Binding of the Low Density Lipoprotein Receptor-Associated Protein (RAP) to Thyroglobulin (Tg): Putative Role of RAP in the Tg Secretory Pathway
Michele Marinò,
Luca Chiovato,
Simonetta Lisi,
Aldo Pinchera and
Robert T. McCluskey
Department of Endocrinology (M.M., L.C., S.L., A.P.), University of
Pisa, 56124 Pisa, Italy; and Pathology Research Laboratory (M.M.,
R.T.M.), Massachusetts General Hospital, Harvard Medical School,
Charlestown, Massachusetts 02129
Address all correspondence and requests for reprints to: Michele Marinò, Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy. E-mail:
m.marino{at}endoc.med.unipi.it
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ABSTRACT
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The 3944 kDa protein known as the receptor-associated
protein binds to members of the low density
lipoprotein receptor family and is found within cells that express
these receptors. The receptor-associated protein has been shown to
prevent premature binding of ligands to the receptors in the
endoplasmic reticulum and to promote proper folding and transport of
the receptors in the secretory pathway. In the thyroid, megalin (a
low-density lipoprotein receptor family member) serves as an endocytic
receptor for thyroglobulin. Here we present evidence that the
receptor-associated protein can bind to thyroglobulin, which suggests a
novel function of the receptor-associated protein, namely binding of
certain megalin ligands possibly during the biosynthetic pathway.
In solid-phase assays thyroglobulin was shown to bind to the
receptor-associated protein with moderately high affinity (mean between
Kd and Ki = 39.8 nM), in a
calcium-dependent and saturable manner. The receptor-associated protein
also bound to a native carboxyl-terminal 230-kDa thyroglobulin
polypeptide, which markedly reduced binding of intact thyroglobulin to
the receptorassociated protein, indicating that the
receptor-associated protein binding sites of thyroglobulin are located
in the carboxyl-terminal portion of the molecule. In addition to
thyroglobulin, the receptor-associated protein specifically bound to
another megalin ligand, namely lipoprotein lipase. Because lipoprotein
lipase markedly reduced receptor-associated protein binding to
thyroglobulin, we concluded that the receptor-associated protein uses
the same binding site/s to bind to thyroglobulin and lipoprotein
lipase. Evidence of thyroglobulin binding to the receptor-associated
protein was also obtained in vivo and in cultured
thyroid cells. Thus, anti-receptor-associated protein antibodies
precipitated intact thyroglobulin from extracts prepared from rat
thyroids and cultured thyroid cells (FRTL-5 cells). Chase
experiments after inhibition of protein synthesis in FRTL-5 cells
showed that thyroglobulin interacts with the receptor-associated
protein shortly after the beginning of thyroglobulin
biosynthesis.
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INTRODUCTION
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THE LOW DENSITY lipoprotein (LDL) receptor
family comprises several cysteine-rich endocytic receptors, including
the LDL receptor itself, the LDL receptor-related protein (LRP),
megalin (gp330), and the VLDL receptor (1, 2, 3, 4, 5). LRP and
megalin, the two largest members of the family, can bind multiple,
unrelated, and, to some extent, overlapping ligands. The expression of
these receptors differs among various organs, and their physiological
function is to internalize ligands present in fluids to which they are
exposed, usually with delivery of ligands to lysosomes
(1, 2, 3, 4, 5). A 39- to 44-kDa receptor-associated protein (RAP)
is present in all epithelial cell types where LRP and megalin are
expressed (1, 2, 3, 4, 5, 6). In vitro RAP can block
binding of virtually all ligands to LRP and megalin
(1, 2, 3, 4, 5, 6). In vivo RAP is an endoplasmic
reticulum resident protein that bears a retention sequence
(HNEL). RAP functions as a molecular chaperone for members of the LDL
receptor family, by preventing premature ligand binding within the
endoplasmic reticulum and by promoting proper folding and assembly of
the receptors, thereby facilitating their transport to the cell surface
along the secretory pathway (1, 2, 3, 4, 5).
