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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 2Go and 3Go) 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.

 
The 125I-labeled T5U-317 antibody was incubated at 4 C with L cells expressing the TSHR. As shown in Fig. 2aGo, 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.

 
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. 2bGo). 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. 3Go, 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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Figure 3. TSHR Internalization and Recycling in TSHR-Expressing L Cells in the Absence of Hormone (•-•). Effect of bTSH Alone ({blacksquare}-{blacksquare}) and bTSH and Monensin ({square}-{square})

TSHR on plasma membrane was labeled with biotinylated R5T-34 antireceptor antibody. The concentration of biotinylated antibody/TSHR complexes present on the plasma membrane was determined at all time points by the binding of [125I]streptavidin.

 
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. 2Go 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. 4Go). 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. 4aGo). 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. 4bGo). At 15 min, they were both present in tubulo-vesicular endosomes (Fig. 4cGo) 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. 4dGo) [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. 4eGo) 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. 4gGo). 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. 4hGo).



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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.

 
At 30 min, the R5T-34-Au5nm particles were observed in smooth vesicles associated with the plasma membrane (Fig. 4fGo). 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. 5Go). 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).

 
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. 6Go).



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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.

 
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. 6AGo; 0 min). After heating at 37 C for 5, 10 (data not shown), and 15 min (Fig. 6AGo), 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. 6BGo). Similarly to TSHR, LHR partially colocalized with TfR at the plasma membrane after incubation at low temperature (Fig. 6BGo; 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. 6BGo; 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. 7Go, 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.

 
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. 8Go, 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. 8Go (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).

 
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. 9aGo). At 10 min they were observed in tubulo-vesicular endosomes (Fig. 9Go, b and c). The hormone-gold complexes were associated with the internal vesicles of the multivesicular bodies (Fig. 9dGo). The sorting of TSH to the degradative pathway was confirmed by its presence in lysosome-like structures after longer incubations at 37 C (Fig. 9eGo). 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.

 
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. 10Go, 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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Figure 10. Internalization of LH-TSHR and TSH-LHR Chimeras

L cells permanently expressing either LH-TSHR ({diamondsuit}{diamondsuit}) or TSH-LHR ({blacksquare}{blacksquare}) were labeled with biotinylated antireceptor antibodies (LHR-38 and R5T-34, respectively). The concentration of biotinylated antibody/chimeric receptor complexes present on the plasma membrane was determined at all time points by measuring the binding of [125I] Streptavidin (see Fig. 3Go).

 
Confocal microscopy was used to compare the cellular trafficking of the chimeras with that of the TfR. At short times after internalization (0–15 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. 11Go). 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. 6Go). Bar, 10 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 (60–90%) of other G protein-coupled receptors such as LH/CG (23), thrombin (49), angiotensin II (62), neuromedin B (63), and {delta}-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{alpha}24H receptors. In contrast, M{alpha}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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 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 {gamma}-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

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 0–60 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 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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