0021-972X/90/7105-1265$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 71, No. 5 Printed in U.S.A.
Transthyretin Receptors on Human Astrocytoma Cells* CELIA M. DIVINO AND GEORGE C. SCHUSSLER Division of Endocrinology, Department of Medicine, State University of New York Health Science Center, Brooklyn, New York 11203
ABSTRACT. Transthyretin (TTR), a transport protein for T4 and retinol-binding protein, is the principal T4-binding protein of cerebrospinal fluid. Its function in regard to the delivery of its ligands and in other respects is unclear. The binding of [125I] TTR to cultured human astrocytoma cells was studied to determine whether these cells carry receptors for TTR. Scatchard analysis was consistent with a single class of binding sites with a Kd of 3 nM. No significant cross-reactivity with transferrin or serum albumin was observed. Internalization of TTR was temperature dependent and proportional to receptor occupancy.
T
RANSTHYRETIN (TTR: T4-binding prealbumin) is the main export protein of the choroid plexus, which secretes most, if not all, of the TTR that enters the cerebrospinal fluid (CSF) (1, 2). The concentration of TTR in CSF is less than 10% of that in plasma, but this is relatively high compared to the concentrations of T4-binding globulin and albumin (3). As a result, although TTR binds only 15% of plasma T4, it is the principal T4-binding protein of the CSF (3, 4). The nature of the interaction between TTR and the cells of the central nervous system is not known. The present study was undertaken to determine whether astrocytes have binding sites for TTR similar to those that have been found on hepatocytes (5). The astrocyte appears to be a target cell for T4's effect and is, therefore, of interest with regard to the hormonal transport function of TTR. It has been shown recently that, in addition to providing substrate for the type II 5'-deiodinase, T 4 directly stimulates actin polymerization in astrocytes (6).
Materials and Methods Purified human TTR was obtained from Calbiochem (La Jolla, CA). [125I]TTR was prepared as previously described (5). Transferrin (Fe-saturated), crystallized human serum albumin (HSA), BSA, bovine 7-globulin, EDTA, 2-deoxyglucose, peniReceived May 4, 1990. Address all correspondence and requests for reprints to: Dr. George C. Schussler, Box 21, State University of New York, Health Science Center, 450 Clarkson Avenue, Brooklyn, New York 11203-2098. * This work was supported by the Boots Co., Inc., Lincolnshire, IL NIH Grant 5R01-AM-32436, and a grant from the Division of Research Resources SUNY-Health Science Center at Brooklyn.
Dilutions of cerebrospinal fluid displaced [125I]TTR in proportion to their content of radioimmunoassayable TTR and in parallel with purified TTR. The uptake and internalization of TTR increased in the presence of high T4 or T 3 concentrations. These results demonstrate that TTR binds to specific high affinity receptors on human astrocytoma cells. Receptor binding of TTR provides a potential mechanism for the delivery of its ligands within the central nervous system. (J Clin Endocrinol Metab 7 1 : 1265-1268, 1990)
cillin, T4, and T 3 were obtained from Sigma Chemical Co. (St. Louis, MO). The HSA was further purified on polyacrylamide gel electrophoresis to remove residual TTR contamination (5). Rabbit antihuman TTR from Behring Diagnostics (La Jolla, CA) was used for RIA at a dilution of 1:20,000. Antibody-bound tracer was precipitated by 14% polyethylene glycol in the presence of 200 mg/dL bovine 7-globulin. Sodium azide was purchased from Fisher Scientific (Pittsburgh, PA). Minimum Essential Medium (MEM), fetal bovine serum, trypsin, and sodium pyruvate were obtained from Gibco (Grand Island, NY). CSF was obtained from the residual of a sample that had been examined by the routine laboratory and found to have no abnormalities. U-673, a human astrocytoma cell line from the American Type Culture Collection (Rockville, MD), was grown in monolayers in MEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/dL sodium pyruvate. The cells were seeded in 24-well cluster dishes and grown for 2-3 days until they were confluent. There were approximately 200,000 cells/well, as determined by counting the detached cells in a hemocytometer. Before the incubations, the monolayers were rinsed three times with ice-cold MEM, leaving the last wash for 30 min. After incubation with [125I]TTR in MEM the supernatant was separated, and the cells were rinsed, twice with ice-cold phosphate-buffered saline (PBS) and once with ice-cold PBS-0.1% BSA. The cells were detached with 250 ML 0.025% trypsin in 1 mM EDTA and then transferred to counting tubes in 1 mL of 1% BSA, with a 500-/tL wash. Uptake was calculated as the ratio of cell-associated activity to the activity in 100 nL supernatant. To measure internalization, monolayers were rinsed once with ice-cold PBS, and the surface-bound tracer was removed by proteolytic digestion (7) with 1 mL 0.25% trypsin for 10 min at 4 C in the presence of 10 mM 2deoxyglucose and 10 mM sodium azide to inhibit further inter-
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nalization. The cells were transferred to Eppendorf centrifuge tubes and centrifuged at 12,000 rpm for 2 min at 4 C. The activity remaining with the pellet was considered to be internalized.