In recent studies (7, 8, 9) we have shown that megalin
is a high-affinity endocytic receptor for thyroglobulin (Tg), the
precursor of thyroid hormones. Tg is synthesized by thyroid cells and
released into the follicle lumen, where it is stored as the major
component of colloid (10, 11, 12, 13, 14, 15). Hormone secretion requires
uptake of Tg by thyrocytes, with transport to lysosomes, where
proteolytic cleavage leads to the release of thyroid hormones
(10, 11, 12, 13, 14, 15). Megalin is expressed on the apical surface of
thyrocytes (6, 16), where it mediates endocytosis of Tg
from the colloid, after which Tg is not transported to lysosomes, as
are most megalin ligands, but is rather transcytosed across thyrocytes
to the basolateral surface, from which Tg is released into the
bloodstream (17, 18, 19). This unusual function of megalin
competes with other mechanisms of Tg endocytosis, probably mainly
nonselective fluid phase micropinocytosis, which lead to the delivery
of Tg to lysosomes (10, 11, 12, 13, 14, 15).
Expression of RAP by thyroid cells has been demonstrated in several
species, including humans and rats (6, 16). Here we show
that RAP binds to Tg in solid-phase assays, as well as in
vivo and in cultured thyroid cells, suggesting a novel function of
RAP, namely binding of certain megalin ligands possibly during the
biosynthetic pathway.
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RESULTS
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Binding of Tg to RAP
Evidence of binding of Tg to RAP was first provided in solid-phase
binding assays. Tg bound to wells coated with RAP, used as a
glutathione-S-transferase (GST) fusion protein (RAP-GST),
whereas there was no binding to wells coated with GST alone (Fig. 1A
). Binding of Tg to RAP-GST-coated
wells is represented as a nonlinear regular fitting plot in Fig. 1A
.
Binding was saturable and markedly reduced by coincubation of Tg with
an excess of RAP-GST. The mean dissociation constant
(Kd), estimated from the midsaturation points of
equilibrium binding experiments, as presented in Fig. 1A
(estimated
apparent affinity, obtained in three experiments), was 22.25
nM for total binding and 16.62
nM for specific binding (total binding -
binding in the presence of an excess RAP-GST). The average
Kd for total and specific binding was 19.40
nM. In addition, we evaluated the inhibition
constant (Ki or K0.5) of Tg
binding to RAP-GST coated wells, by measuring binding of a constant Tg
concentration (75 nM) in the presence of
increasing amounts of RAP-GST (Fig. 1B
). The mean
Ki obtained was 93.50 nM
(calculated in three experiments). Based on Kd
and Ki values, we concluded that binding of Tg to
RAP is of moderately high affinity (mean between
Kd values and Ki = 39.8
nM).

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Figure 1. Binding of Rat Tg to RAP-GST in Solid-Phase Binding
Assays
Microtiter wells coated with RAP-GST or, as a control, with GST alone,
were incubated with Tg followed by a rabbit anti-Tg antibody,
ALP-conjugated antirabbit IgG and
p-nitrophenyl-phosphate. Absorbance (OD) was determined
at 405 nM. A, Increasing concentrations of Tg were added to
RAP-GST or to GST-coated wells, alone or in the presence of RAP-GST
itself (100 µg/ml). Specific binding = total binding -
binding in the presence of RAP-GST (nonspecific binding). The figure is
representative of one of three separate experiments. B, A constant
concentration of Tg (75 nM) was added to RAP-GST-coated
wells in the presence of increasing concentrations of RAP-GST itself.
The figure is representative of one of three separate experiments.
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Because Kd and Ki values
were obtained using an enzyme-linked, indirect binding assay, we
ascertained that the values of absorbance obtained with this assay were
within the linear part of the substrate production curve. For this
purpose, we determined binding of various concentrations of the anti-Tg
antibody (which was used to reveal bound Tg in solid-phase binding
assays) to microtiter wells coated with Tg, followed by the
enzyme-linked secondary antibody [alkaline phosphatase
(ALP)-conjugated antirabbit IgG] and the substrate
(p-nitrophenyl-phosphate). Experiments were performed under
the same conditions used in solid-phase binding assays. As shown in
Fig. 2
, the OD values obtained in the
linear part of the substrate curve ranged from 0.010 to 0.325. All the
OD values obtained in binding experiments were within this range. These
results demonstrate that the Kd and
Ki values calculated in solid-phase binding
assays were reliable.

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Figure 2. Production Curve of the Substrate
(p-Nitrophenyl-phosphate) used in Tg Binding Experiments
Microtiter wells coated with Tg were incubated with various dilutions
of the rabbit anti-Tg antibody, followed by ALP-conjugated antirabbit
IgG and p-nitrophenyl-phosphate. Absorbance (OD) was
determined at 405 nM. The figure is representative of one
of three experiments. Experiments were performed under the same
conditions used in solid-phase binding assays.
