Internalization of Transthyretin

EVIDENCE OF A NOVEL YET UNIDENTIFIED RECEPTOR-ASSOCIATED PROTEIN (RAP)-SENSITIVE RECEPTOR*

Mónica Mendes SousaDagger § and Maria João SaraivaDagger ||

From the Dagger  Amyloid Unit, Instituto de Biologia Molecular e Celular and the  Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4150 Porto, Portugal

Received for publication, December 1, 2000, and in revised form, January 8, 2001




    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transthyretin (TTR) is a plasma carrier of thyroxine and retinol-binding protein (RBP). Though the liver is the major site of TTR degradation, its cellular uptake is poorly understood. We explored TTR uptake using hepatomas and primary hepatocytes and showed internalization by a specific receptor. RBP complexed with TTR led to a 70% decrease of TTR internalization, whereas TTR bound to thyroxine led to a 20% increase. Different TTR mutants showed differences in uptake, suggesting receptor recognition dependent on the structure of TTR. Cross-linking studies using hepatomas and 125I-TTR revealed a ~90-kDa complex corresponding to 125I-TTR bound to its receptor. Given previous evidence that a fraction of TTR is associated with high-density lipoproteins (HDL) and that in the kidney, megalin, a member of the low-density lipoprotein receptor family (LDLr) internalizes TTR, we hypothesized that TTR and lipoproteins could share related degradation pathways. Using lipid-deficient serum in uptake assays, no significant changes were observed showing that TTR uptake is not lipoprotein-dependent or due to TTR-lipoprotein complexes. However, competition studies showed that lipoproteins inhibit TTR internalization. The scavenger receptor SR-BI, a HDL receptor, and known LDLr family hepatic receptors did not mediate TTR uptake as assessed using different cellular systems. Interestingly, the receptor-associated protein (RAP), a ligand for all members of the LDLr, was able to inhibit TTR internalization. Moreover, the ~90-kDa TTR-receptor complex obtained by cross-linking was sensitive to the presence of RAP. To confirm that RAP sensitivity observed in hepatomas did not represent a mechanism absent in normal cells, primary hepatocytes were tested, and similar results were obtained. The RAP-sensitive TTR internalization together with displacement of TTR uptake by lipoproteins, further suggests that a common pathway might exist between TTR and lipoprotein metabolism and that an as yet unidentified RAP-sensitive receptor mediates TTR uptake.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transthyretin (TTR)1 is a serum tetrameric protein of four identical subunits of 14 kDa synthesized early in development and primarily in the liver and choroid plexus of the brain (1). Plasma TTR derives predominantly from the liver and acts as a transport protein for thyroxine (T4) and retinol (vitamin A) (2); in the latter case this is accomplished by the formation of a 1:1 molar complex with retinol-binding protein (RBP) (3). TTR binds virtually all of serum RBP and about 15% of serum T4.

Though it is known that liver, kidney, muscle, and skin are the major sites of TTR degradation in the rat (4), its cellular uptake is poorly understood. Divino and Schussler (5) have reported a specific and saturable TTR uptake, consistent with the existence of a receptor-mediated process in hepatomas, providing the probable mechanism for the in vivo translocation of plasma TTR into the rat liver previously observed by Makover et al. (4). Vieira et al. (6) have investigated the transport of serum TTR into chicken oocytes and showed the existence of a ~115-kDa oocytic TTR receptor, which might be responsible for regulating the uptake of T4 and retinol into the growing embryo. Receptor-mediated uptake of TTR was also shown in astrocytes and ependymomas (7, 8) suggesting a role for delivery of TTR ligands to the central nervous system. All these reports support a receptor-mediated process for TTR internalization in several tissues, but information is lacking on the biochemical nature of the TTR-receptor interaction.

Several point mutations in TTR have been related to the occurrence of familial amyloidotic polyneuropathy (FAP). FAP is an autosomal dominant disease characterized by the extracellular deposition of TTR fibrils in various tissues (9). FAP patients generally present low levels of plasma TTR, which might be related to a different catabolism of the mutant protein; however, because no studies are available on the internalization of different TTR mutants, investigation of cellular uptake of both amyloidogenic and nonamyloidogenic protein can give clues on structural determinants of TTR important for interaction.