Results The uptake of [125I]TTR by astrocytoma cells reached equilibrium at 2 h. No metabolism of [125I]TTR was detected by trichloroacetic acid precipitation or polyacrylamide gel electrophoresis during incubations of up to 4 h. Displacement studies were consistent with a single class of binding sites (Fig. 1). The average Kd, determined by Scatchard analysis in three studies at 37 C, was 3.5 nM (range, 2.9-4.0 nM), with a capacity of 236,000 sites/ cell (range, 212,000-275,000). At 0 C the Kd was 2.8 nM (n = 3; range, 1.9-4.1 nM), and the capacity was 85,000 sites/cell (range, 65,000-119,000). Internalization, determined as the trypsin-sensitive fraction of TTR uptake (7), was reduced, but not completely inhibited, at 0 C. The failure to observe complete inhibition of internalization at 0 C may have been due to internalization occurring during trypsin treatment at 4 C, despite the presence of metabolic inhibitors. Because of some variability in uptake from different seedings of U-673, internalization at 0 and 37 C was compared with simultaneous studies in matched multiwells. In two studies, the proportion of TTR (1.8 nM) that was trypsin insensitive at 37 us. 0 C was 0.57 ± 0.04 (SD) US. 0.20 ± 0.05 (P < 0.001) and 0.36 ± 0.04 us. 0.19 ± 0.04 (P < 0.005). Therefore, the apparently higher capacity at 37 C than at 0 C was at least partially due to greater internalization of TTR at 37 C. Analysis of surface binding, determined as the trypsin-sensitive component of total uptake (7), was consistent with a similar number of cell surface receptors at 0 and 37 C (Fig. 1, inset). Internalization was propor-
JCE & M • 1990 Vol 71 • No 5
tional to surface binding, with a correlation coefficient of 0.93 in the displacement study shown in Fig. 1. The steeper slope of the regression for surface than total binding at 37 C suggests that the proportion of internalized TTR may increase as the surface receptors are saturated. There was greater variability of individual determinations with this technique than with the measurement of whole cell uptake, and it is not certain that the entire difference in the calculated binding capacity at 0 and 37 C can be attributed to internalization. The binding site for TTR did not cross-react with transferrin, and there was little cross-reactivity with repurified HSA (Fig. 2). Dilutions of a normal CSF displaced [125I]TTR in parallel with proportional changes in the concentration of purified TTR (Fig. 3). Displacement by CSF was consistent with its 0.4 jiM radioimmunoassayable TTR content. At concentrations beyond the physiological range, T4 increased the uptake of TTR (Fig. 4). These studies were 1.0
o
Transferrin
0.8
CD
m
on
HSA
TTR 0.6
O 0.4 0.2 0.0
-9
-8
-7
-6
-5
Competitor (log M)
FIG. 2. Binding specificity. There was no significant cross-reactivity of the binding site with human transferrin and only minimal crossreactivity with HSA, which probably represented a very low level of residual TTR contamination. CSF percent 0.04 1.0-
0.10
0.02
0.04
0.06
10.00
• — • TTR O — O CSF nonspecific binding
0.8-
o.oo
1.00
0.08
picomoles/l
FIG. 1. Scatchard analysis of binding at 0 and 37 C. Cells were incubated for 2 h with [125I]TTR and increasing concentrations of nonlabeled TTR. The cell/medium ratios are for approximately 200,000 cells/100 (iL medium. O, Total cell binding at 37 C; A, total cell binding at 0 C. The corresponding filled symbols (inset), indicate cell surface binding, determined as the trypsin-sensitive fraction of total cell uptake.
0.2 ••
0.0 0.4
1.0
10.0
100.0
TTR nM
FIG. 3. Displacement of [125I]TTR by TTR and CSF. The displacement curves are parallel. Nonspecific binding was determined at a TTR concentration of 1 ^M.