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As shown in Fig. 3
, binding of Tg
to RAP-GST was inhibited by EDTA (by
90%), indicating that binding
is calcium dependent. No inhibition was produced by GST or ovalbumin
(OVA) used as negative controls.

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Figure 3. Inhibition of Binding of Rat Tg to RAP-GST in
Solid-Phase Binding Assays
Microtiter wells coated with RAP-GST or, as a control, with GST
alone, were incubated with Tg (75 nM), followed by a rabbit
anti-Tg antibody, ALP-conjugated antirabbit IgG and
p-Nitrophenyl-phosphate. Absorbance (OD) was determined at 405
nM. Tg was added to RAP-GST or to GST coated wells alone or
in the presence of RAP-GST itself (100 µg/ml) or EDTA (20
mM) or, as controls, GST (100 µg/ml) or OVA (100
µg/ml). Values are expressed as mean ± SE % of
total binding (binding of Tg to RAP-GST coated wells in the absence of
competitors or controls) obtained in three separate experiments.
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RAP Binding Sites of Tg Are Located in the Carboxyl-Terminal
Region
Tg binding sites for megalin and heparin are located in the
carboxyl-terminal two-thirds of the molecule (9). To
investigate whether RAP binding sites are also present in this portion
of the Tg molecule, we studied binding of RAP-GST to a 230-kDa Tg
polypeptide, which was previously shown to correspond to the carboxyl
terminal two-thirds of rat Tg (9). As shown in Fig. 4A
, RAP-GST bound to the 230-kDa Tg
polypeptide to a similar extent as to Tg, but not to OVA, used as a
negative control. In addition, binding of RAP-GST to Tg was markedly
reduced by coincubation with the 230-kDa Tg polypeptide (Fig. 4B
), but
not by OVA, providing evidence that Tg binding sites for RAP are
located within the carboxyl-terminal two thirds of the molecule.

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Figure 4. RAP Binding Sites Are Located in the Carboxyl
Terminal Portion of Tg
A, Binding of RAP-GST to a 230-kDa Tg polypeptide corresponding to the
carboxyl-terminal two thirds of rat Tg. Microtiter wells coated with
the 230-kDa Tg polypeptide or with intact Tg, or, as a control, with
OVA, were incubated with RAP-GST (100 µg/ml), followed by a rabbit
anti-RAP antibody, ALP-conjugated antirabbit IgG, and
p-nitrophenyl-phosphate. Absorbance (OD) was determined
at 405 nM. Values are expressed as mean ±
SE obtained in three separate experiments. B, Inhibition of
RAP-GST binding to Tg by the 230-kDa Tg polypeptide. Microtiter wells
coated with intact Tg were incubated with RAP-GST (100 µg/ml) alone
or in the presence of the 230- kDa Tg polypeptide (100 µg/ml) or, as
a control, of OVA (100 µg/ml), followed by a rabbit anti-RAP
antibody, ALP-conjugated antirabbit IgG, and
p-nitrophenyl-phosphate. Absorbance (OD) was determined
at 405 nM. Values are expressed as mean ±
SE obtained in three separate experiments.
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The carboxyl-terminal portion of Tg bears a heparin binding site
contained within a 15-amino acid sequence located from Arg2489 to
Lys2503 in the recently obtained, complete sequence of rat Tg
(20). This heparin binding site was previously shown to be
involved in megalin binding (9). To investigate whether
the Tg heparin binding site is also involved in binding to RAP, we
studied binding of RAP-GST to a synthetic peptide corresponding to the
above mentioned 15-amino acid sequence of rat Tg. However, no
appreciable binding of RAP-GST was seen (not shown) and, in addition,
the Tg synthetic peptide did not reduce binding of RAP-GST to Tg (not
shown), indicating that the Tg heparin binding site is not involved in
binding to RAP.
Binding of RAP to Lipoprotein Lipase (LPL)
To determine whether binding of RAP to megalin ligands is
restricted to Tg, we studied binding of RAP-GST to other megalin
ligands, namely lactoferrin, LPL, and apolipoprotein J (apo J). As
shown in Fig. 5A
, RAP-GST bound to LPL,
but not to lactoferrin or apo J. Binding of RAP-GST to LPL was
specific, as demonstrated by the inhibitory effect of RAP preincubation
with an excess of LPL (Fig. 5B
). Furthermore, binding was calcium
dependent, as demonstrated by the marked inhibition produced by EDTA.
