Internalization and Recycling Pathways of the Thyrotropin Receptor
Catherine Baratti-Elbaz,
Nicolae Ghinea,
Olivier Lahuna,
Hugues Loosfelt,
Christophe Pichon and
Edwin Milgrom
Institut National de la Santé et de la Recherche
Médicale Unité 135 Hormones et Reproduction
Hôpital de Bicêtre 94270 Le Kremlin-Bicêtre,
France
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ABSTRACT
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Scant information is available to date on
the intracellular trafficking of the TSH receptor. In the present study
we have used stably transfected L cells that express the TSH receptor,
125I-labeled TSH, and antireceptor antibodies
as well as gold-conjugated antireceptor monoclonal antibodies and
hormone. The latter allowed us to study, by electron microscopy, the
cellular distribution and endocytosis of TSH receptor. The receptor was
initially localized on the plasmalemma proper and in clathrin-coated
pits but was excluded from smooth vesicles open to the cell surface. It
was internalized through clathrin-coated vesicles. Constitutive
endocytosis represented 10% of cell surface receptor molecules.
Endocytosis was increased 3-fold by incubation with hormone. The
majority of internalized receptor molecules (90%) was recycled to the
cell surface, whereas the hormone was degraded in lysosomes. This
recycling of receptor was inhibited by administration of monensin.
Electron microscopic and confocal microscopic studies were repeated in
primary cultures of human thyroid cells and showed a distribution, and
endocytosis pathways, very similar to those observed in transfected L
cells. A previous study has shown the LH receptor to be endocytosed in
high proportion and to be degraded in lysosomes. Confocal microscopy
and colocalization studies with transferrin receptor confirmed that the
highly homologous LH and TSH receptors exhibit, when expressed in the
same cells, very different cellular trafficking properties. The use of
LH/TSH receptor chimeras showed that transmembrane-intracellular
domains contain information orienting the protein toward recycling or
degradative pathways. The extracellular domain seems to play a role in
the extent of internalization. These observations should now allow the
identification of the molecular signals involved.
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INTRODUCTION
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Cells rapidly internalize surface-bound nutrients and signaling
molecules (hormones and growth factors) via specialized domains of the
plasma membrane, i.e. clathrin-coated pits and caveolae (1).
This ubiquitous process involves cell surface receptors that bind their
ligands with high affinity. Nutrient receptors, such as those for the
low-density lipoproteins (2) and for transferrin (3), are
constitutively endocytosed. These receptors are concentrated in
clathrin-coated pits and rapidly internalized through these structures
in the presence or absence of their ligand (2, 3, 4). Signaling receptors,
on the other hand, are not endocytosed efficiently until an agonist is
bound and transmembrane signaling has occurred (1). Signaling receptors
have been shown to be internalized through either clathrin-coated
vesicles or, more rarely, through caveolae (5, 6). The fate of
endocytosed receptors is variable. In some cases, there is dissociation
from the ligand and the receptor recycles to the cell surface, while in
other cases, the receptor is directed into the lysosomes where it is
degraded (1).
TSH, the main physiological regulator of the thyroid gland, exerts its
cellular effects (secretion, activation of specific gene expression and
of growth) by binding to a membrane receptor (7). The latter is a G
protein-linked receptor whose cloning has allowed the determination of
its sequence and structure: it possesses a characteristic
seven-transmembrane span, which separates a short intracellular segment
from a large extracellular domain (8, 9). The latter is the site of
hormone binding (10). The TSH receptor (TSHR) has a high degree of
homology with the other two receptors for glycoprotein hormones (FSH
and LH). In contrast to LH and FSH receptors, which consist of a single
polypeptide chain (11, 12), the TSHR in the human thyroid gland is
cleaved into two subunits that are held together by disulfide bridges:
an extracellular
-subunit (Mr 53 kDa) and a
transmembrane ß-subunit (Mr 3342 kDa) (13). No
uncleaved receptor could be detected in human thyroids, whereas
cleavage was incomplete in transfected L cells and some monomeric
receptor could be observed (14). Immunocytochemical studies in human
(13), rat, and rabbit thyroids (M.T. Groyer and N. Ghinea, unpublished
observations) have shown the TSHR to be localized in the basolateral
region of the cell plasma membrane. Like other plasma membrane
receptors, the TSHR mediates the internalization of the receptor-bound
ligand (15). However, due to the lack of adequate antibodies, no
immunocytochemical studies of receptor intracellular traffic have been
reported to date. Thus, the fate of the TSHR in thyroid cells is
largely unknown. Furthermore, our knowledge is still relatively limited
on the cellular trafficking of G protein-linked receptors in
general.
The great interest in the study of the TSHR has also been related to
its involvement in several pathological conditions (16). Graves
disease is a relatively frequent condition (1.9% of women) (17) due to
the occurrence of stimulatory anti-TSHR antibodies. The persistence of
this hyperstimulation of the thyroid has raised the question of an
absence of down-regulation of the receptor (18). Hypothyroidism has
been related to the presence of blocking antibodies. More recently,
mutations producing constitutively active receptors have been shown to
occur in toxic adenomas (19) and in nonautoimmune hyperthyroidism (20, 21). A defect in membrane expression of the protein has been shown to
occur in some cases of mutated TSHR (22). To understand these
functional anomalies of the receptor, it was first necessary to analyze
its cellular distribution and endocytosis in physiological conditions.
We used monoclonal antibodies we have prepared against the TSHR (13) to
analyze its distribution and endocytosis mechanisms using confocal and
electron microscopy. We examined both L cells transfected with an
expression vector encoding the human TSHR and primary cultures of human
thyroid cells. The cellular trafficking of the TSHR was compared with
the pattern observed with the LH receptor (LHR) and with chimeras
combining the ectodomain of the LH or TSH receptor with the
transmembrane and intracellular domain of the other receptor.
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RESULTS
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The present studies of intracellular trafficking of TSHR were
performed initially on an L cell line permanently transformed with an
expression vector encoding the receptor (14). They were secondarily
extended to human thyrocytes in primary culture.