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TTR RECEPTORS 125
I TTR B/Bo 3.0
T
2.5 2.0 1.5 1.0
•T
0.5 0.0-1-7
-6
-5
lodothyronine (log M)
FIG. 4. Effects of T4 and T3 on the cellular uptake of TTR. Each value is the mean ± SD of four determinations of TTR uptake, expressed as the ratio of cellular TTR (B) to total cellular TTR in the absence of iodothyronines (Bo). Control values for total uptake are 1 by definition; control values for internalized TTR are shown on the ordinate. Total TTR is significantly increased at all concentrations of T4 shown (P < 0.005 at 1.3 X 10"7 and P < 0.001 at higher concentrations). Internalized TTR is also increased at all concentrations (P < 0.001). The effect of T3 is less pronounced, with a significance of P < 0.001 achieved at 6.4 x 10"7 M for total TTR uptake and at 12.9 x 10"7 M for internalized TTR.
carried out in the presence of 5.5 nM TTR and 2.7 ^M HSA. As might be anticipated on the basis of the higher affinity of TTR for T4 compared to T 3 (8, 9), the latter is less effective than T4. Stimulation of TTR uptake by both T4 and T 3 was associated with an increase in internalized TTR.
Discussion These studies demonstrate that TTR binds to specific high affinity (Kd = 3 nM) sites on human astrocytoma cells. It is likely that similar receptors are present on normal human astrocytes. The internalization of TTR is temperature dependent and proportional to specifically bound TTR. This is consistent with a receptor-mediated energy-requiring process. There is no significant crossreactivity of the TTR-binding site with transferrin or albumin, two structurally unrelated proteins. The minor cross-reactivity with repurified HSA was probably due to a small residual TTR contamination (5). Although TTR carries about 44% of the total CSF T4, only 0.14% of the primary and 0.0013% of the secondary iodothyronine-binding sites on TTR are occupied by T4 at the normal 7.5 X 10~n-M concentration of the free hormone (3).1 The increase in TTR uptake that was 1 The association constants of the primary and secondary TTRbinding sites for T4 are 7 x 107 and 6.7 x 105, respectively (9). From the mass law, the proportion of available sites occupied by T4 can be calculated as: occupied/total sites = ((K^T^)- 1 + I)"1, where K; is the association constant of T4 for site i and [T4] is the concentration of free T4.
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observed at supraphysiological concentrations of T4 may represent amplification of a preferential uptake of the very small proportion of TTR that normally carries T4. A similar effect of T4 has been observed with hepatocytes, where it was temperature dependent and appeared to be specific for TTR (5). Parallel displacement of [125I]TTR by dilutions of purified TTR and CSF indicates that the TTR of the CSF binds to the astrocyte receptor with an affinity similar to that of purified serum TTR. The potency of the CSF relative to purified TTR was consistent with the 0.4-/xM TTR concentration of the sample. With a Kd of 3 nM, receptor saturation should be sensitive to concentrations of TTR in the nanomolar range. The TTR concentration in the microenvironment of the astrocyte is presumably in equilibrium with, but lower than, the TTR concentration of CSF. Thus, in contrast to the hepatic TTR receptor, which is saturated at endogenous TTR concentrations (5), the occupancy of astrocyte TTR receptor may be affected by changes in the TTR concentration of the CSF. There is evidence that the TTR concentration of CSF is physiologically modulated, although unlike serum TTR, it is not responsive to inflammation or caloric deprivation (10, 11). A rapid and specific 2-fold increase in rat choroid plexus TTR mRNA has been found to occur after partial hepatectomy and is thought to be part of an anabolic response supporting liver regeneration (10). In the same study it was observed that the proportion of TTR mRNA in total brain RNA became maximal shortly before maximal brain growth, suggesting that TTR has a function in the development of the central nervous system. TTR binds retinol-binding protein as well as T4 (8, 12). Its role in the delivery of these ligands to target cells remains unclear. Retinol-binding protein is taken up by its own receptor (13,14). Cellular T4 is usually considered to be in equilibrium with the free, rather than the protein-bound, T4 of the extracellular space (15). Nevertheless, specific transport functions of the extracellular T4binding proteins may be of importance. As shown in a model system by Dickson et al. (2), it is likely that a considerable proportion of the T4 that crosses the bloodbrain barrier enters the CSF with TTR synthesized by the choroid plexus. Once in the CSF, the T4-TTR complex probably has a distributive function, assuring availability of T4 to cells throughout the CSF circulation (15, 16). Active uptake of T4 and T 3 by human glioma cells and other neural cell lines has been demonstrated in serum-free medium (17). Although there is little question that free T 4 is available to cells, it has been proposed that there is also direct participation of the binding proteins in T4 uptake (18-20). In vitro studies in our laboratory indicate that in the presence of other T4binding proteins, which reduce the proportion of free
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hormone, TTR increases cellular T4 uptake (19). To a considerable extent, analysis of the exchange of T4 between extracellular and cellular sites has been based on certain characteristics of hormone transport, particularly the capillary transit time, the rate at which T4 dissociates from its protein-binding sites, and cellular uptake from the free T4 pool (15). The interaction of TTR with its receptor should be added to these considerations. The TTR receptor could facilitate T4 uptake by binding the T 4 -TTR complex or by providing a docking function to allow selective release of hormone in the vicinity of the target cell. Such putative functions of the receptor would be of particular importance in the central nervous system, where TTR, rather than T4-binding globulin, is the principal extracellular T4-binding protein.
