Tg in solid-phase assays, as well as in vivo and in cultured thyroid cells, suggesting a novel function of RAP, namely binding of certain megalin ligands possibly during the biosynthetic pathway.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of Tg to RAP
Evidence of binding of Tg to RAP was first provided in solid-phase binding assays. Tg bound to wells coated with RAP, used as a glutathione-S-transferase (GST) fusion protein (RAP-GST), whereas there was no binding to wells coated with GST alone (Fig. 1AGo). Binding of Tg to RAP-GST-coated wells is represented as a nonlinear regular fitting plot in Fig. 1AGo. Binding was saturable and markedly reduced by coincubation of Tg with an excess of RAP-GST. The mean dissociation constant (Kd), estimated from the midsaturation points of equilibrium binding experiments, as presented in Fig. 1AGo (estimated apparent affinity, obtained in three experiments), was 22.25 nM for total binding and 16.62 nM for specific binding (total binding - binding in the presence of an excess RAP-GST). The average Kd for total and specific binding was 19.40 nM. In addition, we evaluated the inhibition constant (Ki or K0.5) of Tg binding to RAP-GST coated wells, by measuring binding of a constant Tg concentration (75 nM) in the presence of increasing amounts of RAP-GST (Fig. 1BGo). The mean Ki obtained was 93.50 nM (calculated in three experiments). Based on Kd and Ki values, we concluded that binding of Tg to RAP is of moderately high affinity (mean between Kd values and Ki = 39.8 nM).



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Figure 1. Binding of Rat Tg to RAP-GST in Solid-Phase Binding Assays

Microtiter wells coated with RAP-GST or, as a control, with GST alone, were incubated with Tg followed by a rabbit anti-Tg antibody, ALP-conjugated antirabbit IgG and p-nitrophenyl-phosphate. Absorbance (OD) was determined at 405 nM. A, Increasing concentrations of Tg were added to RAP-GST or to GST-coated wells, alone or in the presence of RAP-GST itself (100 µg/ml). Specific binding = total binding - binding in the presence of RAP-GST (nonspecific binding). The figure is representative of one of three separate experiments. B, A constant concentration of Tg (75 nM) was added to RAP-GST-coated wells in the presence of increasing concentrations of RAP-GST itself. The figure is representative of one of three separate experiments.

 
Because Kd and Ki values were obtained using an enzyme-linked, indirect binding assay, we ascertained that the values of absorbance obtained with this assay were within the linear part of the substrate production curve. For this purpose, we determined binding of various concentrations of the anti-Tg antibody (which was used to reveal bound Tg in solid-phase binding assays) to microtiter wells coated with Tg, followed by the enzyme-linked secondary antibody [alkaline phosphatase (ALP)-conjugated antirabbit IgG] and the substrate (p-nitrophenyl-phosphate). Experiments were performed under the same conditions used in solid-phase binding assays. As shown in Fig. 2Go, the OD values obtained in the linear part of the substrate curve ranged from 0.010 to 0.325. All the OD values obtained in binding experiments were within this range. These results demonstrate that the Kd and Ki values calculated in solid-phase binding assays were reliable.



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Figure 2. Production Curve of the Substrate (p-Nitrophenyl-phosphate) used in Tg Binding Experiments

Microtiter wells coated with Tg were incubated with various dilutions of the rabbit anti-Tg antibody, followed by ALP-conjugated antirabbit IgG and p-nitrophenyl-phosphate. Absorbance (OD) was determined at 405 nM. The figure is representative of one of three experiments. Experiments were performed under the same conditions used in solid-phase binding assays.

 
As shown in Fig. 3Go, binding of Tg to RAP-GST was inhibited by EDTA (by ~90%), indicating that binding is calcium dependent. No inhibition was produced by GST or ovalbumin (OVA) used as negative controls.