No inhibition of binding of RAP-GST to LPL was produced by negative
controls (GST and OVA).

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Figure 5. Binding of RAP to LPL
A, Binding of RAP-GST to LPL, but not to lactoferrin or clusterin
(apo J). Microtiter wells coated with lactoferrin, LPL, or apo J were
incubated with RAP-GST (100 µg/ml) followed by a rabbit anti-RAP
antibody, ALP-conjugated antirabbit IgG, and
p-nitrophenyl-phosphate. Absorbance (OD) was determined
at 405 nM. Values are expressed as mean ±
SE obtained in three separate experiments. B, Inhibition of
RAP-GST binding to LPL. RAP-GST (100 µg/ml) was added to LPL-coated
wells alone or in the presence of LPL itself (100 µg/ml) or EDTA (20
mM) or, as a control, with OVA (100 µg/ml). Values are
expressed as mean ± SE % of total binding (binding
of RAP-GST to LPL-coated wells in the absence of competitors or
controls) obtained in three separate experiments.
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We then investigated whether binding of RAP to Tg and to LPL
involves the same binding site/s. For this purpose, we investigated the
effect of coincubation with LPL on RAP binding to Tg-coated wells. As
shown in Fig. 6
, coincubation with LPL
markedly reduced RAP binding to Tg, suggesting that RAP uses the same
binding site/s to interact with both Tg and LPL.

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Figure 6. Inhibition of Binding of RAP to Tg by LPL
Microtiter wells coated with Tg were incubated with RAP (100
µg/ml), alone or in the presence of LPL (100 µg/ml) or OVA (100
µg/ml), followed by a rabbit anti-RAP antibody, ALP-conjugated
antirabbit IgG, and p-nitrophenyl-phosphate. Absorbance
(OD) was determined at 405 nM. Values are expressed as
mean ± SE obtained in three separate experiments.
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Interaction between Tg and RAP in Vivo and in
Cultured Thyroid Cells
To determine whether binding of RAP to Tg occurs in
vivo and in cultured thyroid cells, coimmunoprecipitation
experiments were performed. For this purpose, we used frozen rat
thyroids and FRTL-5 cells, an established differentiated rat thyroid
cell line that expresses megalin in a TSH-dependent manner and
synthesizes and secretes Tg (8). Rat thyroid extracts or
FRTL-5 cell extracts were incubated with a rabbit anti-RAP antibody
followed by precipitation with protein A beads. As shown in Fig. 7
, Western blotting with the rabbit
anti-Tg antibody revealed the presence of intact Tg in the material
precipitated by the anti-RAP antibody in both rat thyroids (Fig. 7A
)
and FRTL-5 cells (Fig. 7B
), indicating that Tg was combined with RAP
within cells. The presence of RAP in the immunoprecipitated material
was demonstrated by Western blotting, using the anti-RAP antibody (not
shown). No Tg was precipitated by normal rabbit IgG, used as a negative
control (not shown).

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Figure 7. Immunoprecipitation of Tg with an anti-RAP Antibody
in Rat Thyroid Extracts (A) and in FRTL-5 Cell Extracts (B)
Extracts were incubated with rabbit antibodies coupled with
protein A beads. Beads were then subjected to 515% nonreducing
SDS-PAGE followed by Western blotting, which was performed using a
rabbit anti-Tg antibody. Lanes 1, Extracts not subjected to
immunoprecipitation; lanes 2, extracts precipitated with the rabbit
anti-Tg antibody; lanes 3, extracts precipitated with the rabbit
anti-RAP antibody. The figure is representative of one of three
separate experiments. C, Lack of cross-reactivity of the anti-RAP
antibody with Tg. Purified Tg (10 µg) was incubated with the anti-RAP
antibody coupled with protein A beads, followed by Western blotting,
for Tg. Lane 1, Preparation of rat Tg (1 µg); lane 2, Tg preparation
incubated with the rabbit anti-RAP antibody. The figure is
representative of one of three separate experiments.