Acknowledgment We are grateful for the excellent secretarial assistance of Ms. Anna Marie Jichetti.
References 1. Dickson PW, Aldred AR, Marley PD, Bannister D, Schreiber G. Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver. J Biol Chem. 1986;261:3475-8. 2. Dickson PW, Aldred AR, Menting JGT, Marley PD, Sawyer WH, Schreiber G. Thyroxine transport in choroid plexus. J Biol Chem. 1987;262:13907-15. 3. Hagen GA, Elliott WJ. Transport of thyroid hormones in serum and cerebrospinal fluid. J Clin Endocrinol Metab. 1973;37:415-22. 4. Woeber KA, Ingbar SH. The contribution of thyroxine-binding prealbumin to the binding of thyroxine in human serum, as assessed by immunoadsorption. J Clin Invest. 1968;47:1710-20. 5. Divino CM, Schussler GC. Receptor-mediated uptake and internalization of transthyretin. J Biol Chem. 1990;265:1425-9.
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6. Siegrist-Kaiser CA, Juge-Aubry C, Tranter MP, Ekenbarger DM, Leonard JL. Thyroxine-dependent modulation of actin polymerization in cultured astrocytes: a novel, extranuclear action of thyroid hormone. J Biol Chem. 1990;265:5296-302. 7. Karin M, Mintz B. Receptor-mediated endocytosis of transferrin in developmentally totipotent mouse teratocarcinoma stem cells. J Biol Chem. 1981;256:3245-52. 8. Ingbar SH. Observations concerning the binding of thyroid hormones by human serum prealbumin. J Clin Invest. 1963;42:14360. 9. Robbins J, Edelhoch H. Thyroid hormone transport proteins: their nature, biosynthesis and metabolism. In: Ingbar SH, Braverman LE, eds. The thyroid. Philadelphia: Lippincott; 1986;116-27. 10. Fung WP, Thomas T, Dickson PW, et al. Structure and expression of the rat transthyretin (prealbumin) gene. J Biol Chem. 1988;263:480-8. 11. Wade S, Bleiberg-Daniel F, Le Moullac, B. Rat transthyretin: effects of acute short-term food deprivation and refeeding on serum and cerebrospinal fluid concentration and on hepatic mRNA level. J Nutr. 1988;118:199-205. 12. Kanai M, Raz A, Goodman DS. Retinol binding protein: the transport protein for vitamin A in human plasma. J Clin Invest. 1968;47:2025-44. 13. Heller J. Interactions of plasma retinol-binding protein with its receptor. J Biol Chem. 1975;250:3613-9. 14. Sivaprasadarao A, Findlay JB. The mechanism of uptake of retinol by plasma-membrane vesicles. Biochem J. 1988;255:571-9. 15. Mendel CM. The free hormone hypothesis: a physiologically based mathematical model. Endocr Rev. 1989; 10:232-74. 16. Mendel CM, Weisiger RA, Jones AL, Cavalieri RR. Thyroid hormone-binding proteins in plasma facilitate uniform distribution of thyroxine within tissues: a perfused rat liver study. Endocrinology. 1987;120:1742-9. 17. Goncalves E, Lakshmanan M, Pontecorvi A, Robbins J. Thyroid hormone transport in a human glioma cell line. Mol Cell Endocrinol. 1990;69:157-65. 18. Azimova SS, Umarova GD, Petrova OS, Tukhtaev KR, Abdukarimov A. Nature of thyroid hormone receptors. Translocation of thyroid hormone across plasma membrane. Biokhimiya. 1984;49:1350-6. 19. Divino C, Schussler GC. Binding of thyroxine-transthyretin complexes to specific cell surface receptors [Abstract]. Clin Res. 1989;37:357A. 20. Pardridge WM. Plasma protein-mediated transport of steroid and thyroid hormones. Am J Physiol. 1988;252:E157-64.
