©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transport of Serum Transthyretin into Chicken Oocytes
A RECEPTOR-MEDIATED MECHANISM (*)

(Received for publication, August 31, 1994)

Amandio V. Vieira (1)(§) Esmond J. Sanders (2) Wolfgang J. Schneider (1)(¶)

From the  (1)Department of Molecular Genetics, University and Biocenter, 1030 Vienna, Austria and the (2)Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transthyretin (TTR) is involved in the transport of thyroid hormones and, due to its interaction with serum retinol-binding protein, also of vitamin A. The importance of both ligands in vertebrate embryonic development has prompted us to investigate the molecular details of TTR transport function in a powerful germ cell system, the rapidly growing chicken oocytes. Yolk TTR is derived from the circulatory system, since biotinylated TTR was recovered by immunoaffinity chromatography of yolk obtained from a hen previously infused with invitro biotinylated chicken serum proteins. In concordance with the intraoocytic localization in an endosomal compartment, ligand blotting and chemical cross-linking experiments revealed the presence of a 115-kDa TTR-binding oocyte membrane protein. This putative TTR receptor was not detected in chicken ovarian granulosa cells or embryonic fibroblasts and was different from the previously described oocyte-specific receptor for two estrogen-induced chicken serum lipoproteins, vitellogenin and very low density lipoprotein (Barber, D. L., Sanders, E. J., Aebersold, R., and Schneider, W. J.(1991) J. Biol. Chem. 266, 18761-18770). Furthermore, in contrast to the serum levels of the yolk precursor lipoproteins, those of TTR were not significantly changed by estrogen; thus, TTR represents a newly defined, estrogen-independent class of yolk precursor proteins. These data strongly suggest that oocytic TTR is derived from the circulation, where it is a constitutive component, and deposited into yolk as a result of endocytosis mediated by a specific receptor.


INTRODUCTION

Transthyretin (TTR, (^1)previously called prealbumin) is a 60-kDa homotetrameric protein present in serum and cerebrospinal fluid of vertebrates. The three-dimensional crystal structure is known for normal human TTR (1) as well as for a mutant TTR involved in familial amyloidic polyneuropathic disease(2) . Based on the known primary structure of chicken TTR(3) , the tertiary and quaternary structures of the human and chicken proteins are predicted to be highly similar(3) . The amino acid sequences of TTRs of chicken (130 amino acids) and man (127 amino acids) are 78% identical overall and completely identical in a central channel formed by the tetrameric assembly of the monomers(3) . TTR binds the thyroid hormones, T(3) and T(4), directly in this central channel(1, 4, 5) , and it transports retinol (vitamin A) indirectly by interacting with serum retinol-binding protein (RBP)(6, 7) .

Both thyroid hormones and metabolites of retinol, acting through their respective nuclear receptors, have been shown to be important in the embryonic development of many vertebrates including oviparous (egg laying) species, such as the chicken(8, 9, 10, 11, 12) . But despite these effects, it is not yet known how the oocytes acquire the necessary thyroid hormones and retinoids. Stated differently, many questions remain about the fate of circulatory TTR. Which cell types can interact with TTR? Do the ligands of TTR play any role in its cellular interactions? Does such interaction lead to endocytosis of TTR and/or RBP?

Uptake of TTR has thus far been shown to occur in cultured human HepG2 hepatoma cells and has been proposed to occur in other human and rat cells (e.g. adenocarcinoma, primary hepatocytes, neuroblastoma) by a receptor-mediated mechanism(13) . However, there are also some reports that free T(3) and T(4) can be taken up directly by primary hepatocytes (14) and erythrocytes (15) via cell membrane transporters. In a previous study, we detected TTR in clathrin-coated vesicles of chicken oocytes and proposed that its uptake occurs by receptor-mediated endocytosis(16) . Indeed, the very rapid growth phase of the oocyte has been shown to involve receptor-mediated endocytosis of certain other circulatory proteins and lipoproteins(17, 18, 19, 20, 21) . In this report, we provide the first direct evidence for the uptake of serum transthyretin into growing chicken oocytes, and we identify a specific TTR-binding oocyte membrane protein that may act as a receptor in this process. We show further that TTR differs from the previously characterized chicken proteins that are transported from the serum into the yolk in that its serum levels are not significantly influenced by estrogen action.