Limited Endocytosis and Recycling to the Cell Surface of the
TSHR
To study the TSHR, we used antibodies T5U-317 and R5T-34. These
monoclonal antibodies interact with different epitopes of the
extracellular domain of the receptor. They do not interfere with
hormone binding and do not activate or inhibit adenylate cyclase (not
shown). These antibodies do not modify the cell surface concentration
of the receptor when incubated with TSHR expressing L cells (Fig. 1
). Furthermore, they do not modify
125I-TSH internalization (not shown). T5U-317 and R5T-34
are therefore convenient neutral antibodies allowing the tracing of the
receptor.

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Figure 1. Incubation with Antireceptor Antibodies Does Not
Induce TSHR Internalization
L cells expressing the TSHR were incubated (45 min at 4 C) with the
antibodies and washed. The cells were then incubated for 15 min (time
of maximal internalization, see Figs. 2 and 3 ) at 37 C or 4 C. TSHR
concentration on the cell surface was measured by
[125I]TSH binding. Results are shown as the mean
(±SD) of three measurements.
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The 125I-labeled T5U-317 antibody was incubated at 4 C with
L cells expressing the TSHR. As shown in Fig. 2a
, the binding yielded a linear
Scatchard plot [dissociation constant (KD) = 6
nM] titrating 30,000 surface-associated binding sites per
cell. No saturable binding was observed on control, nontransfected L
cells (not shown).

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Figure 2. Cell Surface TSHR: Effect of Hormone Treatment
L cells expressing the TSHR were incubated at 4 C with
[125I]T5U-317 antireceptor antibody and increasing
concentrations of nonradiolabeled T5U-317. The concentration of bound
and free antibody was determined (a). A Scatchard plot allowed
determination of the concentration of antireceptor antibody binding
sites on the cell surface. The cells were incubated with bTSH for
various time periods at 37 C (b). The concentration of the receptor on
the cell surface was measured for each time point by incubation with
125I-labeled antibody as described above and compared with
that observed before incubation with the hormone (=100%). Results are
shown as the mean (±SD) of three determinations.
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The L cells expressing the receptor were then incubated with bovine TSH
(bTSH) at 37 C, and the cell surface concentration of antireceptor
antibody binding sites was measured at different time periods (Fig. 2b
). In all cases, linear Scatchard plots yielded the same dissociation
constant (KD = 6 nM) for the antibody/TSHR
complexes. At 5 min, 20% of the receptor had been internalized. After
15 min of incubation, hormone treatment had decreased cell surface
concentration of receptor to 70% of control. However, at 20 min,
surface receptor concentration had increased to 85% of control. These
experiments suggested the existence of a limited internalization of
receptor (maximum 30%) followed by its recycling to the cell surface.
An alternative explanation was the possibility of replacement of some
of the internalized receptors by receptors coming from the
intracellular pool of immature mannose-rich precursor (14) or from
intracellular receptors recycling to the surface.
To distinguish between these hypotheses, we incubated the L cells
expressing the TSHR with biotinylated R5T-34 anti-TSHR antibody at 4 C
in the presence or absence of bTSH. This condition allowed us to follow
only one round of internalization of labeled receptors. The
concentration of biotinylated antibody remaining on the cell surface
after incubation at 37 C was then quantified by measuring the binding
of [125I]Streptavidin. [There was no binding of antibody
to control nontransfected L cells (not shown)]. As shown in Fig. 3
, in the absence of hormone about 10%
of the receptor-bound biotinylated antibody was internalized in the
first 5 min. Most of the complexes returned to the cell surface after
20 min. These observations probably correspond to the constitutive
internalization of the receptor occurring in the absence of the
hormone. If the cells were treated with hormone, the internalization
was increased 3-fold, and the receptor-biotinylated antibody complexes
returned to the cell surface after longer incubations (
30 min).
Administration of monensin did not interfere with the internalization
process but completely blocked the reappearance of
receptor-biotinylated antibody complexes on the cell surface. This
result confirmed that the latter phenomenon was due to receptor
recycling toward the cell surface.
This experiment thus showed the existence of a constitutive
internalization of the receptor. It also confirmed that the
hormone-induced changes in receptor concentration on the cell surface
were indeed due to its successive internalization and recycling to the
cell surface.
Furthermore, since cycloheximide was used to prevent protein
neosynthesis in the experiment illustrated in Fig. 2
and did not
inhibit receptor recycling to the cell surface, we can conclude that
the synthesis of new protein is not involved in the recycling
process.
Immunoelectron Microscopic Study of TSHR and Hormone Trafficking in
Transfected L Cells
To follow ultrastructurally the intracellular traffic of both TSH
and its receptor to various compartments involved in the endocytotic
pathway, the hormone (bTSH) was conjugated to 15-nm gold particles,
whereas the antireceptor antibody R5T-34 was coupled to 5-nm gold
particles. Living L cells, permanently expressing the TSHR, were
incubated with both hormone and antibody at 4 C. After washing out the
unbound markers, the temperature was raised to 37 C for various time
periods to allow receptor and hormone internalization. The cells were
analyzed by electron microscopy (Fig. 4
).
In the cells incubated at 4 C, both 5-nm and 15-nm gold particles were
distributed randomly on the plasma membrane, including coated pits
(Fig. 4a
). No smooth vesicle microdomains were labeled in these
conditions. This distribution is similar to that previously observed
for the LHR (23). After 5 min at 37 C, hormone and antireceptor
antibody colocalized in coated vesicles (Fig. 4b
). At 15 min, they were
both present in tubulo-vesicular endosomes (Fig. 4c
) and in
multivesicular bodies. In the latter, the marker localizations were
different: the hormone was present on the internal membrane of the
vesicles, whereas the antireceptor antibody was observed on the
limiting membrane of the multivesicular body (Fig. 4d
) [For
comparison, we show L cells expressing the LHR. In the latter,
gold-labeled hCG and anti-LHR antibodies were both localized on the
membrane of the internal vesicles (Fig. 4e
) and were both directed to
lysosomes (23)]. The localization of TSH and TSHR antibodies in
multivesicular bodies was compatible with the receptor being recycled
and the hormone dissociating from the receptor and being degraded.
Indeed, only the hormone was found associated with lysosome-like
structures where it accumulated (Fig. 4g
). Cationized ferritin has been
shown to be targeted to lysosomes (24). We thus analyzed its
localization and compared it to that of antireceptor antibody. There
was no colocalization, confirming that the latter compound was not
targeted to lysosomes (Fig. 4h
).