We reported that 1-2% of plasma TTR circulates bound to HDL and that the association of TTR to the HDL vesicle occurs through binding to apolipoprotein AI (apoAI) (10). Also, we showed that in the kidney, megalin, a member of the lipoprotein receptor family (LDLr) is responsible for TTR uptake (11). It is thus possible that TTR and lipoproteins share related uptake pathways. The murine scavenger receptor class B type I (SR-BI), has been identified as the receptor that enables cells to capture cholesterol from HDL particles in the bloodstream (12). SR-BI recognizes the apolipoproteins on the surface of the HDL particle and mediates thereby selective HDL lipid uptake (13). Based on the association of TTR with HDL, it would be possible that SR-BI could act as a TTR-receptor. Alternatively, receptors of the LDLr family, other than megalin, and preferentially expressed in the liver (the major site of TTR degradation), such as LDLr and the LDL receptor-related protein (LRP) could mediate TTR internalization. It is interesting to note that all these receptors bind to a chaperone-like molecule, the receptor-associated protein (RAP) (14, 15).

Here we report the various results obtained for TTR uptake using different cell lines and systems and elucidate a possible common pathway between lipoproteins and TTR uptake.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines, Primary Hepatocyte Cultures, and Cell Culture-- LDLr-deficient Chinese hamster ovary cells overexpressing SR-BI (IdLA7-mSRBI) and the equivalent wild-type cells (IdLA7-wt) were a kind gift of Dr. Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA). Wild-type mouse embryonic fibroblasts (MEF1) and equivalent double knockout cells for the LDLr and for LRP mouse embryonic fibroblasts (MEF4) were kindly provided by Dr. Miguel Seabra (Imperial College, London). Single knockout fibroblasts for LRP (MEF2) or LDLr (MEF3) were provided by Dr. Ulrike Beisiegel (Universitatskrankenhaus Eppendorf, Hamburg, Germany). Rat hepatomas (FAO) were obtained from the American Tissue Culture Collection (Manassas, VA). A human hepatoma line not expressing TTR (SAHep) was obtained from Dr. João Monjardino (Imperial College, London). All cell lines were propagated in 25-cm2 flasks in monolayers and maintained at 37 °C in a humidified atmosphere of 95 and 5% CO2. MEF cells were grown in Dulbecco`s minimal essential medium (DMEM, Life Technologies, Inc.); IdLA7, FAO and SAHep were grown in F12-Coon`s (Sigma). All cell culture media were supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 100 units/ml penicillin (Life Technologies, Inc.). When needed, G418 was used at 500 µg/ml. Cultures were kept at a logarithmic phase of growth by splitting 1:3 with 0.025% trypsin-EDTA (Life Technologies, Inc.). Primary hepatocytes from ttr knockout mice (18) were prepared by perfusing the liver of anesthesized animals with 75 ml of liver perfusion medium (Life Technologies, Inc.) containing 200 units/ml penicillin (Life Technologies, Inc.), followed by perfusion with 75 ml of DMEM containing 200 units/ml penicillin and 1 mg/ml collagenase type H (Roche Molecular Biochemicals). Perfused livers were removed, minced using cell scrapers, and subsequently passed through 70-µm nylon cell strainers (Falcon). Liver cells were centrifuged at 400 rpm for 3 min and resuspended in hepatocyte attachment medium (Life Technologies, Inc.). Cells were plated in two 12-well plates/mouse liver and maintained at 37 °C in a humidified atmosphere of 95 and 5% CO2 for 2 h after which the attachment medium was replaced by hepatozyme serum-free medium (Life Technologies, Inc.). Experiments were performed 16 h later.