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Vol. 71, No. 5 Printed in U.S.A.
Transthyretin Receptors on Human Astrocytoma Cells* CELIA M. DIVINO AND GEORGE C. SCHUSSLER Division of Endocrinology, Department of Medicine, State University of New York Health Science Center, Brooklyn, New York 11203
ABSTRACT. Transthyretin (TTR), a transport protein for T4 and retinol-binding protein, is the principal T4-binding protein of cerebrospinal fluid. Its function in regard to the delivery of its ligands and in other respects is unclear. The binding of [125I] TTR to cultured human astrocytoma cells was studied to determine whether these cells carry receptors for TTR. Scatchard analysis was consistent with a single class of binding sites with a Kd of 3 nM. No significant cross-reactivity with transferrin or serum albumin was observed. Internalization of TTR was temperature dependent and proportional to receptor occupancy.
T
RANSTHYRETIN (TTR: T4-binding prealbumin) is the main export protein of the choroid plexus, which secretes most, if not all, of the TTR that enters the cerebrospinal fluid (CSF) (1, 2). The concentration of TTR in CSF is less than 10% of that in plasma, but this is relatively high compared to the concentrations of T4-binding globulin and albumin (3). As a result, although TTR binds only 15% of plasma T4, it is the principal T4-binding protein of the CSF (3, 4). The nature of the interaction between TTR and the cells of the central nervous system is not known. The present study was undertaken to determine whether astrocytes have binding sites for TTR similar to those that have been found on hepatocytes (5). The astrocyte appears to be a target cell for T4's effect and is, therefore, of interest with regard to the hormonal transport function of TTR. It has been shown recently that, in addition to providing substrate for the type II 5'-deiodinase, T 4 directly stimulates actin polymerization in astrocytes (6).
Materials and Methods Purified human TTR was obtained from Calbiochem (La Jolla, CA). [125I]TTR was prepared as previously described (5). Transferrin (Fe-saturated), crystallized human serum albumin (HSA), BSA, bovine 7-globulin, EDTA, 2-deoxyglucose, peniReceived May 4, 1990. Address all correspondence and requests for reprints to: Dr. George C. Schussler, Box 21, State University of New York, Health Science Center, 450 Clarkson Avenue, Brooklyn, New York 11203-2098. * This work was supported by the Boots Co., Inc., Lincolnshire, IL NIH Grant 5R01-AM-32436, and a grant from the Division of Research Resources SUNY-Health Science Center at Brooklyn.
Dilutions of cerebrospinal fluid displaced [125I]TTR in proportion to their content of radioimmunoassayable TTR and in parallel with purified TTR. The uptake and internalization of TTR increased in the presence of high T4 or T 3 concentrations. These results demonstrate that TTR binds to specific high affinity receptors on human astrocytoma cells. Receptor binding of TTR provides a potential mechanism for the delivery of its ligands within the central nervous system. (J Clin Endocrinol Metab 7 1 : 1265-1268, 1990)
cillin, T4, and T 3 were obtained from Sigma Chemical Co. (St. Louis, MO). The HSA was further purified on polyacrylamide gel electrophoresis to remove residual TTR contamination (5). Rabbit antihuman TTR from Behring Diagnostics (La Jolla, CA) was used for RIA at a dilution of 1:20,000. Antibody-bound tracer was precipitated by 14% polyethylene glycol in the presence of 200 mg/dL bovine 7-globulin. Sodium azide was purchased from Fisher Scientific (Pittsburgh, PA). Minimum Essential Medium (MEM), fetal bovine serum, trypsin, and sodium pyruvate were obtained from Gibco (Grand Island, NY). CSF was obtained from the residual of a sample that had been examined by the routine laboratory and found to have no abnormalities. U-673, a human astrocytoma cell line from the American Type Culture Collection (Rockville, MD), was grown in monolayers in MEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/dL sodium pyruvate. The cells were seeded in 24-well cluster dishes and grown for 2-3 days until they were confluent. There were approximately 200,000 cells/well, as determined by counting the detached cells in a hemocytometer. Before the incubations, the monolayers were rinsed three times with ice-cold MEM, leaving the last wash for 30 min. After incubation with [125I]TTR in MEM the supernatant was separated, and the cells were rinsed, twice with ice-cold phosphate-buffered saline (PBS) and once with ice-cold PBS-0.1% BSA. The cells were detached with 250 ML 0.025% trypsin in 1 mM EDTA and then transferred to counting tubes in 1 mL of 1% BSA, with a 500-/tL wash. Uptake was calculated as the ratio of cell-associated activity to the activity in 100 nL supernatant. To measure internalization, monolayers were rinsed once with ice-cold PBS, and the surface-bound tracer was removed by proteolytic digestion (7) with 1 mL 0.25% trypsin for 10 min at 4 C in the presence of 10 mM 2deoxyglucose and 10 mM sodium azide to inhibit further inter-
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DIVINO AND SCHUSSLER
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nalization. The cells were transferred to Eppendorf centrifuge tubes and centrifuged at 12,000 rpm for 2 min at 4 C. The activity remaining with the pellet was considered to be internalized.