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Figure 4. Immunoelectron Microscopic Study of TSHR and
Hormone Trafficking in Transfected L Cells
Living cells were incubated with a mixture of gold-conjugated hormone
(TSH-Au15 nm, arrowheads) and antibody
(R5T-34-Au5 nm, arrows) and processed for
electron microscopy. TSHR and TSH distribution in cells kept at 4 C is
illustrated in panel a. TSHR antibody and TSH were both endocytosed via
clathrin-coated vesicles after 5 min exposure at 37 C (b), and they
colocalized in tubulo-vesicular endosomes (c) after 10 min. At 15 min,
all the TSH and some TSHR were sorted into multivesicular bodies (d) so
that the receptor was localized on the limiting membrane and the
hormone was on the inside vesicles. [Note that in L cells expressing
the LHR (e), both hormone (hCG-Au15 nm,
arrowhead) and receptor (LHR-38-Au5 nm,
arrow) were localized on internal structures of the
multivesicular body.] Only TSH was found in lysosome-like structures
(g) whereas the TSHR was recycled to the cell surface via caveola-like
vesicles (f) after 30 min. The presence of TSHR in a nondegradative
pathway was confirmed by the absence of colocalization of
R5T-34-Au10 nm (arrow) with cationized
ferritin (closed arrowhead), an electron opaque marker
of 5 nm targeted to lysosomes (h). cp, Coated pit; cv, coated vesicle;
lys, lysosome; mvb, multivesicular body; pm, plasma membrane; tve,
tubulo-vesicular endosome; v, caveola-like vesicle. Bar, 0.1 µm.
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At 30 min, the R5T-34-Au5nm particles were observed in
smooth vesicles associated with the plasma membrane (Fig. 4f
). Although
these vesicles resembled caveolae, examination by confocal microscopy
of cells treated with anti-TSHR and anticaveolin-1 antibodies showed
that there was no colocalization of these antibodies (Fig. 5
). Similar uncharacterized small smooth
vesicles have been previously discussed in relation to internalization
of epidermal growth factor and transferrin receptors (TfRs) (25, 26).
These vesicles were probably involved in the recycling of receptor.
However we could not completely eliminate the possibility that they
were related to another receptor internalization mechanism occurring
with slower kinetics.

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Figure 5. Dual Localization of TSHR and Caveolin-1 in
TSHR-Expressing L Cells after 30 min of Incubation at 37 C with bTSH
(10 mU/ml)
Localization of TSHR is compared with that of caveolin-1 by
superposition of both patterns (Col). The fixed cells were incubated
with a mixture of R5T-34 antireceptor antibody and rabbit
anticaveolin-1 polyclonal antibody. Sheep antirabbit IgG-Cy3
antibodies were used as second antibodies. The phase contrast
image of the cell is shown (CI).
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Comparison of Endocytosis Pathways of TSH, LH, and TfRs in
Transfected L Cells Expressing Either the TSHR or the LHR
The experiments described above suggested that TSHRs expressed in
the L cells exhibited cellular trafficking properties very different
from those of LHRs expressed in the same cells (23). The latter, which
undergo a marked hormone-induced endocytosis, are addressed to the
lysosomal degradative pathway and are not recycled to the cell surface.
This was surprising due to the high degree of structural similarity
between both receptors (8). To confirm these observations, we used L
cells to compare the cellular trafficking of either TSHRs or LHRs with
that of TfR, which is known to undergo endocytosis through
clathrin-coated vesicles and to recycle to the cell surface with an
efficiency close to 99% (27, 28).
L cells were incubated at 4 C with anti-TSHR or anti-LHR antibodies and
biotinylated transferrin in the presence of bTSH or hCG, respectively.
After washing, the cells were incubated for different time periods (0,
5, 10, and 15 min) at 37 C. Anti-mouse IgG antibodies were used to
detect the localization of anti-LHR and anti-TSHR antibodies, whereas
streptavidin-Cy3 conjugate allowed visualization of the TfR. Confocal
microscopy was used to compare the localization of both receptors (Fig. 6
).

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Figure 6. Compared Localizations of TSHR and LHR with Those
of TfR
A, Dual localization of TSHR and TfR in TSHR expressing L-cells: cell
surface distribution (0 min) and internalization (15 min at 37
C). Localization of TSHR is compared with that of TfR by the
superposition of both patterns (Col). During internalization both
receptors are endocytosed and sorted to the same endocytotic
compartment (arrows). TSHR and TfR were double labeled
by incubating transfected L cells with R5T-34 and biotinylated
transferrin in the presence of TSH. The TSHR/R5T-34 complex was
detected by an antimouse antibody-FITC conjugate. The
TfR/biotinylated-Tf complex was detected by a streptavidin-Cy3
conjugate. Cellular distributions and colocalization of receptors were
analyzed by confocal microscopy. The cells shown are representative of
a large number of specimens examined randomly at various planes of
focus. B, Dual localization of LHR and TfR in LHR-expressing L cells:
cell surface distribution (0 min) and internalization (15 min at 37 C).
Localization of LHR is compared with that of TfR by the superposition
of both patterns (Col). There was partial colocalization at the plasma
membrane. After 15 min, LHR no longer colocalized with TfR. The cells
were incubated with LHR-38, hCG, and biotinylated transferrin. The
LHR/LHR-38 complexes were detected by an antimouse antibody-FITC
conjugate and the TfR/biotinylated-Tf complexes were detected by a
streptavidin-Cy3 conjugate. Dual cellular distributions and
colocalization of the receptors were analyzed by confocal microscopy.
Cells shown are representative of a large number of specimens examined
randomly at various planes of focus. Bar, 10 µm.
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In L cells expressing the TSHR, the latter was diffusely distributed
over the plasma membrane similarly to TfR. There was partial
superposition of both distribution patterns (Fig. 6A
; 0 min). After
heating at 37 C for 5, 10 (data not shown), and 15 min (Fig. 6A
), both
receptors were internalized and sorted to the same endocytic
compartment (arrows).
A similar experiment was performed in LHR expressing L cells comparing
the trafficking of LHR and TfR (Fig. 6B
). Similarly to TSHR, LHR
partially colocalized with TfR at the plasma membrane after incubation
at low temperature (Fig. 6B
; 0 min). Colocalization was also observed
after 5 and 10 min of incubation at 37 C (not shown). However, after 15
min of incubation at 37 C, LHR and TfR were separated, suggesting that
LHR is sorted to a different endocytic compartment (Fig. 6B
; 15 min).