EXPERIMENTAL PROCEDURES

Materials

Protein A-Sepharose and cyanogen bromide-activated Sepharose were obtained from Pharmacia Biotech Inc. The cross-linker, dithiobis(succinimidyl propionate), and the biotinylation reagent, N-hydroxy-succinimido-biotin were obtained from Sigma. 17beta-Estradiol was obtained from Fluka and dissolved in 1,2-propanediol (Merck) at 20 mg/ml. The sources of other chemicals and materials are listed below or have been previously reported(16) .

Animals

White Leghorn laying hens, approximately 1 year old, and 1-3-month-old roosters were obtained from the Institute of Molecular Pathology, Vienna, and reared in cages until the desired age with a 14-h light period and free access to food and water. Female New Zealand White rabbits (2-3 kg, body weight) were used for the anti-chicken serum TTR antibody production as previously described (16) .

Preparation of TTR and TTRbulletRBP Complex and Radiolabeling Procedures

Purified human serum TTR was purchased from Calbiochem and dissolved in phosphate-buffered saline (PBS, 140 mM NaCl, 3 mM KCl, 8 mM Na(2)HPO(4), pH 7.5) at 1 mg/ml. Chicken serum TTR was prepared by immunoaffinity chromatography as previously described (16) . Chicken serum TTRbulletRBP complex was purified as previously described(16) . TTR was radioiodinated with NaI using the Iodo-Gen (Pierce) method (22) to a specific activity of 600 cpm/ng. Upon analysis by SDS-PAGE, the labeled TTR behaved like the unlabeled ligand in that it migrated as a band with an apparent molecular mass of 33 kDa in unheated samples (presumably a dimeric structure(23, 24) ) and as a 13-kDa monomer when heated.

Ligand Blotting

All operations were performed at room temperature (23 °C). Chicken oocyte membrane detergent extract was prepared exactly as previously described(19) . Aliquots of the extract were subjected to SDS-PAGE under non-reducing conditions and electroblotted onto nitrocellulose as previously described(22, 25) . Two nitrocellulose strips were cut from each lane and blocked for 3 h in 3% (w/v) bovine serum albumin dissolved in TBSC (50 mM Tris-HCl, 100 mM NaCl, 1 mM CaCl(2), pH 7.5). I-TTR (4 µg/ml) with or without a 30-fold molar excess of unlabeled TTR was added to 0.5 ml of the TBSC-bovine serum albumin and incubated for 2 h. After 5 washes in TBSC, the strips were allowed to dry and were subjected to autoradiography. As a control, one of the strips was probed with I-vitellogenin (VTG) as previously described (22) to reveal the 95- and 380-kDa chicken oocyte lipoprotein receptors(21, 22, 26) .

Chicken ovarian granulosa cells and embryonic fibroblasts were cultured, and membrane extracts were prepared from them as described in (25) .

Cross-linking of TTR and Oocyte Membranes

Chicken oocyte membrane extract (300 µl containing 1.5 mg of protein) and 30 µl (6 times 10^6 cpm, 10 µg) of I-TTR were added to 1.5-ml Eppendorf tubes containing 80 µl of PBS with or without 800 µg of purified human TTR. The tubes were incubated at room temperature with occasional mixing for 1 h. Then, dithiobis(succinimidyl propionate) cross-linker (2 µl; final concentration, 1 mM) dissolved in dimethyl sulfoxide was added to both tubes and allowed to react for 20 min on ice before quenching with 30 µl of 1 M Tris-HCl, pH 7.5. Purified preimmune rabbit IgG (33 µl, 350 µg) was then added to both tubes and allowed to bind for 3 h before the addition of 100 µl of protein A-Sepharose (50% (v/v) suspension in PBS) and incubation for 1 h. The tubes were then microcentrifuged at full speed for 20 s, and the supernatants were transferred to new tubes. The pellets were washed twice with 1% (w/v) Triton X-100 in PBS, first with 150 µl and then with 500 µl, by gentle vortexing followed by microcentrifugation as above. The final washed pellets are designated ``non-immune'' precipitates. Each of the first washes (150 µl) was combined with its respective supernatant; purified anti-chicken serum TTR IgG (33 µl, 360 µg) was added and incubated overnight at 4 °C. The subsequent protein A-Sepharose precipitation and wash steps were performed as above to yield the two final pellets designated ``immune'' precipitates.