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Figure 3. Inhibition of Binding of Rat Tg to RAP-GST in Solid-Phase Binding Assays

Microtiter wells coated with RAP-GST or, as a control, with GST alone, were incubated with Tg (75 nM), followed by a rabbit anti-Tg antibody, ALP-conjugated antirabbit IgG and p-Nitrophenyl-phosphate. Absorbance (OD) was determined at 405 nM. Tg was added to RAP-GST or to GST coated wells alone or in the presence of RAP-GST itself (100 µg/ml) or EDTA (20 mM) or, as controls, GST (100 µg/ml) or OVA (100 µg/ml). Values are expressed as mean ± SE % of total binding (binding of Tg to RAP-GST coated wells in the absence of competitors or controls) obtained in three separate experiments.

 
RAP Binding Sites of Tg Are Located in the Carboxyl-Terminal Region
Tg binding sites for megalin and heparin are located in the carboxyl-terminal two-thirds of the molecule (9). To investigate whether RAP binding sites are also present in this portion of the Tg molecule, we studied binding of RAP-GST to a 230-kDa Tg polypeptide, which was previously shown to correspond to the carboxyl terminal two-thirds of rat Tg (9). As shown in Fig. 4AGo, RAP-GST bound to the 230-kDa Tg polypeptide to a similar extent as to Tg, but not to OVA, used as a negative control. In addition, binding of RAP-GST to Tg was markedly reduced by coincubation with the 230-kDa Tg polypeptide (Fig. 4BGo), but not by OVA, providing evidence that Tg binding sites for RAP are located within the carboxyl-terminal two thirds of the molecule.



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Figure 4. RAP Binding Sites Are Located in the Carboxyl Terminal Portion of Tg

A, Binding of RAP-GST to a 230-kDa Tg polypeptide corresponding to the carboxyl-terminal two thirds of rat Tg. Microtiter wells coated with the 230-kDa Tg polypeptide or with intact Tg, or, as a control, with OVA, were incubated with RAP-GST (100 µg/ml), followed by a rabbit anti-RAP antibody, ALP-conjugated antirabbit IgG, and p-nitrophenyl-phosphate. Absorbance (OD) was determined at 405 nM. Values are expressed as mean ± SE obtained in three separate experiments. B, Inhibition of RAP-GST binding to Tg by the 230-kDa Tg polypeptide. Microtiter wells coated with intact Tg were incubated with RAP-GST (100 µg/ml) alone or in the presence of the 230- kDa Tg polypeptide (100 µg/ml) or, as a control, of OVA (100 µg/ml), followed by a rabbit anti-RAP antibody, ALP-conjugated antirabbit IgG, and p-nitrophenyl-phosphate. Absorbance (OD) was determined at 405 nM. Values are expressed as mean ± SE obtained in three separate experiments.

 
The carboxyl-terminal portion of Tg bears a heparin binding site contained within a 15-amino acid sequence located from Arg2489 to Lys2503 in the recently obtained, complete sequence of rat Tg (20). This heparin binding site was previously shown to be involved in megalin binding (9). To investigate whether the Tg heparin binding site is also involved in binding to RAP, we studied binding of RAP-GST to a synthetic peptide corresponding to the above mentioned 15-amino acid sequence of rat Tg. However, no appreciable binding of RAP-GST was seen (not shown) and, in addition, the Tg synthetic peptide did not reduce binding of RAP-GST to Tg (not shown), indicating that the Tg heparin binding site is not involved in binding to RAP.

Binding of RAP to Lipoprotein Lipase (LPL)
To determine whether binding of RAP to megalin ligands is restricted to Tg, we studied binding of RAP-GST to other megalin ligands, namely lactoferrin, LPL, and apolipoprotein J (apo J). As shown in Fig. 5AGo, RAP-GST bound to LPL, but not to lactoferrin or apo J. Binding of RAP-GST to LPL was specific, as demonstrated by the inhibitory effect of RAP preincubation with an excess of LPL (Fig. 5BGo). Furthermore, binding was calcium dependent, as demonstrated by the marked inhibition produced by EDTA. No inhibition of binding of RAP-GST to LPL was produced by negative controls (GST and OVA).