Indeed, TfR is known to recycle to the cell surface (29), whereas LHR
has been shown to be localized to the lysosomes where it is degraded
(23).
Comparison of Receptor-Mediated Endocytosis of
[125I]bTSH and [125I]hCG in L Cells
Expressing Either the TSH or the LH/CG Receptor
In L cells expressing either the TSHR or the LHR, we also compared
the internalization of the corresponding hormone. As illustrated in
Fig. 7
, the internalization of
[125I]bTSH by L cells expressing the TSHR was maximal
after 20 min and represented only 35% of the receptor-bound hormone.
In parallel, we studied the internalization of [125I]hCG
in L cells expressing the LHR. The kinetics of endocytosis were
identical to those of bTSH, but the proportion of receptor-bound
hormone that was internalized was markedly higher (70%). Neither the
[125I]bTSH nor the [125I]hCG were
internalized by nontransfected L cells (not shown).

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Figure 7. Comparison of Receptor-Mediated Endocytosis of
[125I]TSH and [125I]hCG in L Cells
Permanently Expressing Either the TSHR or the LHR
The cells were incubated with radiolabeled hormones, washed, and placed
at 37 C for various periods of time. The cells were recovered by
incubation with trypsin. Trypsin-resistant radioactivity was considered
as representing the internalized hormone and expressed as a percentage
of the iodinated hormone bound to the cells.
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It was also noted that when [125I]bTSH and
[125I]hCG were bound to their respective receptors and,
subsequently, the cells were incubated at 37 C, the radioactivity found
in the cell culture medium increased with the time of incubation. Most
of this radioactivity could not be precipitated by trichloroacetic
acid, and therefore probably represents degraded hormone as previously
demonstrated for LHR (30, 31, 32) and epidermal growth factor receptor
(33). Indeed, the recycling properties of the TSHRs and LHRs were
different, but in both cases the endocytosed hormone was targeted to
the lysosomes.
Intracellular Trafficking of TSHR and TSH in Cultured Human
Thyrocytes
Use of permanently transfected L cells to study intracellular
trafficking of TSHR and hormone has several advantages. It allows
comparison with the LHR under the same cellular conditions. The number
of receptor molecules expressed on the cell surface is relatively high,
allowing easier observations. Finally, these cells will allow future
studies of the expression of mutated receptors. However, the
possibility exists that observations made in L cells cannot be extended
to physiological conditions, i.e. to normal thyroid cells.
To examine this possibility we used primary human thyroid cell
cultures.
Confocal microscopy showed that the TSHR was expressed by all
thyrocytes on the basolateral domain of the plasma membrane (Fig. 8
, a and b). This is similar to the
observations made on human thyroid sections (13). Endocytosis of TSHR
was observed as previously described for the L cells and compared with
that of TfR. Colocalization of both receptors was observed at all time
periods. As shown in Fig. 8
(c, d, and e), after 15 min all the
internalized TSHR was localized in the same vesicles as the TfR. This
result, identical to the observations made in L cells, suggests that
the TSHR in thyrocytes is also present in vesicles involved in
recycling to the plasma membrane.

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Figure 8. Cell Surface Distribution (0 min) (a and b) and
Intracellular Trafficking (15 min) (c, d, and e) of TSHR in Primary
Culture of Human Thyroid Cells
TSHR was expressed on the basolateral domain of the plasma membrane (a
and b). When the thyrocytes were incubated with TSH at 37 C, TSHR was
internalized (c). Confocal analysis demonstrated that TSHR colocalizes
in the same endosomes with TfR (d and e). To visualize the TSHR, the
human thyrocytes were incubated with antibody R5T-34, which was
detected by an antimouse antibody-FITC conjugate. To follow the
endocytosis of TfR, biotinylated transferrin was added to the
incubation medium. The TfR/biotinylated-Tf complexes were visualized by
addition of the streptavidin-Cy3 conjugate (d). Confocal microscopy
observations are shown. Cellular organization was examined by
brightfield phase, and the localization of the nucleus is shown (N).
Bar, 5 µm (a and b) and 0.25 µm (c, d, and e).
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Ultrastructural studies were performed using an anti-TSHR antibody
conjugated to 5-nm gold particles and TSH conjugated to 15-nm gold
particles. Both tracers were bound to the basolateral domain of the
cell membrane, with a distribution similar to that observed in
transfected L cells, i.e. plasmalemma proper and coated pits
but not smooth vesicles open to the cell surface. The cells were
incubated at 37 C for varying periods of time before electron
microscopic processing. Observations were made difficult by the low
concentration of receptors in human thyrocytes (34).
After incubation at 4 C, both tracers were localized in clathrin-coated
vesicles (Fig. 9a
). At 10 min they were
observed in tubulo-vesicular endosomes (Fig. 9
, b and c). The
hormone-gold complexes were associated with the internal vesicles of
the multivesicular bodies (Fig. 9d
). The sorting of TSH to the
degradative pathway was confirmed by its presence in lysosome-like
structures after longer incubations at 37 C (Fig. 9e
). The anti-TSHR
antibody was not observed in these structures, suggesting that the
recycling of the receptor occurred from early endosomes where it seemed
to be concentrated.

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Figure 9. Immunoelectron Microscopic Study of TSHR and
Hormone Trafficking in Primary Cultures of Human Thyroid Cells
Thyrocytes were prepared as described in Materials and
Methods and incubated with a mixture of TSH-Au15 nm
(arrowheads) and R5T-34-Au5 nm
(arrows) at 4 C for 45 min. The unbound markers were
washed, and the cells were incubated for various time periods at 37 C
before final processing for electron microscopy. Both tracers were
bound only to the basolateral domain of thyroid cells. After incubation
at 4 C, anti-TSHR antibody and TSH were both present in clathrin-coated
pits (a), and after 10 min at 37 C in tubulo-vesicular endosomes (b and
c). After an incubation of 15 min at 37 C, TSH was observed in
multivesicular bodies on the internal structures (d). After 20 min, TSH
was present in lysosome-like structures (e). bl, Basolateral domain;
cp, coated pit; cv, coated vesicle; lys, lysosome; mvb, multivesicular
body; pm, plasma membrane; tbv, tubulo-vesicular endosome. Bar, 0.1
µm.