The precipitates were mixed with 150 µl of SDS-PAGE sample buffer (non-reducing), heated at 95 °C for 2 min to release the bound IgG and ligand complexes, and briefly microcentrifuged at maximum speed. Maximum volumes of supernatants (60,000 cpm from the immune and 11,000 cpm from the non-immune reactions) were then loaded on a 4.5-18% SDS-polyacrylamide gel and subjected to electrophoresis under non-reducing conditions. The gels were subsequently stained, destained, dried, and subjected to autoradiography(22) .

Biotinylation of Serum and Injection into Laying Hens

N-Hydroxy-succinimido-biotin (40 µmol) dissolved in 1 ml of dimethylformamide was added to 5 ml of laying hen serum. After incubation for 1 h at room temperature, the mixture was dialyzed at 4 °C overnight against two changes of TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). Blotting with streptavidin-horseradish peroxidase (see below) demonstrated that a wide size range of proteins had been biotinylated. Furthermore, it was confirmed that purified human and chicken TTR were indeed biotinylated under the conditions used to biotinylate serum and that the biotin-TTRs were still recognized by the respective anti-human and anti-chicken TTR IgG (data not shown, cf. Fig. 1). The biotinylated serum was filtered through 0.8-µm Millipore (Millex-HA) filters and then injected (4.5 ml) via a wing vein into the circulatory system of the hen.


Figure 1: Detection of intravenously administered biotinylated chicken serum TTR in yolk. Whole laying hen serum was biotinylated and then reinjected into a laying hen as described under ``Experimental Procedures.'' Subsequently, oocytic yolk from the injected hen (lanes1 and 3) and a control hen (lanes2 and 4) was subjected to anti-TTR immunoaffinity chromatography. The column eluates were analyzed for biotinylated proteins with streptavidin-horseradish peroxidase (A) and for TTR with anti-TTR IgG (B); detection was performed by the ECL method. The positions of migration of the two expected TTR species (monomer, 13 kDa; dimer, 33 kDa) are indicated by arrowheads. The two larger size protein bands observed in lane1 likely represent biotinylated proteins that interact nonspecifically with the IgG-Sepharose immunoaffinity matrix (see ``Results'').



Preparation of Yolk and Immunoaffinity Chromatography

The hen was sacrificed 42 h post-injection, and the ovary was transferred to ice-cold PBS. Working on ice over a period of 30-60 min, the large follicles (15-30 mm in diameter) were dissected from the ovary; each was punctured once with a needle, and the yolk was allowed to flow into a clean vessel. Contact of the yolk with any external part of the follicle was avoided. The yolk (``biotin yolk'') was collected and pooled, and an aqueous yolk extract was prepared exactly as previously described(16) . In the same way, an aqueous yolk extract was prepared from the 15-30-mm follicles of an uninjected chicken (``control yolk'').

The anti-chicken TTR IgG-Sepharose immunoaffinity column for isolation of TTR has been previously described(16) . Control and biotin yolk (15 ml each) were mixed with 1.5 ml of 10 times PBS and subjected to TTR immunoaffinity chromatography as described(16) , except that the final low pH column eluate was collected in 1-ml aliquots into tubes containing 100 µl of 2 M Tris-HCl, pH 8. The contents of the tubes containing the eluted protein peak (A > 0.1) were pooled and concentrated using Centricon 10 microconcentrators (Amicon). The TTR component of the eluted biotin-TTR was detected by immunoblotting with anti-TTR IgG followed by protein A-horseradish peroxidase, and its biotin moiety was detected by probing the blots with streptavidin-horseradish peroxidase. For both blots, the final step involved the horseradish peroxidase-dependent generation and detection of ECL according to the instructions supplied with the ECL kit (Amersham Corp.).

Estrogen Treatment of Roosters and Preparation of Serum

A total of three roosters were injected into the leg muscle with 20 mg of 17beta-estradiol/kg of body weight. At various times post-injection (6-125 h), they were bled from a wing vein. The blood was allowed to clot for 2-4 h at 4 °C and then subjected to a 3-min centrifugation. The serum was removed, diluted with 4 volumes of PBS, and kept frozen at -20 °C. The same procedure was used for the preparation of control rooster and laying hen serum.

Other Methods

Electroblotting was performed in a solution containing 25 mM Tris and 190 mM glycine. Protein concentrations were determined by the method of Lowry et al.(27) .