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Figure 5. Binding of RAP to LPL

A, Binding of RAP-GST to LPL, but not to lactoferrin or clusterin (apo J). Microtiter wells coated with lactoferrin, LPL, or apo J were incubated with RAP-GST (100 µg/ml) followed by a rabbit anti-RAP antibody, ALP-conjugated antirabbit IgG, and p-nitrophenyl-phosphate. Absorbance (OD) was determined at 405 nM. Values are expressed as mean ± SE obtained in three separate experiments. B, Inhibition of RAP-GST binding to LPL. RAP-GST (100 µg/ml) was added to LPL-coated wells alone or in the presence of LPL itself (100 µg/ml) or EDTA (20 mM) or, as a control, with OVA (100 µg/ml). Values are expressed as mean ± SE % of total binding (binding of RAP-GST to LPL-coated wells in the absence of competitors or controls) obtained in three separate experiments.

 
We then investigated whether binding of RAP to Tg and to LPL involves the same binding site/s. For this purpose, we investigated the effect of coincubation with LPL on RAP binding to Tg-coated wells. As shown in Fig. 6Go, coincubation with LPL markedly reduced RAP binding to Tg, suggesting that RAP uses the same binding site/s to interact with both Tg and LPL.



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Figure 6. Inhibition of Binding of RAP to Tg by LPL

Microtiter wells coated with Tg were incubated with RAP (100 µg/ml), alone or in the presence of LPL (100 µg/ml) or OVA (100 µg/ml), followed by a rabbit anti-RAP antibody, ALP-conjugated antirabbit IgG, and p-nitrophenyl-phosphate. Absorbance (OD) was determined at 405 nM. Values are expressed as mean ± SE obtained in three separate experiments.

 
Interaction between Tg and RAP in Vivo and in Cultured Thyroid Cells
To determine whether binding of RAP to Tg occurs in vivo and in cultured thyroid cells, coimmunoprecipitation experiments were performed. For this purpose, we used frozen rat thyroids and FRTL-5 cells, an established differentiated rat thyroid cell line that expresses megalin in a TSH-dependent manner and synthesizes and secretes Tg (8). Rat thyroid extracts or FRTL-5 cell extracts were incubated with a rabbit anti-RAP antibody followed by precipitation with protein A beads. As shown in Fig. 7Go, Western blotting with the rabbit anti-Tg antibody revealed the presence of intact Tg in the material precipitated by the anti-RAP antibody in both rat thyroids (Fig. 7AGo) and FRTL-5 cells (Fig. 7BGo), indicating that Tg was combined with RAP within cells. The presence of RAP in the immunoprecipitated material was demonstrated by Western blotting, using the anti-RAP antibody (not shown). No Tg was precipitated by normal rabbit IgG, used as a negative control (not shown).



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Figure 7. Immunoprecipitation of Tg with an anti-RAP Antibody in Rat Thyroid Extracts (A) and in FRTL-5 Cell Extracts (B)

Extracts were incubated with rabbit antibodies coupled with protein A beads. Beads were then subjected to 5–15% nonreducing SDS-PAGE followed by Western blotting, which was performed using a rabbit anti-Tg antibody. Lanes 1, Extracts not subjected to immunoprecipitation; lanes 2, extracts precipitated with the rabbit anti-Tg antibody; lanes 3, extracts precipitated with the rabbit anti-RAP antibody. The figure is representative of one of three separate experiments. C, Lack of cross-reactivity of the anti-RAP antibody with Tg. Purified Tg (10 µg) was incubated with the anti-RAP antibody coupled with protein A beads, followed by Western blotting, for Tg. Lane 1, Preparation of rat Tg (1 µg); lane 2, Tg preparation incubated with the rabbit anti-RAP antibody. The figure is representative of one of three separate experiments.

 
To rule out the possibility that Tg was precipitated by the anti-RAP antibody because the antibody cross-reacted with Tg epitopes, we assessed whether the anti-RAP antibody would react with Tg. For this purpose we performed immunoprecipitation experiments with purified rat Tg and found that Tg was not precipitated by the anti-RAP antibody (Fig. 7CGo). Additional evidence that the anti-RAP antibody did not react with Tg was provided by immunofluorescence staining of paraformaldehyde-L-lysine-sodium periodate (PLP)-fixed rat thyroid sections with this antibody, as compared with staining of the rat thyroid sections with an anti-Tg antibody. As expected, Tg staining was found to be very intense in the colloid, with only very faint intracellular staining of thyrocytes (Fig. 8AGo). In contrast, staining for RAP was confined to the intracellular compartment, with granular staining distributed throughout the cytoplasm, whereas no staining of the colloid was observed (Fig. 8BGo).