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These studies suggest that the endocytosis and membrane recycling
mechanisms of the TSHR are very similar in cultured human thyrocytes
and transfected L cells.
Internalization Patterns of TSHR and LHR Chimeras
We constructed two chimeras to explore the molecular basis
of differences between TSHR and LHR intracellular trafficking: LHR
extracellular domain/TSHR transmembrane and intracellular domains
(LH-TSH.R) and TSHR extracellular domain/LHR transmembrane and
intracellular domains (TSH-LH.R). L cells permanently expressing each
chimera were obtained. As in the case of wild-type receptors, the
internalization pattern of chimeras was analyzed by using biotinylated
antireceptor ectodomain antibodies and [125I]Streptavidin
and by immunofluorescence colocalization with the TfR. In both
experiments the markers were added at 4 C in the presence of 10 mU/ml
of either bTSH or hCG. After washing of unbound markers, the cells were
incubated at 37 C for various periods of time.
As illustrated in Fig. 10
, the
concentration of TSH-LH.R and LH-TSH.R associated with the cell surface
decreased during the first 20 min. LH-TSH.R and TSH-LH.R appeared to be
internalized with similar kinetics. After 20 min, the plasma membrane
concentration of TSH-LH.R continued to decrease, reaching about 60% of
its initial value. As in the case of LHR (23), the TSH-LH.R was not
recycled to the cell surface. In contrast, the plasma membrane
concentration of LH-TSH.R increased back to the initial value. Thus the
transmembrane-intracellular domains of these receptors determine
whether they recycle or not to the cell surface. However, the extent of
internalization of the TSH-LH.R chimera is lower than that of the LHR,
suggesting that the extracellular domain plays a role in this
respect.
Confocal microscopy was used to compare the cellular trafficking of the
chimeras with that of the TfR. At short times after internalization
(015 min), TfR, LH-TSH.R, and TSH-LH.R were all localized in the same
structures (not shown). At longer times (20 min), LH-TSH.R colocalized
with TfR, which is known to recycle to the cell surface, whereas
TSH-LH.R showed a different localization (Fig. 11
). This experiment confirmed that
addressing of receptor to the recycling pathway is determined by
signals present in the transmembrane-intracellular domain.

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Figure 11. Dual Localization of LH-TSHR and TSH-LHR Chimeras
and of TfRs
L cells expressing either LH-TSHR or TSH-LHR chimeras were labeled with
antireceptor ectodomain antibodies in the presence of hormone and with
biotinylated transferrin. The antibody/receptor complexes were detected
by an antimouse antibody-FITC conjugate, and the biotinylated-Tf/TfR
complexes were detected by a streptavidin-Cy3 conjugate (see Fig. 6 ).
Bar, 10 µm.
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DISCUSSION
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Very little information has been available on the membrane
distribution of the TSHR and its intracellular trafficking. Two
experimental approaches have been used to date. Receptor concentration
has been measured after challenging the thyroid cells with hormone.
This has led to varying results, and to the overall conclusion that
"TSH had either a positive or a negative modulatory role on its own
receptor according to both its concentration and thyroid gland
activity" (35). In transfected heterologous cell lines,
[125I]TSH binding was decreased in NIH3T3 cells, but only
after several hours of incubation (36). It was not decreased in Chinese
hamster ovary (CHO) cells (36). The molecular mechanisms involved in
these variations have been debated. Both down-regulation (37) and
up-regulation (38) of TSHR mRNA have been described, the latter
being perhaps mediated by a posttranscriptional nuclear event (39). The
only studies on the internalization of the TSHR have used either
[125I]TSH (36, 40, 41, 42) or fluorochrome-TSH conjugate
(15). No immunocytochemical study, even at the level of optical
microscopy, has been performed to date to analyze TSHR intracellular
trafficking.
For G protein-coupled receptors in general, studies have been performed
using mainly radioactivity, fluorochrome, or enzyme-labeled ligands
and, in some rare cases, antireceptor antibodies. Only a few studies
(5, 23, 43, 44, 45) have been performed at the electron microscopic level.
Initially, Raposo et al. (43) studied the muscarinic
acetylcholine receptor and the ß-adrenergic receptor (5). Since both
receptors were internalized via caveolae, it was proposed (46) that all
G protein-coupled receptors would follow this pathway, in contrast to
tyrosine kinase-coupled receptors, which were known to be internalized
through clathrin-coated vesicles.
However, using gold-conjugated anti-LHR antibodies and
ultrastructural analysis, we demonstrated the LH/CG receptor to be
internalized via clathrin-coated vesicles (23). Recently,
internalization through clathrin-coated vesicles has been described in
some cell types for ß-adrenergic (47) and muscarinic receptors (48).
The pathway of internalization of several other G protein-coupled
receptors was also studied recently, using mainly high-sucrose
concentration sensitivity of membrane-associated clathrin lattices
(49). In most cases [thrombin (45, 50), cholecystokinin (44),
gastrin-releasing peptide (51, 52), and PTH (53) receptors], the
endocytosis seemed to involve clathrin-coated vesicles. Among these
receptors, some were recycled to the cell surface, e.g. GnRH
(54) and TRH (55, 56) receptors in pituitary cells,
ß2-adrenergic receptor (57), and angiotensin II receptors
(58) in transfected 293 cells and gastrin-releasing peptide receptor in
transfected epithelial cells (52). Other G protein-coupled receptors
were, on the contrary, mostly degraded once endocytosed. This was the
case with LH/CG (23), thrombin (45, 50), and yeast pheromone (59)
receptors. In transfected CHO cells permanently expressing the
cholecystokinin receptor, internalization of the receptor has been
analyzed by using the green fluorescent protein (60) or by electron
microscopy (44). In the latter case, two pathways were observed.
Clathrin-coated vesicles directed the receptor toward the degradative
pathway leading to the lysosomes, whereas caveolae remained associated
with the plasma membrane and allowed a rapid recycling of the
receptor.
These differences in receptor recycling or degradation may explain
different effects of the hormones. The degradation in the lysosomes of
the LHR may explain the negative regulation of the receptor observed in
previous biochemical studies (30, 31). On the other hand, the recycling
of the TSHR may explain why stimulating autoantibodies maintain their
activity and provoke longstanding hyperthyroidism in Graves
disease.