Proteins and Antibodies-- Serum TTR was purified as described (16). Recombinant TTR was purified from Escherichia coli D1210 transformed with plasmids carrying either wt TTR (pINTRwt) or the suitable mutant TTR cDNA (pINTR30, pINTR119, and pINTR55) also according to Almeida et al. (16). Serum RBP was isolated by affinity chromatography in a TTR column and saturated with all-trans retinol (Sigma) as described (10). TTR was iodinated as previously reported (10). Specific activities were determined after each iodination, and characteristic specific activities were about 104 cpm/ng. 125I-TTR was complexed with either RBP or T4 according to Sousa et al. (10). An expression vector for rat RAP was a kind gift from Dr. Miguel Seabra (Imperial College, London). Recombinant RAP was expressed and purified as previously described (17). Plasma lipoproteins either from human plasma or from ttr knockout mice plasma (18) were isolated by vertical rotor ultracentrifugation (Beckman) in KBr density gradient: LDL (d = 1.019-1.063) and HDL (d = 1.063-1.21) (19). Bottom fractions (delipidated serum) were dialyzed overnight for subsequent studies. Human apolipoproteins AI, AII, and E were from CalBiochem. Goat anti-apoAI and anti-apoB were from CalBiochem. Polyclonal antibodies against megalin and cubilin were a kind gift from Dr Pierre Verroust (INSERM, Paris, France).

Binding Assays-- Approximately 200,000 cells/well, counted in a hemacytometer, were seeded in 12-well cluster dishes and used for experiments 2-3 days after plating (~80% confluency). Prior to the uptake experiments, cells were left in the appropriate medium without fetal bovine serum for 16 h. Before the incubations, the monolayers were rinsed two times with ice-cold DMEM. After incubation for the appropriate time with the indicated amount of 125I-TTR (200,000 cpm/well unless otherwise stated) in 0.5 ml of DMEM containing 0.2% ovalbumin, the supernatant was separated and the cells were detached with 250 µl of 0.025% trypsin in 1 mM EDTA (Life Technologies, Inc.) for 5 min at 37 °C. Cells were separated by centrifugation at 14,000 rpm for 5 min after which the activities in the pellet (internalized) and in the supernatant (surface bound) were counted. Specific internalization or surface-bound radioactivity was obtained by subtracting the value obtained in the same conditions but in the presence of 100-fold molar excess of unlabeled TTR. Degradation of labeled protein was measured by precipitation of the incubation medium in 10% trichloroacetic acid. In all experiments, a control was included in which the amount of degradation was assessed in the absence of cells. The counted radioactivity in each experiment was corrected according to the specific activity of the protein used in each assay. Values shown are the mean of duplicates of three independent experiments. In displacement experiments, apoAI, apoAII, apoE, RAP, RBP, LDL, and HDL were used to a final concentration of 1 µM unless otherwise stated. Dextran sulfate (final concentration of 10 mg/ml) and heparin (0.1 mg/ml) were added in specific experiments.