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To rule out the possibility that Tg was precipitated by the anti-RAP
antibody because the antibody cross-reacted with Tg epitopes, we
assessed whether the anti-RAP antibody would react with Tg. For this
purpose we performed immunoprecipitation experiments with purified rat
Tg and found that Tg was not precipitated by the anti-RAP antibody
(Fig. 7C
). Additional evidence that the anti-RAP antibody did not react
with Tg was provided by immunofluorescence staining of
paraformaldehyde-L-lysine-sodium periodate (PLP)-fixed rat
thyroid sections with this antibody, as compared with staining of the
rat thyroid sections with an anti-Tg antibody. As expected, Tg staining
was found to be very intense in the colloid, with only very faint
intracellular staining of thyrocytes (Fig. 8A
). In contrast, staining for RAP was
confined to the intracellular compartment, with granular staining
distributed throughout the cytoplasm, whereas no staining of the
colloid was observed (Fig. 8B
).

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Figure 8. Immunofluorescence Staining for Tg (A) and RAP (B)
of Frozen Rat Thyroid Sections
PLP-fixed thyroid sections were incubated with either the rabbit
anti-Tg antibody or the rabbit anti-RAP antibody, followed by
FITC-conjugated antirabbit IgG. A, Intense Tg staining is seen in the
colloid, with only very faint intracellular staining of thyrocytes. B,
Granular intracellular staining for RAP is seen, distributed throughout
the cytoplasm, whereas no staining of the colloid is observed.
Magnification, x100.
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The Interaction between Tg and RAP in Cultured Thyroid Cells
Occurs Early in the Secretory Pathway
The results presented above clearly demonstrate that RAP binds to
Tg in solid- phase assays and that binding can also occur in thyroid
cells. Based on the known function of RAP as a molecular chaperone of
LDL receptors, we postulated that RAP-Tg interactions may occur during
Tg biosynthesis. Therefore, we investigated whether Tg-RAP interactions
take place early during the Tg biosynthetic pathway. For this purpose,
we performed immunoprecipitation experiments with FRTL-5 cell extracts
at various time points after inhibition of protein synthesis with
cyclohexamide. As shown in Fig. 9A
, treatment for 48 h with medium containing cyclohexamide abolished
endogenous Tg synthesis by FRTL-5 cells. Immunoprecipitation
experiments with an anti-Tg antibody demonstrated that Tg synthesis had
begun as early as 15 min after replacement of medium without
cyclohexamide (Fig. 9B
). As shown in Fig. 9C
, the anti-RAP antibody
precipitated Tg in FRTL-5 cell extracts 15 min after restoration of
protein synthesis, indicating that RAP interacts with Tg shortly after
biosynthesis.

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Figure 9. Tg Interacts with RAP Shortly After Its
Biosynthesis
A, Inhibition of Tg synthesis by FRTL-5 cells. Cells were cultured for
24 h (lane 2) or 48 h (lane 3) with medium containing
cyclohexamide (10 µg/ml). Cell extracts were prepared and subjected
to immunoprecipitation with a rabbit anti-Tg antibody, followed by
Western blotting for Tg. Lane 1, Extract from untreated cells. B and C,
After treatment of FRTL-5 cells with cyclohexamide for 48 h, cells
were cultured in medium without cyclohexamide. Cell extracts were
prepared at various time points and then subjected to
immunoprecipitation with the anti-Tg antibody (B), or with the anti-RAP
antibody (C), followed by Western blotting for Tg.
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DISCUSSION
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In the present study we show that Tg interacts with the LDL
receptor-related protein RAP. Evidence of binding of Tg to RAP was
first obtained in solid-phase binding assays. Binding of Tg to RAP was
saturable, calcium dependent, and of moderately high affinity (mean
between Kd values and Ki =
39.8 nM). Evidence that RAP binds to Tg within thyroid
cells was provided by the demonstration that anti-RAP antibodies
precipitated intact Tg from thyroid glands and cultured thyrocytes
(FRTL-5 cells). Furthermore, we showed that RAP-Tg interactions occur
early in the Tg secretory pathway. Thus, Tg was precipitable by an
anti-RAP antibody within 15 min after restoration of protein synthesis
after its suppression by cyclohexamide treatment.