Hormone and receptor endocytosis were compared. Large gold particles
(15 nm) were coupled to TSH, whereas smaller particles were coupled to
antireceptor antibodies. Theoretically, although such large particles
may, in some cases, modify the intracellular trafficking of proteins,
the results obtained with this method were substantiated by the study
of internalization of [125I]TSH. Furthermore, in the case
of the highly homologous hormone hCG, the size of gold particles did
not influence cellular trafficking (23).
The very limited endocytosis of the TSHR (30% of receptor molecules)
is similar to that observed for the GnRH receptor at physiological
hormone concentrations (61) but very different from the high
endocytosis rate (6090%) of other G protein-coupled receptors such
as LH/CG (23), thrombin (49), angiotensin II (62), neuromedin B (63),
and
-opioid (64) receptors. A constitutive endocytosis of TSHR was
observed in the absence of hormone. Similar observations have
previously been made for the LDL (2), the transferrin (3), the LH (23),
and the cholecystokinin receptors (59).
The LH, FSH, and TSH receptors share a high degree of homology (65). It
is, therefore, surprising that the cellular trafficking of TSHRs and
LHRs is so different. A different pH sensitivity for dissociation of
the bTSH-TSHR complex vs. the hCG-LHR complex has not been
observed (31, 66). It was found recently in fibroblasts transfected
with different subtypes of adrenergic receptors, that different
subcellular sorting occurred (67). The ß2-receptors were
internalized to intracellular vesicles that were different from those
of internalized M
24H receptors. In contrast,
M
210H receptors remained on the cell surface.
Cellular trafficking of TSHR and LHR in L cells closely resembles their
trafficking in thyroid and Leydig cells, respectively. This allowed us
to perform comparative studies in a single cell type. The analysis of L
cell lines expressing chimeric receptors of LH and TSH allowed us to
assert that the transmembrane and intracellular domains determine the
intracellular fate of the internalized receptor. However, the
extracellular domain seems to modulate the extent of internalization.
Indeed, the TSH-LH.R chimera was less internalized than the LHR (23).
These experiments are the first step in the identification of signals
that specify the distinct intracellular sorting patterns of these
receptors. Such experiments should also allow us to establish the
relationship between the signals involved in basolateral localization
of TSH (and FSH) receptors in polarized cells and the endocytosis
signals. The role of receptor phosphorylation in its cellular
trafficking will also have to be considered.
 |
MATERIALS AND METHODS
|
---|
Chemicals
Cell culture products were obtained from the following sources.
DMEM, nutrient mixture F12 (Ham), Geneticin (G-148 Sulfate),
antibiotics mixture (penicillin, streptomycin, and neomycin), FCS, and
human plasma fibronectin were from Life Technologies, Inc.
(Gaithersburg, MD). Lab-Tek culture chambers were from Nunc Inc.
(Naperville, IL), and cell culture flasks were from Costar
(Cambridge, MA). Human insulin was from Eli Lilly & Co.
(Saint Cloud, France); soybean trypsin inhibitor was from Roche Molecular Biochemicals (Mannheim, Germany). Crude collagenase
type IA (1,000 U/mg), BSA, holo-transferrin, monensin, biotin-labeled
transferrin, cationized ferritin, sheep antimouse IgG-fluorescence
[fluorescein isothiocyanate (FITC)] conjugate, sheep antirabbit
IgG-Cy3 conjugate, streptavidin-Cy3 conjugate, mouse-purified Igs,
and sheep serum were from Sigma Chemical Co. (St.Louis,
MO). Rabbit antihuman caveolin-1 (N-20) antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Electron microscopic
reagents were purchased from various sources: paraformaldehyde extra
pure, glutaraldehyde, lead (II) citrate, and uranyl acetate dihydrate
were from Merck & Co., Inc. (Darmstadt, Germany); osmium
tetroxyde was from Interchim (Montluçon, France). Poly/Bed 812
embedding medium was from Polysciences Inc. (Warrington, PA), and
colloidal gold solutions were from BioCell Research Laboratories
(Cardiff, UK). [125I]Streptavidin was from Amersham International (Buckinghamshire, UK). Plasmid pSG5 was from
Stratagene Cloning Systems (Montigny le Bretonneux,
France).
Monoclonal Antibodies and Hormones
Three distinct monoclonal antibodies were used: T5U-317 and
R5T-34 are directed against the extracellular domain of the human TSHR
(13), and LHR-38 is directed against the extracellular domain of the
porcine LHR (11). T5U-317 was radiolabeled with 125I
and Iodo-Gen reagent from Pierce Chemical Co. (Oud
Beijerland, Netherlands). The specific activity was 6.2 µCi/µg and
the biological activity of the radiolabeled antibody was 30%.
[125I]bTSH (70 µCi/µg) was purchased from ERIA
Diagnostics Pasteur (Marnes la Coquette, France). Its ability to bind
to the human TSHR expressed in L cells was determined to be 57%.
[125I]hCG (90 µCi/µg) was from NEN Life Science Products (Dupont de Nemours, Belgium). Its biological
activity was found to be 40%. Monoclonal antibodies were biotinylated
by using the ECL protein biotinylation module following the procedure
recommended by Amersham International. R5T-34-Au5
nm, R5T-34-Au10 nm, and bTSH-Au15 nm
were prepared according to standard methods (68). The highly purified
bTSH was a generous gift from the NIH. bTSH used to stimulate the TSHR
was from Sigma Chemical Co., and hCG (Gonadotropine
Chorionique "Endo") was from Organon (Eragny sur Epte,
France).
All receptor/antibody complexes (i.e. with nonmodified,
biotinylated, radiolabeled, and gold-conjugated antibodies) were stable
at pH
4.5 (data not shown), which is the pH of the endocytic
system (69). This suggested that antibody/receptor complexes did not
dissociate in intracellular compartments during internalization.
Cell Culture
L Cells Permanently Expressing the Human TSHR and the Porcine
LHR
The immortalized L cell lines, permanently expressing the human TSHR
and the porcine LHR, were obtained as previously described (11, 14, 23). Two clones expressing high levels of each receptor were selected
and used in this study. Cells were cultured for 4872 h before each
experiment in DMEM containing 10% FCS and Geneticin (0.25 mg/ml).