RESULTS

In previous biochemical analyses of the distribution of TTR by immunoaffinity chromatography and Western blotting, we have shown its presence in yolk(16) . In fact, TTR and RBP could be demonstrated in oocytic coated vesicles(16) , indicating a specific uptake pathway. Moreover, analysis of chicken yolk by immuno-electron microscopy has revealed that other serum components, for which specific receptors have been identified, are found in the electron-lucent phase of yolk(19, 20) , which represents a compartment derived from endocytic transport vesicles(16, 18, 20) .

Thus, we first wanted to determine directly whether circulatory TTR is the precursor of TTR found in the yolk of growing oocytes. To this end, whole laying hen serum was biotinylated and reinjected into the hen, and biotin-TTR was recovered from yolk. Biotinylation was selected instead of radioiodination because of greater sensitivity in combination with ECL detection and to avoid in vivo and reisolation experiments with large amounts of radioactivity. The biotinylation reaction was highly efficient as determined by probing control and biotinylated serum proteins, after SDS-PAGE and transfer to nitrocellulose, with streptavidin-horseradish peroxidase (data not shown). Chromatography of yolk from oocytes of the hen injected with biotinylated serum proteins on an anti-TTR IgG-Sepharose column and blotting of the immunoaffinity column eluate with streptavidin-horseradish peroxidase revealed the presence of serum biotin-TTR in the oocyte (Fig. 1A, lane1 and B, lane3). The control yolk from an uninjected hen, as expected, was only positive for the TTR analysis with the antibody (Fig. 1B, lane4) but not for biotin-TTR (Fig. 1A, lane2).

With evidence from other studies (17, 18, 19, 20, 21) that oocytic uptake of certain serum components in oviparous organisms is a specific, receptor-mediated process, we tested oocyte membrane proteins for the presence of TTR-binding components by ligand blotting ( Fig. 2and Fig. 3) and chemical cross-linking (Fig. 4). The ligand blots revealed a I-TTR-binding component with a relative molecular mass of approximately 115 kDa (Fig. 2, lane1). The binding of I-TTR to the 115-kDa protein could be competed for by unlabeled TTR (Fig. 2, lane2) but not by the serum albumin present in the blotting buffer. This component clearly was distinct from the abundant and well characterized 95- and 380-kDa oocytic lipoprotein receptors (19, 26) (Fig. 2, lane3). Membranes from cultured chicken granulosa cells (somatic cells that enclose the oocyte in the ovarian follicle) (Fig. 2, lane4) and chicken embryo fibroblasts (lane5) were also tested. However, no TTR-binding component could be detected, even though much higher amounts of somatic cell membrane proteins were analyzed than used for oocytes. Recognition of TTR and/or the TTRbulletRBP complex by the 115-kDa oocyte membrane protein was confirmed by ligand blotting using the isolated unlabeled ligand complex, followed by detection with anti-TTR IgG (Fig. 3).


Figure 2: Ligand blotting analysis of cellular membrane proteins for the presence of TTR-binding components. The blots were performed as described under ``Experimental Procedures.'' Membrane proteins from chicken oocytes (lanes1-3, 75 µg each), granulosa cells (lane4, 200 µg), and embryo fibroblasts (lane5, 200 µg) were transferred to nitrocellulose and probed with I-TTR (4 µg/ml, 600 cpm/ng) in the absence (lanes1, 4, and5) or presence (lane2) of a 30-fold molar excess of unlabeled TTR. Lane3 was incubated with I-vitellogenin (20 µg/ml, 50 cpm/ng). Molecular mass standards (kDa) are indicated to the left of lane1.




Figure 3: Ligand blot analysis using isolated TTRbulletRBP complex. Oocyte membrane proteins (75 µg/lane) were separated by SDS-PAGE, transferred to nitrocellulose, and incubated with chicken TTRbulletRBP complex (lane1, none; lane2, 4 µg/ml) for 3 h at 24 °C. Bound TTR was detected by incubation with anti-chicken TTR IgG followed by I-labeled protein A and autoradiography. The position of migration of molecular mass standards are indicated on the right (from top to bottom, in kDa: 200, 116, 97, 68, and 45).