Results The uptake of [125I]TTR by astrocytoma cells reached equilibrium at 2 h. No metabolism of [125I]TTR was detected by trichloroacetic acid precipitation or polyacrylamide gel electrophoresis during incubations of up to 4 h. Displacement studies were consistent with a single class of binding sites (Fig. 1). The average Kd, determined by Scatchard analysis in three studies at 37 C, was 3.5 nM (range, 2.9-4.0 nM), with a capacity of 236,000 sites/ cell (range, 212,000-275,000). At 0 C the Kd was 2.8 nM (n = 3; range, 1.9-4.1 nM), and the capacity was 85,000 sites/cell (range, 65,000-119,000). Internalization, determined as the trypsin-sensitive fraction of TTR uptake (7), was reduced, but not completely inhibited, at 0 C. The failure to observe complete inhibition of internalization at 0 C may have been due to internalization occurring during trypsin treatment at 4 C, despite the presence of metabolic inhibitors. Because of some variability in uptake from different seedings of U-673, internalization at 0 and 37 C was compared with simultaneous studies in matched multiwells. In two studies, the proportion of TTR (1.8 nM) that was trypsin insensitive at 37 us. 0 C was 0.57 ± 0.04 (SD) US. 0.20 ± 0.05 (P < 0.001) and 0.36 ± 0.04 us. 0.19 ± 0.04 (P < 0.005). Therefore, the apparently higher capacity at 37 C than at 0 C was at least partially due to greater internalization of TTR at 37 C. Analysis of surface binding, determined as the trypsin-sensitive component of total uptake (7), was consistent with a similar number of cell surface receptors at 0 and 37 C (Fig. 1, inset). Internalization was propor-
JCE & M • 1990 Vol 71 • No 5
tional to surface binding, with a correlation coefficient of 0.93 in the displacement study shown in Fig. 1. The steeper slope of the regression for surface than total binding at 37 C suggests that the proportion of internalized TTR may increase as the surface receptors are saturated. There was greater variability of individual determinations with this technique than with the measurement of whole cell uptake, and it is not certain that the entire difference in the calculated binding capacity at 0 and 37 C can be attributed to internalization. The binding site for TTR did not cross-react with transferrin, and there was little cross-reactivity with repurified HSA (Fig. 2). Dilutions of a normal CSF displaced [125I]TTR in parallel with proportional changes in the concentration of purified TTR (Fig. 3). Displacement by CSF was consistent with its 0.4 jiM radioimmunoassayable TTR content. At concentrations beyond the physiological range, T4 increased the uptake of TTR (Fig. 4). These studies were 1.0
o
Transferrin
0.8
CD
m
on
HSA
TTR 0.6
O 0.4 0.2 0.0
-9
-8
-7
-6
-5
Competitor (log M)
FIG. 2. Binding specificity. There was no significant cross-reactivity of the binding site with human transferrin and only minimal crossreactivity with HSA, which probably represented a very low level of residual TTR contamination. CSF percent 0.04 1.0-
0.10
0.02
0.04
0.06
10.00
• — • TTR O — O CSF nonspecific binding
0.8-
o.oo
1.00
0.08
picomoles/l
FIG. 1. Scatchard analysis of binding at 0 and 37 C. Cells were incubated for 2 h with [125I]TTR and increasing concentrations of nonlabeled TTR. The cell/medium ratios are for approximately 200,000 cells/100 (iL medium. O, Total cell binding at 37 C; A, total cell binding at 0 C. The corresponding filled symbols (inset), indicate cell surface binding, determined as the trypsin-sensitive fraction of total cell uptake.
0.2 ••
0.0 0.4
1.0
10.0
100.0
TTR nM
FIG. 3. Displacement of [125I]TTR by TTR and CSF. The displacement curves are parallel. Nonspecific binding was determined at a TTR concentration of 1 ^M.