The physiological significance of RAP binding to Tg is unknown. Because
RAP is known to function as a chaperone in the biosynthetic pathway of
megalin and of other members of the LDL receptor family
(1, 2, 3, 4, 5), one hypothesis is that RAP may have a similar role
in the Tg biosynthetic pathway. This hypothesis is indirectly supported
by the knowledge that RAP is an endoplasmic reticulum resident protein
(1, 2, 3, 4, 5) and by results presented here which demonstrate
that RAP interacts with Tg shortly after biosynthesis. Further studies
are needed to characterize the function of RAP in the Tg biosynthetic
pathway. It would be of particular interest to study the kinetics of
dissociation of RAP-Tg complexes in thyroid cells after biosynthesis,
through pulse-chase experiments after inhibition of protein synthesis,
followed by immuno-electron microscopy to track RAP-Tg complexes in
cell organelles after their synthesis.
Although further studies are clearly needed to investigate whether RAP
functions as a Tg chaperone, the present study may provide a way to
gain new insights into the Tg biosynthetic pathway. The role of
chaperones and other factors involved in the maturation and transport
of Tg in the endoplasmic reticulum of thyrocytes have been extensively
investigated in recent years by Arvan and associates
(21, 22, 23, 24). Newly synthesized Tg proceeds through a series
of folding intermediates, including aggregates with and without
interchain disulfide bonds, unfolded free monomers, folded monomers,
and finally dimers. Multiple molecular chaperones, including calnexin,
calreticulin, BiP (a member of the heat shock protein 70 class),
endoplasmic reticoloru protein 72, glucose-regulated protein 78 have
all been implicated in interactions with unfolded forms of Tg
(21, 22, 23, 24). Different chaperones may interact with folding
intermediates of Tg, both concurrently and sequentially. If a
chaperoning function of RAP in the Tg biosynthetic pathway is
demonstrated, it will be interesting to study the relationship of RAP
with known Tg chaperones.
Another hypothesis concerning the role of RAP-Tg interactions is that
RAP may prevent premature binding of Tg to megalin during the
biosynthetic pathway, not only by binding to megalin, as it is known to
occur for other megalin ligands (1, 2, 3, 4, 5), but also by
binding to Tg. However, in view of results presented here and of
results obtained in previous studies (7, 8, 17), this
possibility is unlikely. Thus, although we found that RAP binds to the
carboxyl-terminal portion of Tg, where megalin binding sites are
located (9), RAP did not bind to a carboxyl-terminal
synthetic peptide corresponding to a heparin binding sequence of Tg
(Arg2489-Lys2503) that is functionally involved in megalin binding
(9). In addition, it was previously shown that RAP binds
to megalin with higher affinity than to Tg (1, 2, 3, 4, 5) and that
the degree of inhibition of Tg-megalin interactions produced by other
megalin competitors, including a monoclonal antimegalin antibody, is
similar to that produced by RAP (7, 8, 17). Taken
together, these results indicate that inhibition by RAP of Tg-megalin
interactions is due to occupation of megalin binding sites and not of
Tg binding sites. Nevertheless, this issue requires further
investigation, aimed at the precise identification of megalin and RAP
binding sites within the Tg molecule.
To our knowledge, the present report is the first to show that RAP can
interact with a ligand of the LDL receptor family. Moreover, we found
that RAP can interact not only with Tg, but also with LPL, another
megalin ligand. Further studies are needed to investigate whether RAP
can bind to megalin ligands other than Tg and LPL. However, two
other megalin ligands tested, namely lactoferrin and apo J, did
not bind to RAP.
It has been shown that the functions of RAP in promoting proper folding
of LRP and in the prevention of premature interaction of ligands with
the receptor are independent and are mediated by different regions of
RAP (1, 2, 3, 4, 5). Although the function of RAP binding to
megalin ligands is unknown, the finding that RAP uses the same binding
site(s) for binding to both Tg and LPL suggests that it may serve a
similar function for both megalin ligands.
In conclusion, we found that Tg binds to the megalin-related protein
RAP in solid- phase assays, in vivo, and in cultured thyroid
cells. The physiological function of Tg-RAP interactions remains to be
investigated, although, in view of the knowledge that RAP is an
endoplasmic reticulum resident protein, the most likely role played by
RAP is in the Tg biosynthetic pathway.
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MATERIALS AND METHODS
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Materials
Thyroglobulin (Tg) was purified from rat thyroids by ammonium
sulfate precipitation and column fractionation, as previously described
(7, 8, 9, 17). Tg preparations were analyzed both by
nonreducing and reducing SDS-PAGE, followed by Coomassie staining or by
Western blotting, as previously reported (7, 8, 9, 17). Under
nonreducing conditions two bands were seen at about 660 and 330 kDa.