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Figure 8. Immunofluorescence Staining for Tg (A) and RAP (B) of Frozen Rat Thyroid Sections

PLP-fixed thyroid sections were incubated with either the rabbit anti-Tg antibody or the rabbit anti-RAP antibody, followed by FITC-conjugated antirabbit IgG. A, Intense Tg staining is seen in the colloid, with only very faint intracellular staining of thyrocytes. B, Granular intracellular staining for RAP is seen, distributed throughout the cytoplasm, whereas no staining of the colloid is observed. Magnification, x100.

 
The Interaction between Tg and RAP in Cultured Thyroid Cells Occurs Early in the Secretory Pathway
The results presented above clearly demonstrate that RAP binds to Tg in solid- phase assays and that binding can also occur in thyroid cells. Based on the known function of RAP as a molecular chaperone of LDL receptors, we postulated that RAP-Tg interactions may occur during Tg biosynthesis. Therefore, we investigated whether Tg-RAP interactions take place early during the Tg biosynthetic pathway. For this purpose, we performed immunoprecipitation experiments with FRTL-5 cell extracts at various time points after inhibition of protein synthesis with cyclohexamide. As shown in Fig. 9AGo, treatment for 48 h with medium containing cyclohexamide abolished endogenous Tg synthesis by FRTL-5 cells. Immunoprecipitation experiments with an anti-Tg antibody demonstrated that Tg synthesis had begun as early as 15 min after replacement of medium without cyclohexamide (Fig. 9BGo). As shown in Fig. 9CGo, the anti-RAP antibody precipitated Tg in FRTL-5 cell extracts 15 min after restoration of protein synthesis, indicating that RAP interacts with Tg shortly after biosynthesis.



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Figure 9. Tg Interacts with RAP Shortly After Its Biosynthesis

A, Inhibition of Tg synthesis by FRTL-5 cells. Cells were cultured for 24 h (lane 2) or 48 h (lane 3) with medium containing cyclohexamide (10 µg/ml). Cell extracts were prepared and subjected to immunoprecipitation with a rabbit anti-Tg antibody, followed by Western blotting for Tg. Lane 1, Extract from untreated cells. B and C, After treatment of FRTL-5 cells with cyclohexamide for 48 h, cells were cultured in medium without cyclohexamide. Cell extracts were prepared at various time points and then subjected to immunoprecipitation with the anti-Tg antibody (B), or with the anti-RAP antibody (C), followed by Western blotting for Tg.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we show that Tg interacts with the LDL receptor-related protein RAP. Evidence of binding of Tg to RAP was first obtained in solid-phase binding assays. Binding of Tg to RAP was saturable, calcium dependent, and of moderately high affinity (mean between Kd values and Ki = 39.8 nM). Evidence that RAP binds to Tg within thyroid cells was provided by the demonstration that anti-RAP antibodies precipitated intact Tg from thyroid glands and cultured thyrocytes (FRTL-5 cells). Furthermore, we showed that RAP-Tg interactions occur early in the Tg secretory pathway. Thus, Tg was precipitable by an anti-RAP antibody within 15 min after restoration of protein synthesis after its suppression by cyclohexamide treatment.

The physiological significance of RAP binding to Tg is unknown. Because RAP is known to function as a chaperone in the biosynthetic pathway of megalin and of other members of the LDL receptor family (1, 2, 3, 4, 5), one hypothesis is that RAP may have a similar role in the Tg biosynthetic pathway. This hypothesis is indirectly supported by the knowledge that RAP is an endoplasmic reticulum resident protein (1, 2, 3, 4, 5) and by results presented here which demonstrate that RAP interacts with Tg shortly after biosynthesis. Further studies are needed to characterize the function of RAP in the Tg biosynthetic pathway. It would be of particular interest to study the kinetics of dissociation of RAP-Tg complexes in thyroid cells after biosynthesis, through pulse-chase experiments after inhibition of protein synthesis, followed by immuno-electron microscopy to track RAP-Tg complexes in cell organelles after their synthesis.