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TTR RECEPTORS 125
I TTR B/Bo 3.0
T
2.5 2.0 1.5 1.0
•T
0.5 0.0-1-7
-6
-5
lodothyronine (log M)
FIG. 4. Effects of T4 and T3 on the cellular uptake of TTR. Each value is the mean ± SD of four determinations of TTR uptake, expressed as the ratio of cellular TTR (B) to total cellular TTR in the absence of iodothyronines (Bo). Control values for total uptake are 1 by definition; control values for internalized TTR are shown on the ordinate. Total TTR is significantly increased at all concentrations of T4 shown (P < 0.005 at 1.3 X 10"7 and P < 0.001 at higher concentrations). Internalized TTR is also increased at all concentrations (P < 0.001). The effect of T3 is less pronounced, with a significance of P < 0.001 achieved at 6.4 x 10"7 M for total TTR uptake and at 12.9 x 10"7 M for internalized TTR.
carried out in the presence of 5.5 nM TTR and 2.7 ^M HSA. As might be anticipated on the basis of the higher affinity of TTR for T4 compared to T 3 (8, 9), the latter is less effective than T4. Stimulation of TTR uptake by both T4 and T 3 was associated with an increase in internalized TTR.
Discussion These studies demonstrate that TTR binds to specific high affinity (Kd = 3 nM) sites on human astrocytoma cells. It is likely that similar receptors are present on normal human astrocytes. The internalization of TTR is temperature dependent and proportional to specifically bound TTR. This is consistent with a receptor-mediated energy-requiring process. There is no significant crossreactivity of the TTR-binding site with transferrin or albumin, two structurally unrelated proteins. The minor cross-reactivity with repurified HSA was probably due to a small residual TTR contamination (5). Although TTR carries about 44% of the total CSF T4, only 0.14% of the primary and 0.0013% of the secondary iodothyronine-binding sites on TTR are occupied by T4 at the normal 7.5 X 10~n-M concentration of the free hormone (3).1 The increase in TTR uptake that was 1 The association constants of the primary and secondary TTRbinding sites for T4 are 7 x 107 and 6.7 x 105, respectively (9). From the mass law, the proportion of available sites occupied by T4 can be calculated as: occupied/total sites = ((K^T^)- 1 + I)"1, where K; is the association constant of T4 for site i and [T4] is the concentration of free T4.
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observed at supraphysiological concentrations of T4 may represent amplification of a preferential uptake of the very small proportion of TTR that normally carries T4. A similar effect of T4 has been observed with hepatocytes, where it was temperature dependent and appeared to be specific for TTR (5). Parallel displacement of [125I]TTR by dilutions of purified TTR and CSF indicates that the TTR of the CSF binds to the astrocyte receptor with an affinity similar to that of purified serum TTR. The potency of the CSF relative to purified TTR was consistent with the 0.4-/xM TTR concentration of the sample. With a Kd of 3 nM, receptor saturation should be sensitive to concentrations of TTR in the nanomolar range. The TTR concentration in the microenvironment of the astrocyte is presumably in equilibrium with, but lower than, the TTR concentration of CSF. Thus, in contrast to the hepatic TTR receptor, which is saturated at endogenous TTR concentrations (5), the occupancy of astrocyte TTR receptor may be affected by changes in the TTR concentration of the CSF. There is evidence that the TTR concentration of CSF is physiologically modulated, although unlike serum TTR, it is not responsive to inflammation or caloric deprivation (10, 11). A rapid and specific 2-fold increase in rat choroid plexus TTR mRNA has been found to occur after partial hepatectomy and is thought to be part of an anabolic response supporting liver regeneration (10). In the same study it was observed that the proportion of TTR mRNA in total brain RNA became maximal shortly before maximal brain growth, suggesting that TTR has a function in the development of the central nervous system. TTR binds retinol-binding protein as well as T4 (8, 12). Its role in the delivery of these ligands to target cells remains unclear. Retinol-binding protein is taken up by its own receptor (13,14). Cellular T4 is usually considered to be in equilibrium with the free, rather than the protein-bound, T4 of the extracellular space (15). Nevertheless, specific transport functions of the extracellular T4binding proteins may be of importance. As shown in a model system by Dickson et al. (2), it is likely that a considerable proportion of the T4 that crosses the bloodbrain barrier enters the CSF with TTR synthesized by the choroid plexus. Once in the CSF, the T4-TTR complex probably has a distributive function, assuring availability of T4 to cells throughout the CSF circulation (15, 16). Active uptake of T4 and T 3 by human glioma cells and other neural cell lines has been demonstrated in serum-free medium (17). Although there is little question that free T 4 is available to cells, it has been proposed that there is also direct participation of the binding proteins in T4 uptake (18-20). In vitro studies in our laboratory indicate that in the presence of other T4binding proteins, which reduce the proportion of free
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DIVINO AND SCHUSSLER
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hormone, TTR increases cellular T4 uptake (19). To a considerable extent, analysis of the exchange of T4 between extracellular and cellular sites has been based on certain characteristics of hormone transport, particularly the capillary transit time, the rate at which T4 dissociates from its protein-binding sites, and cellular uptake from the free T4 pool (15). The interaction of TTR with its receptor should be added to these considerations. The TTR receptor could facilitate T4 uptake by binding the T 4 -TTR complex or by providing a docking function to allow selective release of hormone in the vicinity of the target cell. Such putative functions of the receptor would be of particular importance in the central nervous system, where TTR, rather than T4-binding globulin, is the principal extracellular T4-binding protein.