Chimeric Receptors
To create expression vectors encoding the TSH-LH.R and LH-TSH.R
chimeric receptors, we initially introduced an EcoR V
restriction site into the pSG5-hTSHR and pSG5-pLHR expression plasmids
(8). This was done by PCR- based site-directed mutagenesis (70). Silent
point mutations were introduced in the aspartate and isoleucine codons
located at positions 410 and 411 of the hTSHR and in the isoleucine
codon located at position 356 of the pLHR (8). The mutated expression
plasmids were then digested with EcoRI and EcoR V
to isolate the ectodomains. After purification, the DNA fragments
encoding the ectodomains were exchanged and subcloned to get the
recombinant expression plasmids pSG5-TSH-LH.R and pSG5-LH-TSH.R. All
constructs were verified by sequencing. The pSG5-TSH-LH.R expression
plasmid encodes a protein of 751 amino acids consisting of the 410
amino acids of the hTSHR ectodomain linked to the 341 amino acids of
the pLHR transmembrane and intracellular domains. The pSG5-LH-TSH.R
expression plasmid encodes a protein of 709 amino acids consisting of
the 355 amino acids of the pLHR ectodomain linked to the 354 amino
acids of the hTSHR transmembrane and intracellular domains.
Cell lines permanently expressing the TSH-LH.R and LH-TSH.R chimeric
receptors were obtained through a calcium phosphate cotransfection of
the pSG5-TSH-LH.R or pSG5-LH-TSH.R together with the pSV2-neo vector,
which confers resistance to the antibiotic G418, in the mouse L cell
line. Colonies were selected with G418 (0.8 mg/ml) and screened for
expression by immunocytochemistry using the monoclonal anti-TSHR or
anti-LHR antibodies, T5U-51 and LHR-38, respectively. Positive colonies
were finally subcloned to get clonal cells by dilution limit.
Human Thyroid Cells
Human thyroid tissues were obtained from euthyroid patients undergoing
surgery for a goiter. Thyroid follicles were prepared as previously
described (71) with the following modifications: the minced thyroid
tissue was digested for 30 min at 37 C with collagenase (25 µg/ml) in
DMEM containing 1% BSA (buffer A), soybean trypsin inhibitor (80
µg/ml), and a mixture of antibiotics (see above). The incubate was
filtered successively through two screen cups from the Cell
Dissociation Sieve-Tissue Grinder Kit (Sigma Chemical Co.). After washing, any tissue remaining on the first screen
(380-µm mesh) underwent a new digestion cycle. The follicles retained
on the second screen (140 µm mesh) were resuspended in DMEM
containing 10% FCS. Follicles were dissociated by pipetting and plated
on plastic dishes or glass Lab-Tek precoated with human fibronectin (10
µg/ml). They were cultured in a 1:1 mixture of DMEM and Ham F12
supplemented with holo-transferrin (5 µg/ml), human insulin (15
mU/ml), 10% FCS, and soybean trypsin inhibitor (80 µg/ml). On the
second day of culture, the soybean trypsin inhibitor was washed out and
FCS concentration was lowered to 1%.
Studies Using Radiolabeled Antibodies or Hormones
Effect of Anti-TSHR Antibodies on Receptor Internalization
TSHR-expressing L cells were incubated at 4 C for 45 min with T5U-317,
R5T-34, or R5T-34-biotin (10 µg/ml), rinsed with cold PBS (3 x
5 min), and then incubated for 15 min at 37 C. Cells maintained at 4 C
were used as control. Cells were then incubated at 4 C for 45 min with
a saturating concentration of [125I]TSH (0.1 µg/ml),
rinsed with cold PBS (4 x 5 min), and recovered by addition of
trypsin (2.5 mg/ml)-EDTA (5 mM) in PBS. The radioactivity
of the suspension was counted. Nonspecific binding was measured in the
presence of an excess of bTSH (0.1 mg/ml) and subtracted from the total
binding. Antibody-induced internalization of TSHR was compared with
that induced by mock incubation with PBS (control). Results are given
as mean of ± SD of three different experiments.
Scatchard Analysis of the TSHR Expressed on the Cell Surface
Cells were incubated for 45 min at 4 C with [125I]T5U-317
(6 pM) and increasing concentrations (02
µM) of T5U-317 in DMEM containing 1% BSA (buffer A).
Each incubation was performed in triplicate. The unbound antibody was
washed out. Cells were detached by treatment with 5 mM EDTA
for 10 min at 37 C. The radioactivity associated with the cell
suspension was counted in a
-counter. The results were normalized
according to the number of cells (900,000 cells/ml). Nonspecific
binding was determined by using control nontransfected L cells and
subtracted from the total binding. An excess of noniodinated T5U-317 (1
mg/ml) abolished the binding of [125I]T5U-317. Scatchard
analysis of the binding data yielded the number of binding sites
present on the cell surface. A similar method was used to determine the
concentration of TSHR on the plasma membrane after incubation at 37 C
for 5, 15, and 20 min with bTSH (10 mU/ml). During this experiment, the
neosynthesis of the receptor was blocked by cycloheximide (10
µg/ml).
Internalization of TSHR in the Presence of TSH and Monensin
Cells were rinsed twice with cold DMEM and preincubated for 45 min at 4
C with 5 µg/ml of biotinylated R5T-34 in buffer A. TSH (10 mU/ml) and
monensin (50 µM) were added to some incubations. Cells
were washed at 4 C and incubated at 37 C for 040 min. The
biotinylated R5T-34 present on the plasma membrane was quantified by
incubation for 45 min at 4 C with [125I]Streptavidin (2
ng/ml, 40 µCi/µg). The excess of [125I]Streptavidin
was washed out with PBS. The cells were recovered and the radioactivity
counted. The binding of [125I]Streptavidin was negligible
in the absence of biotinylated R5T-34. The nonspecific binding to
nontransfected L cells was subtracted from the total binding. Each
experiment was performed in triplicate.