Although further studies are clearly needed to investigate whether RAP functions as a Tg chaperone, the present study may provide a way to gain new insights into the Tg biosynthetic pathway. The role of chaperones and other factors involved in the maturation and transport of Tg in the endoplasmic reticulum of thyrocytes have been extensively investigated in recent years by Arvan and associates (21, 22, 23, 24). Newly synthesized Tg proceeds through a series of folding intermediates, including aggregates with and without interchain disulfide bonds, unfolded free monomers, folded monomers, and finally dimers. Multiple molecular chaperones, including calnexin, calreticulin, BiP (a member of the heat shock protein 70 class), endoplasmic reticoloru protein 72, glucose-regulated protein 78 have all been implicated in interactions with unfolded forms of Tg (21, 22, 23, 24). Different chaperones may interact with folding intermediates of Tg, both concurrently and sequentially. If a chaperoning function of RAP in the Tg biosynthetic pathway is demonstrated, it will be interesting to study the relationship of RAP with known Tg chaperones.

Another hypothesis concerning the role of RAP-Tg interactions is that RAP may prevent premature binding of Tg to megalin during the biosynthetic pathway, not only by binding to megalin, as it is known to occur for other megalin ligands (1, 2, 3, 4, 5), but also by binding to Tg. However, in view of results presented here and of results obtained in previous studies (7, 8, 17), this possibility is unlikely. Thus, although we found that RAP binds to the carboxyl-terminal portion of Tg, where megalin binding sites are located (9), RAP did not bind to a carboxyl-terminal synthetic peptide corresponding to a heparin binding sequence of Tg (Arg2489-Lys2503) that is functionally involved in megalin binding (9). In addition, it was previously shown that RAP binds to megalin with higher affinity than to Tg (1, 2, 3, 4, 5) and that the degree of inhibition of Tg-megalin interactions produced by other megalin competitors, including a monoclonal antimegalin antibody, is similar to that produced by RAP (7, 8, 17). Taken together, these results indicate that inhibition by RAP of Tg-megalin interactions is due to occupation of megalin binding sites and not of Tg binding sites. Nevertheless, this issue requires further investigation, aimed at the precise identification of megalin and RAP binding sites within the Tg molecule.

To our knowledge, the present report is the first to show that RAP can interact with a ligand of the LDL receptor family. Moreover, we found that RAP can interact not only with Tg, but also with LPL, another megalin ligand. Further studies are needed to investigate whether RAP can bind to megalin ligands other than Tg and LPL. However, two other megalin ligands tested, namely lactoferrin and apo J, did not bind to RAP.

It has been shown that the functions of RAP in promoting proper folding of LRP and in the prevention of premature interaction of ligands with the receptor are independent and are mediated by different regions of RAP (1, 2, 3, 4, 5). Although the function of RAP binding to megalin ligands is unknown, the finding that RAP uses the same binding site(s) for binding to both Tg and LPL suggests that it may serve a similar function for both megalin ligands.

In conclusion, we found that Tg binds to the megalin-related protein RAP in solid- phase assays, in vivo, and in cultured thyroid cells. The physiological function of Tg-RAP interactions remains to be investigated, although, in view of the knowledge that RAP is an endoplasmic reticulum resident protein, the most likely role played by RAP is in the Tg biosynthetic pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Thyroglobulin (Tg) was purified from rat thyroids by ammonium sulfate precipitation and column fractionation, as previously described (7, 8, 9, 17). Tg preparations were analyzed both by nonreducing and reducing SDS-PAGE, followed by Coomassie staining or by Western blotting, as previously reported (7, 8, 9, 17). Under nonreducing conditions two bands were seen at about 660 and 330 kDa. The 660-kDa band corresponded to covalently linked Tg dimers. Size exclusion gel chromatography showed that almost all (~95%) of the 330-kDa band represented monomers derived from noncovalently associated Tg dimers that had been dissociated by SDS-PAGE, with a small fraction (~5%) of free Tg monomers. Under reducing conditions, two bands, one slower (S) and one faster (F), were seen, as previously described (7, 8, 9, 17). Other Tg products with lower molecular masses were present in minimal amounts.