Figure 4: Cross-linking analysis for TTR-binding components on chicken oocyte membranes. Detergent-solubilized membranes were incubated with chicken I-TTR in the absence (lanes1 and 3) or presence (lanes2 and 4) of 100-fold excess of unlabeled human TTR, cross-linked, and immunoprecipitated with rabbit anti-chicken TTR IgG (lanes1 and 2) or non-immune IgG (lanes3 and 4) as described under ``Experimental Procedures.'' Electrophoresis was performed under nonreducing conditions. Molecular mass standards (kDa) and the top of the separating gel are indicated to the left of lane1.



Finally, we addressed the characterization of the putative TTR receptor by an alternative method. With oocyte membrane proteins in the soluble phase, we performed cross-linking reactions with I-TTR followed by immunoprecipitation with anti-TTR IgG (Fig. 4). The cross-linking reaction between TTR and oocyte membrane proteins resulted in a ligand-membrane protein complex of 175-180 kDa and a very minor, less defined component of approximately 160 kDa. Possibly, this minor component is a breakdown product of the major reactive protein or represents a different TTR-binding species. Other minor bands seen in the immunoprecipitates (lanes1 and 2) but not in the nonimmune controls (lanes3 and 4) may represent higher order TTR structures (e.g. tetramers and/or products of the oxidative action of Iodo-Gen). Since under these conditions TTR can be expected to exist as a tetramer of 60 kDa, the apparent size of the cross-linked complex is compatible with the binding of a TTR tetramer to the 115-kDa protein identified in ligand blots. The major bands (13 and 33 kDa) seen in Fig. 4, lane1, represent the TTR monomer and dimer, respectively, which are stable in SDS under nonreducing conditions (described in (23) and (24) ). Because the anti-chicken TTR antibody does not recognize human TTR, these bands were undiminished when excess unlabeled human TTR was included in the incubations (Fig. 4, lane2). Thus, by a variety of methods using human or chicken TTR, the 115-kDa protein was consistently identified as a specific TTR-binding oocyte membrane protein.

At the onset of egg laying, the sudden rise in circulatory estrogen levels causes a dramatic induction of hepatic synthesis and serum levels of egg yolk precursor proteins and lipoproteins(28, 29, 30, 31, 32, 33) . The effect of estrogen is readily observed and typically studied in roosters by injecting them with 17beta-estradiol(28, 29, 30, 31, 32) . Previously characterized proteins and lipoproteins, e.g. VTG (Fig. 5), which are induced in the hen, taken up by the oocyte, and deposited into yolk, are also induced rapidly (within 24-48 h) (cf. (31) ) in the rooster model(28, 29, 30, 32, 33) . In contrast, Fig. 5shows that estrogen treatment does not significantly change serum TTR levels relative to those in control roosters over a period of at least 125 h (Fig. 5, upperpanel). As expected, serum vitellogenin is strongly induced, with peak values reached approximately 60 h following hormone administration (Fig. 5, lowerpanel).


Figure 5: Effects of estrogen treatment on serum TTR (upper panel) and VTG (lower panel) levels in roosters. Each lane of the Western blots contains the proteins of 2 µl of rooster serum (prepared as described under ``Experimental Procedures'') treated with 50 mM dithiothreitol. Polyclonal antibodies against TTR and VTG were used to detect the two respective proteins at the following times for post-estrogen treatment: lane1, 12 h; lane2, 26 h; lane3, 33 h; lane4, 57 h; lane5, 81 h; lane6, 125 h. Lane7 represents serum of the untreated control. Molecular mass standards are indicated to the left of the respective panels.




DISCUSSION

Our experiments provide direct evidence for the endocytosis of circulatory transthyretin by growing oocytes in an oviparous animal, the chicken. We show, by the detection of biotinylated TTR in the yolk of growing oocytes following intravenous administration of biotinylated serum proteins, that efficient TTR uptake occurs in vivo. This represents the first such demonstration for a non-lipoprotein precursor and suggests a general procedure to study the formation of yolk from serum components amenable to biotin tagging.