Internalization of [125I]bTSH and
[125I]hCG
L cells expressing TSHR and LHR were processed similarly. They were
rinsed twice with cold buffer A and preincubated for 45 min at 4 C with
either [125I]hCG or [125I]bTSH (1
mU/ml). The unbound hormones were washed out with cold buffer A. The
cells were then placed in a water bath at 37 C for varying time periods
(050 min). The incubation medium was discarded, and the cells were
recovered by addition of trypsin (2.5 mg/ml)-EDTA (5 mM) in
PBS for 10 min at 4 C, and the total radioactivity of the suspension
was counted. This value represented the total binding of the hormone.
After four centrifugations and washes with cold PBS, the radioactivity
was counted again. This trypsin-resistant radioactivity was considered
as representative of the internalized fraction of the hormone.
(Preliminary experiments have shown that the trypsin-EDTA treatment of
the cells preincubated at 4 C with radiolabeled probes solubilized
100% of the cell surface-bound hormone or antibody.) Each experiment
was performed in triplicate. Nonspecific binding was determined on
nontransfected L cells or by incubating transfected L cells with a
100-fold excess of nonlabeled hormone. In all cases, nonspecific
binding was less than 5% of the specific binding.
The radioactivity present in the cell incubation medium (buffer A) was
also measured, and the fraction precipitable with 10% trichloroacetic
acid (overnight at 4 C) was determined as previously described
(30, 31, 32, 33).
Microscopic Studies
Confocal Immunofluorescence
Human thyrocytes and L cells expressing the TSHR, the LHR, and the
chimeric receptors were processed similarly. Cells were chilled at 4 C
and incubated in buffer A for 45 min with biotinylated transferrin (20
µg/ml) and R5T-34 or LHR-38 (20 µg/ml) in the presence of 1 mU/ml
of bTSH or hCG, respectively. Unbound ligands were washed out and the
cells were warmed to 37 C for 020 min. The cells were fixed with 3%
paraformaldehyde in PBS, pH 7.3, for 15 min at 20 C and permeabilized
by incubation for 10 min with PBS-Triton X-100 (0.4%). Nonspecific
binding sites were quenched by incubating the cells three times for 5
min at 20 C in PBS containing 50 mM NH4Cl, and
for 45 min at 20 C in PBS containing 1% BSA, 0.2% Tween 20, and 0.1%
sheep serum (buffer B). The monoclonal antibodies were revealed by
sheep antimouse IgG-FITC conjugate (diluted 1:60) and the biotinylated
transferrin was revealed by streptavidin-Cy3 conjugate (diluted 1:100)
in buffer B, for 60 min at 20 C. The cells were washed with buffer B
(three times for 5 min), with PBS (twice for 5 min) and with distilled
water (twice for 5 min). Nontransfected L cells were used as control.
Colocalization was assessed by a confocal system (LSM410) on an
Axiovert 135 M microscope (Carl Zeiss, Thornwood, NY).
Cells were randomly selected and observed morphologically by bright
field observation, and horizontal sections (1 µm) were scanned for
colocalization.
In some experiments L cells expressing the TSHR were incubated for 30
min at 37 C with bTSH (1 mU/ml), and the distribution of TSHR and
caveolin-1 was compared. After cell fixation, permeabilization, and
quenching (see above) the cells were incubated for 60 min at room
temperature with a mixture of R5T-34 (1 µg/ml) and rabbit
anticaveolin-1 antibody (0.5 µg/ml). Sheep antimouse IgG-FITC
(diluted 1:60) and sheep anti-rabbit IgG-Cy3 (diluted 1:100) were used
as secondary antibodies. Confocal microscopy was used as described
above.
Electron Microscopy
Human thyrocytes and TSHR-expressing L cells were washed twice at
20 C and chilled at 4 C in buffer A. The cells were incubated at this
temperature for 45 min with a mixture of R5T-34-Au5 nm
and of
bTSH-Au15 nm
in buffer A in
the presence of mouse IgG (10 µg/ml). The cells were rinsed twice
with buffer A at 4 C and warmed to 37 C for 060 min. Two washes with
0.1 M cacodylate buffer, pH 7.3 (buffer C), at 20 C were
performed before fixing the cells with 2.5% glutaraldehyde and 5%
paraformaldehyde in buffer C. The cells were then processed for
electron microscopy as follows: after fixation with 1%
OsO4 in buffer C, staining with 0.5% uranyl acetate in
distilled water, dehydration in ethanol, and embedding of the cell
monolayers in poly/Bed 812 resin. Ultrathin sections (
50 nm thick)
were cut on a Reichert Jung ultracut microtome and stained with uranyl
acetate and lead citrate. Observations and photographs were taken at 80
kV on a Jeol JEM-1010 electron microscope (JEOL Ltd.,
Tokyo, Japan). To ensure the specificity of the tracers,
we verified the absence of binding to nontransfected L cells and that
no gold particles were associated with transfected L cells in the
presence of an excess of nonconjugated tracers.
Experiments were also performed to compare the endocytic pathway of
TSHR with that of a macromolecule (cationized ferritin) known to be
targeted to the lysosomes (24). In this experiment, R5T-34 was coupled
to 10-nm gold particles, not to be confused with cationized ferritin
particles (5 nm). L cells expressing TSHR were incubated with 1 mg/ml
of cationized ferritin in 0.15 mM NaCl for 10 min at 37 C.
The unbound cationized ferritin was washed out and the cells cultured
for 16 h. The L cells expressing the TSHR were then incubated at 4
C with R5T-34-Au10 nm
in buffer A in
the presence of bTSH (10 mU/ml) and mouse IgG (10 µg/ml). The cells
were rinsed twice with buffer A and warmed to 37 C for 1 h. The
cells were then fixed and processed for electron microscopy as
described above.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. M. Misrahi and Dr. M. VuHai-LuuThi for
the gift of transfected L cells permanently expressing the TSHR and the
LHR, respectively. We thank Philippe Leclerc for help with confocal
microscopy.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. E. Milgrom, Hôpital de Bicêtre 3 me niveau, 94270 Le Kremlin Bicêtre, France.
This work was supported in part by lInstitut National de la
Santé et de la Recherche Médicale, lInstitut
Fédératif de Recherche du Kremlin Bicêtre,
lUniversité Paris Sud, lAssociation pour la Recherche sur le
Cancer, la Ligue Nationale Contre le Cancer, and la Fondation pour la
Recherche Médicale Française.
Received for publication June 26, 1998.
Revision received May 28, 1999.
Accepted for publication July 8, 1999.
 |
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