Cross-linking of TTR to Intact Cells-- SAHep were grown as previously described using 75-cm2 flasks until 80% confluence. Cells were left 16 h prior to the experiment in F12 Coon`s medium without fetal bovine serum. Cell monolayers were washed twice with ice-cold PBS and incubated with Hank`s balanced salt solution (Life Technologies, Inc.) with 50 mM HEPES and ~106 cpm of 125I-TTR or 100 µg/ml TTR for 3 h at room temperature. After incubation with tracer, cell monolayers were washed five times with ice-cold PBS with 1 mM phenylmethylsulfonyl fluoride (Sigma) and 0.1 mM leupeptin (Roche Molecular Biochemicals). 2 ml of PBS with 1 mM 3,3'-dithiobis (propionic acid N-hydroxysuccinimide ester) (DTSP) (Sigma), made 100-fold concentrated in dimethylsulfoxide (Me2SO) was added to cell monolayers to a final concentration of 1 µM to cross-link TTR to its cell surface receptor. Incubation proceeded 1 h at room temperature. As negative controls, flasks without added cross-linker or with 100× molar excess of unlabeled TTR for experiments using 125I-TTR were used. For competition with RAP, this protein was used at a final concentration of 5 µM. The cross-linking reaction was quenched by addition of Tris to a final concentration of 10 mM. Cells were scraped from flasks in 2 ml of PBS with 1 mM phenylmethylsulfonyl fluoride and pelleted by centrifugation 5 min at 3,000 rpm. Cell pellets were resuspended in 100 µl of membrane solubilization buffer (50 mM TrisCl, pH 7.4; 0.5% octylglucoside; 1% Nonidet-P40; 1 mM CaCl2; 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM leupeptin), passed through a 26-G syringe and left on ice for 1 h. Solubilized components were separated from cell debris by centrifugation at 14,000 rpm for 10 min. Supernatants were collected, SDS sample buffer was added, and 30 µl of each sample were separated on 12% SDS-PAGE gels. For experiments with 125I-TTR, gels were dried and exposed at -70 °C for 5 days for autoradiography. For experiments with unlabeled TTR, electrophoresis was followed by electrotransfer to nitrocellulose membranes (Hybond), and TTR was detected by Western blots using rabbit anti-human TTR (DAKO).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Saturation and Temperature Dependence of TTR Uptake-- Initial experiments tested time, temperature, and concentration dependence of 125I-TTR uptake in the different cell lines FAO and MEF1, which correspond to the main tissues of TTR degradation in the rat, i.e. liver and skin, respectively. Iodinated protein migrated as a monomer and dimer on 15% SDS-PAGE and as a single band on native gels (not shown). As expected considering that the liver is the major site of TTR catabolism, 125I-TTR uptake by FAO was much higher when compared with MEF1 (Fig. 1A). A maximum TTR uptake in FAO occurred at 2.5 h (Fig. 1A) with specific internalization of 40% relative to total activity. Specific internalization at each time point was determined by subtracting internalized 125I-TTR from the values obtained under the same conditions but in the presence of a 100-fold molar excess of unlabeled TTR. More than 80% of internalization inhibition was observed in the presence of 100-fold molar excess unlabeled TTR, proving that TTR uptake by FAO and MEF1 cells is specific. Saturable high affinity binding to FAO at 37 °C was demonstrated by Scatchard analysis and indicated a single class of sites with a Kd of 4.3 nM (Fig. 1B). Interspecies validation was obtained by testing the uptake of TTR by human hepatoma cells (SAHep), and essentially the same results as for the rat hepatomas were obtained (Fig. 1C). The similar uptake of human TTR by human and rat hepatomas is consistent with the high evolutionary conservation of the protein. Degradation experiments were performed with SAHep and in accordance with an endocytic process, radiolabeled degradation products appeared in the medium (Fig. 1C). To confirm that results obtained with the hepatoma lines represent a mechanism found in normal hepatocytes, the binding studies were repeated using primary cultures of hepatocytes from ttr knockout mice. In these hepatocytes, essentially the same results that had been previously observed with SAHep and FAO cell lines were obtained (Fig. 1C).



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Fig. 1.   A, time dependence of TTR uptake in MEF1 and FAO cell lines. B, Scatchard analysis of 125I-TTR internalization in FAO at 37 °C. Cells were incubated for 3 h with 125I-TTR and increasing concentrations of nonisotopic TTR. Data were corrected for nonspecific binding. C, time course of internalization of 125I-TTR (black-square) and increase in trichloroacetic acid-soluble 125I-labeled degradation products () in the medium using SAHep (---) and primary hepatocytes from ttr knockout mice (---) at 37 °C.

Influence of TTR Ligands (T4 and Retinol) on the Uptake of Protein-- To characterize the influence of TTR ligands on the internalization of the protein, we performed comparative studies of TTR uptake using either TTR alone or complexed to T4 and RBP (Fig. 2A). Only data obtained with FAO are shown although SAHep and MEF1 were also tested and presented similar results. RBP saturated with retinol and complexed with 125I-TTR led to a 70% decrease of TTR entry into cells suggesting that this ligand inhibits binding of TTR to its cellular receptor. On the other hand, 125I-TTR·T4 led to a 20% increased uptake. It is interesting to note that a similar inhibitory effect of RBP on the uptake of 125I-TTR complexed to T4 was observed (data not shown). This result was expected, given the high inhibitory effect of RBP on TTR uptake when compared with the increase produced by T4.



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Fig. 2.   A, influence of T4 and RBP on TTR uptake. FAO cells were incubated with 125I-TTR alone (TTR) or complexed to T4 (TTR-T4) or RBP (TTR-RBP) for 3 h at 37 °C after which specific internalization of 125I-TTR was measured. B, uptake of different mutant TTRs. FAO cells were incubated with different mutant TTRs at 37 °C for 3 h. Data were corrected for the specific activity of each of the iodinated proteins.