A 230-kDa Tg polypeptide, corresponding to the carboxyl-terminal two-thirds of rat Tg, was purified by ammonium sulfate precipitation and column fractionation, as previously described (9). A previously described 15-amino acid peptide designated Tg peptide 1, corresponding to a sequence (RELPSRRLKRPLPVK, Arg2489-Lys2503) in the carboxyl-terminal portion of rat Tg (20), was synthesized by Genemed Biotechnologies (South San Francisco, CA).

RAP was used in the form of a GST fusion protein. DH5{alpha} 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 (25).

Lactoferrin and LPL were obtained from Sigma (St. Louis, MO). Human apo J (also known as clusterin) was obtained from Quidel (San Diego, CA).

A rabbit antihuman Tg antibody cross-reactive with Tg from other species was obtained from Axle (Westbury, NY). A rabbit antibody against RAP-GST was previously described (6). Alkaline phosphatase (ALP)-conjugated goat antirabbit IgG and horseradish peroxidase-conjugated goat antirabbit IgG were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG was obtained from Cappel (Durham, NC).

Cell Cultures
Fisher rat thyroid cells (FRTL-5) (American Type Culture Collection, Manassas, VA) were cultured as described (8, 17, 18, 19), in Coon’s F12 medium containing 5% FCS and a mixture of six hormones. We previously showed that the FRTL-5 cells cultured under these conditions synthesize and secrete intact Tg after radiometabolic labeling (17) and produce cAMP after TSH stimulation (18), which indicates that they maintain functions of differentiated thyroid cells.

Solid-Phase Binding Assays
Solid-phase assays were performed as previously described (7, 9, 26). Microtiter plates (96-well) were coated overnight at 4 C with RAP-GST, Tg, 230-kDa Tg polypeptide, Tg peptide 1, lactoferrin, apo J, LPL, OVA, or GST, at a concentration of 100 µg/ml in PBS. Wells were blocked for 3 h at 4 C with BSA at a concentration of 1 mg/ml, washed with Tris-buffered saline (TBS), 0.05% Tween-50, and incubated for 2 h at room temperature with ligands (Tg or RAP-GST) at various concentrations in binding buffer (TBS, 5 mM CaCl2, 0.5 mM MgCl2, 0.05% Tween 20, 0.5% BSA). To reveal bound ligands, wells were washed and incubated with primary rabbit antibodies (rabbit anti-Tg, diluted 1:500, or rabbit anti-RAP at a concentration of 10 µg/ml). After further washing, bound primary antibodies were revealed using ALP-conjugated antirabbit IgG (1:3,000), followed by p-nitrophenyl-phosphate. OD was determined at 405 nM using an E1–311 ELISA microplate reader. The background, obtained by incubating coated wells with the primary and secondary antibody, was subtracted from the results.

For inhibition experiments, either RAP-GST or Tg were preincubated overnight at 4 C in the presence of competitors and was added to the wells in the presence of the competitors. The competitors used were: RAP-GST itself (various concentrations), LPL (100 µg/ml), 230-kDa Tg polypeptide (100 µg/ml), Tg peptide 1 (100 µg/ml), EDTA (20 mM), GST (100 µg/ml), or OVA (100 µg/ml).

The Kd and Ki values were estimated using Prism (PPC) (GraphPad Software, Inc., San Diego, CA). We performed experiments to ascertain that the values of absorbance in the experiments in which the Kd and the Ki were calculated were within the linear portion of the substrate production curve. For this purpose, microtiter wells coated with Tg (100 µg/ml) were incubated with various concentrations of the rabbit anti-Tg antibody, followed by ALP-conjugated antirabbit IgG and p-nitrophenyl-phosphate. Absorbance was determined at 405 nM. Experiments were performed under the same conditions used for solid-phase binding assays.