The 660-kDa band corresponded to covalently linked Tg dimers. Size
exclusion gel chromatography showed that almost all (
95%) of the
330-kDa band represented monomers derived from noncovalently associated
Tg dimers that had been dissociated by SDS-PAGE, with a small fraction
(
5%) of free Tg monomers. Under reducing conditions, two bands, one
slower (S) and one faster (F), were seen, as previously described
(7, 8, 9, 17). Other Tg products with lower molecular masses
were present in minimal amounts.
A 230-kDa Tg polypeptide, corresponding to the carboxyl-terminal
two-thirds of rat Tg, was purified by ammonium sulfate precipitation
and column fractionation, as previously described (9). A
previously described 15-amino acid peptide designated Tg peptide 1,
corresponding to a sequence (RELPSRRLKRPLPVK, Arg2489-Lys2503) in the
carboxyl-terminal portion of rat Tg (20), was synthesized
by Genemed Biotechnologies (South San Francisco, CA).
RAP was used in the form of a GST fusion protein. DH5
bacteria
harboring the pGEX-RAP expression construct were kindly provided by Dr.
Joachim Herz (University of Texas Southern Medical Center, Dallas, TX).
The production of RAP-GST and GST was performed as described
(25).
Lactoferrin and LPL were obtained from Sigma (St. Louis,
MO). Human apo J (also known as clusterin) was obtained from Quidel
(San Diego, CA).
A rabbit antihuman Tg antibody cross-reactive with Tg from other
species was obtained from Axle (Westbury, NY). A rabbit antibody
against RAP-GST was previously described (6). Alkaline
phosphatase (ALP)-conjugated goat antirabbit IgG and horseradish
peroxidase-conjugated goat antirabbit IgG were obtained from
Bio-Rad Laboratories, Inc. (Hercules, CA). Fluorescein
isothiocyanate (FITC)-conjugated goat antirabbit IgG was obtained from
Cappel (Durham, NC).
Cell Cultures
Fisher rat thyroid cells (FRTL-5) (American Type Culture Collection, Manassas, VA) were cultured as described (8, 17, 18, 19), in Coons F12 medium containing 5% FCS and a mixture
of six hormones. We previously showed that the FRTL-5 cells cultured
under these conditions synthesize and secrete intact Tg after
radiometabolic labeling (17) and produce cAMP after TSH
stimulation (18), which indicates that they maintain
functions of differentiated thyroid cells.
Solid-Phase Binding Assays
Solid-phase assays were performed as previously described
(7, 9, 26). Microtiter plates (96-well) were coated
overnight at 4 C with RAP-GST, Tg, 230-kDa Tg polypeptide, Tg peptide
1, lactoferrin, apo J, LPL, OVA, or GST, at a concentration of 100
µg/ml in PBS. Wells were blocked for 3 h at 4 C with BSA at a
concentration of 1 mg/ml, washed with Tris-buffered saline (TBS),
0.05% Tween-50, and incubated for 2 h at room temperature with
ligands (Tg or RAP-GST) at various concentrations in binding buffer
(TBS, 5 mM CaCl2, 0.5 mM
MgCl2, 0.05% Tween 20, 0.5% BSA). To reveal
bound ligands, wells were washed and incubated with primary rabbit
antibodies (rabbit anti-Tg, diluted 1:500, or rabbit anti-RAP at a
concentration of 10 µg/ml). After further washing, bound primary
antibodies were revealed using ALP-conjugated antirabbit IgG (1:3,000),
followed by p-nitrophenyl-phosphate. OD was determined at
405 nM using an E1311 ELISA microplate reader.
The background, obtained by incubating coated wells with the primary
and secondary antibody, was subtracted from the results.
For inhibition experiments, either RAP-GST or Tg were preincubated
overnight at 4 C in the presence of competitors and was added to the
wells in the presence of the competitors. The competitors used were:
RAP-GST itself (various concentrations), LPL (100 µg/ml), 230-kDa Tg
polypeptide (100 µg/ml), Tg peptide 1 (100 µg/ml), EDTA (20
mM), GST (100 µg/ml), or OVA (100 µg/ml).
The Kd and Ki values were
estimated using Prism (PPC) (GraphPad Software, Inc., San
Diego, CA). We performed experiments to ascertain that the values of
absorbance in the experiments in which the Kd and
the Ki were calculated were within the linear
portion of the substrate production curve. For this purpose, microtiter
wells coated with Tg (100 µg/ml) were incubated with various
concentrations of the rabbit anti-Tg antibody, followed by
ALP-conjugated antirabbit IgG and p-nitrophenyl-phosphate.