Uptake of Different TTR Mutants-- Different TTR mutants, both amyloidogenic nonamyloidogenic were assayed. Nonamyloidogenic TTRs included TTR T119M; mildly amyloidogenic, TTR V30M/T119M; amyloidogenic, TTR V30M and highly amyloidogenic TTR L55P. Only data obtained with FAO are shown although SAHep and MEF1 were also tested and showed similar results. No direct correlation was observed between TTR amyloidogenicity and TTR uptake (Fig. 2B). It is though interesting to note that L55P, the most aggressive FAP-related TTR mutation did not enter cells whereas T119M, the nonamyloidogenic mutant presented the highest degree of internalization. The possibility that the 125I-TTR L55P variant could be in an aggregated form (therefore not able to bind the cellular membrane) was ruled out because it presented a similar migration pattern under native conditions as wt 125I-TTR protein (data not shown). Another interesting feature was that the compound heterozygote V30M/T119M showed approximately a mean internalization of V30M and T119M when tested separately (Fig. 2B). Thus, the conformation of the protein seems to be important for cell interaction.

Chemical Cross-linking of 125I-TTR to SAHep Cellular Membranes-- Cross-linking of 125I-TTR to intact SAHep was carried out followed by subsequent membrane isolation and detection of cell-surface components covalently bound to 125I-TTR. The cross-linker used in this assay was DTSP, a thiol-cleavable, membrane insoluble, homobifunctional N-hydroxysuccinimide ester. By using this approach, we visualized a complex of ~90 kDa corresponding to TTR bound to its receptor (Fig. 3, lane 4). The specificity of the cross-linked band was shown by performing the same experiment both in the absence of cross-linker (Fig. 3, lane 1) and in the presence of 100× molar excess unlabeled TTR (Fig. 3, lane 3).



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Fig. 3.   12% SDS-PAGE analysis under nonreducing conditions of crude membrane extracts of SAHep cross-linked to 125I-TTR. SAHep were incubated with 125I-TTR in the absence of cross-linker (lane 1); 125I-TTR (50,000 cpm) cross-linked with 1 µM DTSP (lane 2); SAHep incubated with 125I-TTR and 1 µM DTSP in the presence (lane 3) and absence (lane 4) of 100× molar excess unlabeled TTR.

Effect of Lipoproteins on TTR Uptake-- We have previously established that a fraction of TTR circulates in plasma associated with HDL, and that binding of the protein to this lipoprotein vesicle occurs through apoAI (10). To assess the influence of lipoproteins on uptake and/or affinity of TTR to different lipoprotein receptors, we performed experiments in the presence and absence of isolated lipoproteins. We first assessed if major lipoproteins and/or lipids were present in cell culture medium despite the serum-free conditions used in the assays. We measured apoAI and apoB by enzyme-linked immunosorbent assay and determined cholesterol levels in conditioned medium from SAHep cells. No detectable amounts were observed by these sensitive assays. In addition, we used lipoprotein-deficient serum from ttr knockout mice, and no significant inhibition in uptake was observed (Fig. 4) corroborating that TTR uptake is not lipid- or lipoprotein-dependent or caused by TTR-lipoprotein complexes. However, in all cell lines both LDLs and HDLs were able to displace TTR binding, with the HDLs as more efficient (Fig. 4). To ascertain if the observed competition was attributed to the lipoprotein vesicle itself or to its TTR content, we performed the same assays in FAO using HDLs isolated from ttr knockout mice plasma and observed that the lipoprotein vesicle depleted of TTR was still able to inhibit TTR internalization (Fig. 4). As TTR association with HDL occurs through binding to apoAI, we tested the ability of isolated apoAI to displace TTR uptake but no effect was observed. Further, we also tested apoAII and apoE (other apolipoproteins present in HDL) and again no competition with TTR internalization was observed (data not shown). These results suggest that the integrity of the lipoprotein vesicle is crucial for displacement of TTR binding to cellular surfaces.



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Fig. 4.   Effect of lipoproteins on TTR uptake. FAO cells were incubated with 125I-TTR in the presence of either 1 µM HDL, LDL, lipoprotein-deficient serum from ttr knockout mice (lip def) or HDL from ttr knockout mice (ttr def HDL) for 3 h at 37 °C after which TTR internalization was assessed.

Involvement of Lipoprotein Receptors in TTR Uptake-- As SR-BI is a specific HDL receptor, we next investigated the hypothesis that this receptor might act as a mediator of TTR internalization. By comparing a cell line overexpressing SRBI (IdLA7-SRBI) with a wt control cell line (IDLA7-wt), no differences in the kinetics and extent of internalization were observed (data not shown). Therefore, despite its association with HDL and the ability of HDL to displace TTR uptake, TTR internalization is not occurring through SR-BI.