Previous studies have indicated that most of the yolk precursors present in serum are taken up by the oocyte via specific, receptor-mediated processes(17, 18, 19, 20, 21, 26) . We have shown that there are two (95 and 380 kDa) receptors on the chicken oocyte membrane that interact with the major yolk components very low density lipoprotein, VTG, and possibly alpha(2)-macroglobulin(19, 21, 22, 26) . Here, we show in two differently designed ligand-blotting experiments ( Fig. 2and Fig. 3) that free as well as RBP-complexed TTR interacts with a different oocyte component, i.e. a 115-kDa membrane protein. In addition, chemical cross-linking (Fig. 4) confirmed the binding of a similarly sized membrane protein to the 60-kDa TTR tetramer. In this context, it is noted that a considerable portion of circulatory TTR in vertebrates is present complexed to RBP ( (16) and Refs. therein), and in addition to TTR, TTRbulletRBP complexes can be isolated from yolk(16) . It would be of interest to address TTRbulletRBP transport by the biotinylation technique described here for TTR; unfortunately, however, it cannot be applied to complexed RBP, as it is not readily labeled by biotinylation (nor by iodination). (^2)Future studies are necessary to determine if the TTR receptor we have identified is involved in the oocyte-directed uptake of free TTR as well as of TTRbulletRBP complexes.

Estrogen treatment of roosters mimics the laying hen situation in that it dramatically increases the levels of serum components that, in the laying hen, are known to be massively endocytosed by the oocyte (28, 29, 30, 31, 32, 33) . Our present results, however, do not show an increase in the serum levels of TTR after estrogen administration. Furthermore, in Western blots of serum, laying hen TTR levels do not appear higher than in roosters (data not shown). Although we have not ruled out that estrogen affects both the rate of synthesis and catabolism (or clearance) of serum TTR, it is clear that TTR belongs to a class of proteins whose serum levels are not significantly changed by estrogen but which nevertheless are efficiently transported to the oocyte. One possible explanation for this difference between TTR and most major yolk proteins is that large fluctuations in the levels of TTR could adversely affect the controlled delivery of the important regulatory ligands, thyroid hormones and retinol, to peripheral cells. Consistent with this idea, preliminary results suggest that serum levels of RBP are similarly refractory to estrogen. (^3)Thus, cell-specific receptors such as the 115-kDa protein identified here are likely mediating the accumulation of regulatory molecules in growing oocytes.

It is not known whether the TTR protein itself plays any role in oogenesis and embryogenesis. However, the hormones that TTR transports from the serum into the oocyte have been shown to have important functions in embryogenesis in a wide variety of species. In the chicken, thyroid hormones have been reported to accumulate in the yolk during oogenesis(34) . Although the role of TTR was not examined in that study, it was noted that the highest accumulation of the hormones per unit mass occurs in the smallest oocytes(34) . We have previously reported that TTR levels relative to total yolk protein are also highest in the smaller oocytes(16) . The thyroid hormone, T(3), as well as various derivatives of retinol (e.g. all-trans retinoic acid and 9-cis retinoic acid) affect expression of important regulatory genes through their respective nuclear receptors(8, 10, 12, 35, 36, 37) . Furthermore, the nuclear receptors for T(3) and retinoids can affect each others' activities through their common heterodimerization partner, retinoid X receptor, another nuclear retinoid-binding receptor (38, 39, 40, 41) . In this respect, it is interesting to speculate that the uptake of both retinol and T(3) into the oocyte may be co-regulated as a result of their common interaction with TTR and the subsequent interaction of the complex with the oocytic TTR receptor.


FOOTNOTES

*
These studies were supported by the Alberta Heritage Foundation for Medical Research and the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (Project P-9040-MOB). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
This work represents partial fulfillment of the requirements for the Ph.D. degree from The University of Alberta. Current address: Dept. of Cell Biology, Scripps Research Institute, La Jolla, CA 92037-1027.

To whom correspondence should be addressed: Dept. of Molecular Genetics, University and Biocenter Vienna, Dr. Bohrgasse 9/2, Vienna, Austria. Tel.: 43-1-79515-2113; Fax: 43-1-79515-2013.

(^1)
The abbreviations used are: TTR, transthyretin; RBP, retinol-binding protein; T(3), 3,5,3`-L-triiodothyronine; T(4), 3,5,3`,5`-L-tetraiodothyronine; VTG, vitellogenin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
A. V. Vieira and W. J. Schneider, unpublished observations.

(^3)
A. V. Vieira, K. Kuchler, and W. J. Schneider, unpublished observations.


ACKNOWLEDGEMENTS

We thank Rita Lo for excellent technical assistance and Dr. P. M. Vieira for helpful discussion. Dr. S. Stifani kindly provided the rabbit anti-VTG IgG.


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