Acknowledgment We are grateful for the excellent secretarial assistance of Ms. Anna Marie Jichetti.
References 1. Dickson PW, Aldred AR, Marley PD, Bannister D, Schreiber G. Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver. J Biol Chem. 1986;261:3475-8. 2. Dickson PW, Aldred AR, Menting JGT, Marley PD, Sawyer WH, Schreiber G. Thyroxine transport in choroid plexus. J Biol Chem. 1987;262:13907-15. 3. Hagen GA, Elliott WJ. Transport of thyroid hormones in serum and cerebrospinal fluid. J Clin Endocrinol Metab. 1973;37:415-22. 4. Woeber KA, Ingbar SH. The contribution of thyroxine-binding prealbumin to the binding of thyroxine in human serum, as assessed by immunoadsorption. J Clin Invest. 1968;47:1710-20. 5. Divino CM, Schussler GC. Receptor-mediated uptake and internalization of transthyretin. J Biol Chem. 1990;265:1425-9.
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6. Siegrist-Kaiser CA, Juge-Aubry C, Tranter MP, Ekenbarger DM, Leonard JL. Thyroxine-dependent modulation of actin polymerization in cultured astrocytes: a novel, extranuclear action of thyroid hormone. J Biol Chem. 1990;265:5296-302. 7. Karin M, Mintz B. Receptor-mediated endocytosis of transferrin in developmentally totipotent mouse teratocarcinoma stem cells. J Biol Chem. 1981;256:3245-52. 8. Ingbar SH. Observations concerning the binding of thyroid hormones by human serum prealbumin. J Clin Invest. 1963;42:14360. 9. Robbins J, Edelhoch H. Thyroid hormone transport proteins: their nature, biosynthesis and metabolism. In: Ingbar SH, Braverman LE, eds. The thyroid. Philadelphia: Lippincott; 1986;116-27. 10. Fung WP, Thomas T, Dickson PW, et al. Structure and expression of the rat transthyretin (prealbumin) gene. J Biol Chem. 1988;263:480-8. 11. Wade S, Bleiberg-Daniel F, Le Moullac, B. Rat transthyretin: effects of acute short-term food deprivation and refeeding on serum and cerebrospinal fluid concentration and on hepatic mRNA level. J Nutr. 1988;118:199-205. 12. Kanai M, Raz A, Goodman DS. Retinol binding protein: the transport protein for vitamin A in human plasma. J Clin Invest. 1968;47:2025-44. 13. Heller J. Interactions of plasma retinol-binding protein with its receptor. J Biol Chem. 1975;250:3613-9. 14. Sivaprasadarao A, Findlay JB. The mechanism of uptake of retinol by plasma-membrane vesicles. Biochem J. 1988;255:571-9. 15. Mendel CM. The free hormone hypothesis: a physiologically based mathematical model. Endocr Rev. 1989; 10:232-74. 16. Mendel CM, Weisiger RA, Jones AL, Cavalieri RR. Thyroid hormone-binding proteins in plasma facilitate uniform distribution of thyroxine within tissues: a perfused rat liver study. Endocrinology. 1987;120:1742-9. 17. Goncalves E, Lakshmanan M, Pontecorvi A, Robbins J. Thyroid hormone transport in a human glioma cell line. Mol Cell Endocrinol. 1990;69:157-65. 18. Azimova SS, Umarova GD, Petrova OS, Tukhtaev KR, Abdukarimov A. Nature of thyroid hormone receptors. Translocation of thyroid hormone across plasma membrane. Biokhimiya. 1984;49:1350-6. 19. Divino C, Schussler GC. Binding of thyroxine-transthyretin complexes to specific cell surface receptors [Abstract]. Clin Res. 1989;37:357A. 20. Pardridge WM. Plasma protein-mediated transport of steroid and thyroid hormones. Am J Physiol. 1988;252:E157-64.
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