To test if one of the members of the LDLr family expressed in the liver (LDLr and LRP) was involved in TTR internalization, comparative studies of TTR uptake using different cell lines of mouse embryonic fibroblasts were performed: MEF1 (wt fibroblasts), MEF2 (LRP-deficient fibroblasts), MEF3 (LDLr-deficient fibroblasts) and MEF4 (deficient for both the LDLr and LRP). Again, no difference was seen in the kinetics and extent of uptake between the different cell lines (data not shown), suggesting that none of these receptors participates in TTR uptake.

Despite the negative results obtained for the LDLr and LRP and to further investigate the possible involvement of lipoproteins in TTR cell internalization, we studied the effect of RAP on this process. RAP is a protein that is known to bind all the members of the LDLr family. TTR uptake was found to be RAP-sensitive, since the internalization of the protein was virtually abolished by the presence of 1 µM RAP in the cell culture medium (Fig. 5A). To confirm that RAP sensitivity observed in cell lines does not represent a mechanism not present in normal cells, primary cultures of hepatocytes from ttr knockout mice were also tested. In these normal hepatocytes, essentially the same results that had been previously observed with hepatomas were obtained, including RAP sensitivity (Fig. 5A). It is also noteworthy that MEF1, MEF2, MEF3, and MEF4 presented similar inhibition of TTR uptake in the presence of RAP (data not shown) suggesting not only that LDLr and LRP are not involved in TTR uptake but also that an alternate RAP-sensitive pathway exists in these cell lines. To further ascertain that TTR uptake is RAP-sensitive, we repeated the cross-linking experiments of TTR with hepatomas, but now using RAP in the cell culture medium as a competitor. Using these conditions (Fig. 5B), no complex between TTR and its receptor was found, thus showing the RAP sensitivity of the internalization process.



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Fig. 5.   A, displacement of 125I-TTR internalization by RAP in MEF1, FAO, and primary hepatocytes from ttr knockout mice. Increasing concentrations of RAP were added to cells incubated with 125I-TTR for 3 h at 37 °C, after which specific internalization was determined. B, 12% SDS-PAGE analysis of crude membrane extracts of SAHep cross-linked to TTR. SAHep were incubated with TTR in the absence (lane 1), or presence of cross-linker and run under nonreducing (lane 4) or reducing (lane 2) conditions. Competition with 5 µM RAP was performed (lane 3), leading to disappearance of the ~90-kDa TTR-receptor complex. C, effect of dextran sulfate and heparin on TTR internalization. MEF1 or FAO were incubated with 125I-TTR in the presence of either 10 mg/ml dextran sulfate or 0.1 mg/ml heparin for 3 h at 37 °C. Representative results obtained for internalization of 125I-TTR with FAO are shown.

The receptor-mediated catabolism of several ligands of RAP-sensitive receptors is thought to be a two step process in which initial cell-association of the ligand is mediated by cell-surface proteoglycans (20, 21). This step is followed by subsequent receptor-mediated internalization. The initial step is inhibited by certain molecules such as heparin and dextran sulfate, which bind to cell-surface proteoglycans. We therefore examined the effect of heparin and dextran sulfate in 125I-TTR-cell interaction (Fig. 5C). The results revealed that the presence of heparin and dextran sulfate prevents TTR binding to the cellular surface, suggesting that cell-surface proteoglycans may contribute to the cellular uptake of the protein.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our experiments provide direct evidence for the existence of a specific receptor for TTR on hepatocytes and fibroblasts. Because the liver is the major site of catabolism of the protein, the existence of high affinity and saturable binding sites on hepatomas was expected. TTR had already been found to bind to HepG2 (a human hepatoma cell line) by a receptor-mediated process (5). However, in this cell line, TTR is synthesized and secreted into the cell medium (22) and could interfere with a proper assessment of the binding capacity and affinity because the amount of iodinated TTR used is on the nanogram range as are the levels of TTR secreted by HepG2 (~150 ng TTR/106 cells) (22). For this reason, all our assays were performed under serum-free conditions and with cells that do not produce TTR. Interestingly, our results in hepatomas that do not produce TTR and in primary cultures of hepatocytes from ttr knockout mice showed a single class of high affinity binding sites with a Kd of ~4 nM at 37 °C, which is effectively in the same range as the one previously reported (~10 nM). This Kd indicates that the receptor is saturated with the normal plasma TTR concentration of 5 µM and that TTR can be taken up efficiently from much lower concentrations existing in the extracellular fluid.

