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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 33–42 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(13), rat, and rabbit thyroids (M.T. Groyer and N. Ghinea, unpublished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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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, 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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). 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.
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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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). 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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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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; 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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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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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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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.
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). 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (60–90%) 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 M24H receptors. In contrast,
M210H 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 48–72 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 (0–2
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 0–40 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
(0–50 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 0–20 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
 |
 |
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
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported in part by l’Institut National de la Santé et de la Recherche Médicale, l’Institut Fédératif de Recherche du Kremlin Bicêtre, l’Université Paris Sud, l’Association 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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