Absorbance was determined at 405 nM. Experiments
were performed under the same conditions used for solid-phase binding
assays.
Immunoprecipitation Experiments
Immunoprecipitation experiments were performed with both FRTL-5
cell extracts and rat thyroid extracts or as a control, with purified
rat Tg. Cell extracts were prepared using 1% Triton-X-100, 1%
deoxycholate (both from Fisher Scientific, Springfield,
NJ) and briefly sonicated before use. Rat thyroid extracts were
prepared using frozen thyroids (Pel-Freez Biologicals,
Rogers, AK), which were minced with a surgical razor blade and
solubilized overnight at 4 C in 1% Triton X-100, 1% deoxycholate in
TBS (pH 8.0) containing 2 mM phenylmethylsulfonyl fluoride,
2 mM N-ethylmaleimide, 5
mM e-amino-n-caproic acid, 5
mM benzamidine (all from Sigma) and
10 mM EDTA (Fisher Scientific).
Insoluble materials were pelleted by centrifugation.
Samples were precleared with protein A beads (Pierce Chemical Co., Rockford, IL; 50 µl of beads for 500 µl of sample),
washed with PBS, and incubated overnight at 4 C with the rabbit anti-Tg
antibody (1:500), with the rabbit anti-RAP antibody (5 µg), or, as a
control, with normal rabbit IgG (5 µg). Fifty microliters of protein
A beads were then added to each sample, for 1 h at 4 C and then
washed eight times with PBS. The beads were resuspended in nonreducing
Laemmli buffer, boiled, and spun by centrifugation and supernatants
were subjected to 515% SDS-PAGE and blotted onto nitrocellulose
membranes, which were incubated with the rabbit anti-Tg antibody
(1:500) followed by horseradish peroxidase-conjugated antirabbit IgG
(1:2,500).
In certain experiments, before preparing cell extracts, FRTL-5 cells
were cultured for 2448 h with medium containing cyclohexamide (10
µg/ml) to abolish protein synthesis. Cells were then washed and
cultured with medium without cyclohexamide for 15120 min. Extracts
were prepared and subjected to immunoprecipitation as described
above.
Immunofluorescence Staining of Rat Thyroid Sections
Six female Lewis rats weighing 100120 g were purchased from
Charles River Laboratories, Inc. (Wilmington, MA). Animal
care and sacrifice procedures were in accordance with institutional
guidelines. To obtain in situ fixed tissue, rats were
perfused through the aorta under ether anesthesia with 200 ml of PBS,
followed by 200 ml of PLP (Sigma). The thyroid was
harvested and immersed in PLP fixative overnight, washed with PBS, and
immersed in 30% sucrose overnight at 4 C before being embedded in
O.C.T. compound (Miles, Inc., Elkhart, IN) and frozen in
liquid nitrogen. Sections (4-µm) were prepared, dried, blocked, and
incubated for 1 h with the rabbit anti-Tg antibody (1:500) or the
rabbit anti-RAP antibody (20 µg/ml), followed by FITC-conjugated
antirabbit IgG (1:1,000).
 |
FOOTNOTES
|
---|
This work was supported by the 1999 American Thyroid Association
Research Grant (M.M.), by NIDDK Grant 46301 (R.T.M.) and by Grants from
the National Research Council (Consiglio Nazionale Ricerche, Roma,
Italy), Target Project Biotechnology and Bioinstrumentation (Grant
91.01219), and Target Project Prevention and Control of Disease Factors
(Grant 93.00437) and by European Economic Community Stimulation
Action-Science Plan Contract SC1-CT91-0707.
Abbreviations: ALP, Alkaline phosphatase; apo J, apolipoprotein
J; FITC, fluorescein isothiocyanate; GST,
glutathione-S-transferase; LDL, low-density lipoprotein;
LPL, lipoprotein lipase; LRP, LDL receptor-related protein; OVA,
ovalbumin; PLP, paraformaldehyde-L-lysine-sodium periodate;
RAP, receptor-associated protein; TBS, Tris-buffered saline; Tg,
thyroglobulin.
Received for publication December 1, 2000.
Accepted for publication June 25, 2001.
 |
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