To assess the influence of lipoproteins on TTR internalization, some uptake experiments were performed using lipoprotein-deficient serum from ttr knockout mice and as expected, no significant inhibition of uptake was observed nor differences in binding constants were obtained. We therefore concluded that the measured TTR uptake does not represent TTR-lipoprotein complex internalization, although both HDL and LDL are able to inhibit TTR uptake. It is also noteworthy that only a small fraction of TTR (1-2% of the total plasma TTR pool) circulates bound to HDLs and that there are even some individuals that are devoid of TTR bound to lipoproteins (10). It is therefore unlikely that this small fraction of TTR bound to lipoproteins significantly accounts for the physiological turnover of the protein. At this point we cannot however exclude that the TTR·HDL complex might be internalized by an alternative pathway, namely involving one of the members of the LDLr. However, in this event, the physiological relevance would have to be explained because of the small amount of TTR bound to lipoproteins.

In this study we show that RAP, a multifunctional ligand binding all members of the LDLr family antagonizing their ligand binding activity, inhibits TTR uptake. The inhibition was observed in different cell lines and in primary hepatocytes. This finding was reinforced by the fact that the ~90-kDa TTR-receptor complex observed by cross-linking on hepatoma cells was also RAP-sensitive. We had already demonstrated (11) that in the kidney, TTR uptake occurring through megalin, is a RAP-sensitive process. However we observed that in the case of cells from liver origin, none of the candidate members of the LDLr family were involved in TTR uptake. We conclude that TTR uptake is occurring via another yet unidentified RAP-sensitive receptor. It has been suggested (23) that the only RAP-sensitive receptors present in the liver are the LDLr and the LRP. This was based on the evidence that in LDLr and LRP double knockout mice, no RAP binding could be detected in the liver using both membrane binding studies and ligand-blotting experiments. However it is possible that RAP-sensitive receptors were not identified because of low receptor binding affinities. For instance, isolated membranes may present partially destroyed and/or functionally defective receptors. The involvement of other RAP-sensitive receptors such as megalin and cubilin, which are expressed on the apical surfaces of many specialized absorptive epithelia (24, 25), was highly unlikely because these receptors are not physiologically expressed in the liver. We did however perform Western blot analysis for megalin and cubilin on cellular extracts of both hepatoma cell lines used and found that these cell lines do not express megalin nor cubilin (data not shown). It is also noteworthy that the high molecular weight of megalin and cubilin is not compatible with the ~90-kDa band we obtained by cross-linking for the receptor here reported. The ~90-kDa TTR-receptor complex identified, corresponds to a lower molecular weight receptor than the ~100-kDa TTR receptor previously described (6, 8). Because TTR is a homotetramer (determined by cross-linking assays), it is difficult to predict which form of the protein will preferentially bind the receptor. Although the kinetics of the interaction of TTR with its receptor are identical to the ones described for other cell types (5-8), we cannot exclude at this point that the TTR receptor here described is different from the receptors reported in oocytes and ependymomas.

We observed that complexation of TTR to T4 led to a 20% increased cell surface binding, which agrees with previous studies (5). The mechanism by which T4 increases TTR uptake is probably due to slight conformational changes that this ligand induces in TTR (26). Because only a small proportion of TTR molecules carry T4, a supraphysiological concentration of the hormone would be required to demonstrate stimulation of TTR uptake. Moreover, TTR does not appear to play a role in the delivery of T4 to the tissues; thus, studies performed on ttr knockout mice revealed that TTR is not essential for T4 to reach tissues, namely the liver (27). Given the 20% increased uptake of the TTR·T4 complex, we would predict that the addition of lipoproteins that induce a high inhibition of TTR uptake would still be able to reduce the uptake of the TTR·T4 complex. However, methodologically as T4 also binds lipoproteins, competition experiments using the TTR·T4 complex, and lipoproteins would be difficult to interpret.