Immunoprecipitation Experiments
Immunoprecipitation experiments were performed with both FRTL-5 cell extracts and rat thyroid extracts or as a control, with purified rat Tg. Cell extracts were prepared using 1% Triton-X-100, 1% deoxycholate (both from Fisher Scientific, Springfield, NJ) and briefly sonicated before use. Rat thyroid extracts were prepared using frozen thyroids (Pel-Freez Biologicals, Rogers, AK), which were minced with a surgical razor blade and solubilized overnight at 4 C in 1% Triton X-100, 1% deoxycholate in TBS (pH 8.0) containing 2 mM phenylmethylsulfonyl fluoride, 2 mM N-ethylmaleimide, 5 mM e-amino-n-caproic acid, 5 mM benzamidine (all from Sigma) and 10 mM EDTA (Fisher Scientific). Insoluble materials were pelleted by centrifugation.

Samples were precleared with protein A beads (Pierce Chemical Co., Rockford, IL; 50 µl of beads for 500 µl of sample), washed with PBS, and incubated overnight at 4 C with the rabbit anti-Tg antibody (1:500), with the rabbit anti-RAP antibody (5 µg), or, as a control, with normal rabbit IgG (5 µg). Fifty microliters of protein A beads were then added to each sample, for 1 h at 4 C and then washed eight times with PBS. The beads were resuspended in nonreducing Laemmli buffer, boiled, and spun by centrifugation and supernatants were subjected to 5–15% SDS-PAGE and blotted onto nitrocellulose membranes, which were incubated with the rabbit anti-Tg antibody (1:500) followed by horseradish peroxidase-conjugated antirabbit IgG (1:2,500).

In certain experiments, before preparing cell extracts, FRTL-5 cells were cultured for 24–48 h with medium containing cyclohexamide (10 µg/ml) to abolish protein synthesis. Cells were then washed and cultured with medium without cyclohexamide for 15–120 min. Extracts were prepared and subjected to immunoprecipitation as described above.

Immunofluorescence Staining of Rat Thyroid Sections
Six female Lewis rats weighing 100–120 g were purchased from Charles River Laboratories, Inc. (Wilmington, MA). Animal care and sacrifice procedures were in accordance with institutional guidelines. To obtain in situ fixed tissue, rats were perfused through the aorta under ether anesthesia with 200 ml of PBS, followed by 200 ml of PLP (Sigma). The thyroid was harvested and immersed in PLP fixative overnight, washed with PBS, and immersed in 30% sucrose overnight at 4 C before being embedded in O.C.T. compound (Miles, Inc., Elkhart, IN) and frozen in liquid nitrogen. Sections (4-µm) were prepared, dried, blocked, and incubated for 1 h with the rabbit anti-Tg antibody (1:500) or the rabbit anti-RAP antibody (20 µg/ml), followed by FITC-conjugated antirabbit IgG (1:1,000).


    FOOTNOTES
 
This work was supported by the 1999 American Thyroid Association Research Grant (M.M.), by NIDDK Grant 46301 (R.T.M.) and by Grants from the National Research Council (Consiglio Nazionale Ricerche, Roma, Italy), Target Project Biotechnology and Bioinstrumentation (Grant 91.01219), and Target Project Prevention and Control of Disease Factors (Grant 93.00437) and by European Economic Community Stimulation Action-Science Plan Contract SC1-CT91-0707.

Abbreviations: ALP, Alkaline phosphatase; apo J, apolipoprotein J; FITC, fluorescein isothiocyanate; GST, glutathione-S-transferase; LDL, low-density lipoprotein; LPL, lipoprotein lipase; LRP, LDL receptor-related protein; OVA, ovalbumin; PLP, paraformaldehyde-L-lysine-sodium periodate; RAP, receptor-associated protein; TBS, Tris-buffered saline; Tg, thyroglobulin.

Received for publication December 1, 2000. Accepted for publication June 25, 2001.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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