On the other hand, RBP·TTR led to a 70% decreased uptake, which shows that TTR is preferentially taken up in its non-RBP complexed form. This can be explained by several hypotheses: steric hindrance by RBP of TTR binding to its receptor or even the possibility that the interaction sites of TTR with RBP and with its receptor are overlapping. RBP had the same inhibitory effect on the TTR·T4 complex, which was predictable given the high inhibitory effect of RBP on TTR uptake when compared with the increase produced by T4. Conflicting results have been published with regard to the effect of TTR on RBP binding to its receptor; Sivaprasadarao et al. (28) reported TTR inhibition of RBP uptake whereas Bavik et al. (29) showed that TTR has no effect on RBP internalization. Our results on TTR uptake are in agreement with the first observation. Under physiological conditions where TTR and RBP circulate as a complex, prior to TTR and/or RBP uptake, it is probable that the two proteins dissociate because they are internalized by distinct pathways.

As already mentioned, several point mutations in TTR are related to the extracellular deposition of amyloid fibrils. TTR plasma levels are decreased in FAP V30M patients (30), despite an equal expression of the variant and normal TTR in the liver (31). This suggested that TTR metabolism could be related to the amyloid formation process. Moreover, higher TTR levels were found in carriers of the nonpathogenic TTR T119M mutation. Comparative in vivo clearance studies of TTR V30M and TTR T119M have been performed (32) and showed a slower clearance for TTR T119M and a faster one for TTR V30M. When the TTR V30M/T119M was studied, it showed a similar behavior as normal TTR. This led to the hypothesis that, at least in part, a different clearance could account for the differences in circulating plasma levels observed for each of the mutations. In our uptake studies however, no direct correlation was observed between TTR amyloidogenicity and TTR uptake. This can be explained by the fact that an in vivo system comprises complex and different pools not represented in a cellular system such as the ones used in our assays. TTR mutants were used to evaluate binding to megalin (11). It is interesting to note that the mutant with a faster uptake in the kidney (V30M) corresponds in this study to a mutant with a low cell association. This not only shows that the receptors involved in TTR uptake in the kidney and liver are different, but also suggests that the turnover of the protein in the body is probably the overall result of processes mediated by different organ-specific receptors.

The membrane protein bound to TTR is now under study to further isolate the TTR receptor. The final identification of a TTR receptor should be useful in further studies of transport function and metabolic effects of TTR and possibly with respect to the pathological deposition of TTR variants. The reason why both LDL and HDL particles are able to inhibit TTR uptake, despite the lack of interaction of TTR with any of the described LDL or HDL receptors present in the liver, remains to be further elucidated. Recently, RAP has been shown to interact with a growing number of proteins, including several that are unrelated to the LDL receptor family as well as new members of this rapidly expanding family (33).


    ACKNOWLEDGEMENTS

We thank Paul Moreira for the production and purification of recombinant TTR and Dr. Isabel Alves for expertise in preparing mouse primary hepatocyte cultures.


    FOOTNOTES

* This work was supported by Grant 2/2.1/BIA/459/94 from PRAXIS XXI in Portugal. Part of this work was presented as an oral communication at the VIII International Symposium on Amyloidosis; de Sousa, M., and Saraiva, M. J. Characterization of Receptor-mediated Internalization of Transthyretin, Mayo Clinic, August, 1998.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.

§ Recipient of Postdoctoral Fellowship PRAXIS XXI/BPD/22027/99 from Fundação para a Ciência e Tecnologia from Portugal.

|| To whom correspondence should be addressed: Amyloid Unit-IBMC, R. Campo Alegre, 823 4150 Porto, Portugal. Tel.: 351-22-6074900; Fax: 351-22-6099157; mjsaraiv@ibmc.up.pt.

Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M010869200


    ABBREVIATIONS

The abbreviations used are: TTR, transthyretin; FAP, familial amyloidotic polyneuropathy; HDL, high-density lipoprotein; LDLr, low-density lipoprotein receptor; LRP, LDLr-related protein; RAP, receptor-associated protein; RBP, retinol-binding protein; SR-BI, murine scavenger receptor type B class I; T4, thyroxine; DMEM, Dulbecco's modified Eagle's medium; wt, wild-type; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; MEF, mouse embryonic fibroblasts.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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