TSH Receptor Interaction with the Extracellular Matrix: Role on Constitutive Activity and Sensitivity to Hormonal Stimulation
Nicolae Ghinea,
Catherine Baratti-Elbaz,
Angelo De Jesus-Lucas and
Edwin Milgrom
Institut National de la Santé et de la Recherche Médicale Unité135, Hormones, Gènes et Reproduction, Hôpital de Bicêtre, 94275 Le Kremlin-Bicêtre, France
Address all correspondence and requests for reprints to: Nicolae Ghinea, Ph.D., Institut National de la Santé et de la Recherche Médicale Unité 135, Centre Hospitalier Universitaire de Bicêtre, 3eme niveau, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France. E-mail: ghinea{at}infobiogen.fr.
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ABSTRACT
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Using immunocytochemistry, we have observed that the TSH receptor (TSHR) is concentrated at the leading edge of lamellipodia in both cultured human thyroid cells and in various transfected cells. This segregation of the receptor is due to its interaction with extracellular matrix (ECM) and specially with fibronectin. The TSHR, which interacts with the ECM, is known to undergo cleavage by a matrix metalloprotease. The homologous LH receptor, which does not interact with ECM, is not cleaved.
The attachment to the ECM modifies the functional properties of the receptor: it increases adenylate cyclase stimulation by hormone, whereas PLC stimulation is not modified. Furthermore, the constitutive activity of the TSHR is only observed in attached cells, suggesting that it is dependent on TSHR interaction with the ECM. Thus, aside from its classical properties of hormone binding and signalization through G proteins, the TSHR is also involved in cell-matrix interactions, which modulate its functional properties.
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INTRODUCTION
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CELLS CARRY ON their surface mainly two types of proteins: adhesion proteins involved in interactions with the extracellular matrix (ECM) and with neighboring cells and receptors that recognize diffusible informational molecules (hormones and growth factors). After binding their cognate ligand, the receptors elicit intracellular signals. The cell adhesion proteins belong to several categories: the immunoglobulin superfamily, the cadherins, the selectins, the proteoglycans, and the integrins (1). For most of them, and especially for the integrins, it has been shown that aside from their role in cell adhesion, they also function as signal-transmitting molecules modulating protein tyrosine phosphorylation, G protein function, Ca2+ influx, cytoplasmic alkalinization, and phosphatidylinositol turnover (2, 3, 4, 5, 6). Thus, the difference between adhesion molecules and signal transmitting receptors has become blurred. However, there are few examples of the converse situation where a hormone or growth factor receptor is involved directly in cell-cell or cell-matrix interactions (7, 8, 9, 10, 11). We have obtained evidence that this is the case for the THSR. It is a G protein-linked receptor whose cloning has allowed the determination of its sequence and structure: it possesses a characteristic seven transmembrane span that separates a short intracellular segment from a very large extracellular domain (12, 13). The TSHR ectodomain is constituted by a 9-fold repetition of a leucine-rich motif (14) and is responsible for the binding of the hormone. The latter activates both adenylate cyclase and PLCß1 via TSHR (15).
Immunopurification and immunoblotting studies (16) have shown that the TSHR is cleaved into two subunits that are held together by disulfide bridges: an extracellular
-subunit (53 kDa) and a transmembrane ß-subunit (3342 kDa). No uncleaved receptor could be detected in human thyroid glands, whereas cleavage was incomplete in transfected L cells where some monomeric receptor could be observed (17). Immunocytochemical studies in human (16) and rat and rabbit thyroids (Ghinea, N., unpublished data), as well as in transfected MDCK cells (18) and in primary cultures of human thyroid cells (19) have shown the TSH receptor to be localized in the basolateral segment of the cellular membrane.
We have examined in more detail TSHR localization and observed that it is concentrated at the sites of close contact between cells and the extracellular matrix. Binding to fibronectin plays a major role in this interaction. Receptor-ECM interaction modulates the receptor response to hormone and is also involved in ligand-independent activation of the receptor.
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RESULTS
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TSHR Is Concentrated at Sites of Close Contact between Cells and ECM, Particularly at the Leading Edge of Lamellipodia
Initial experiments were performed on an L cell line stably expressing the TSHR. Immunofluorescence microscopy of nonconfluent cells showed a streaky distribution of TSHR at the leading edge of lamellipodia (Fig. 1A
). The presence of TSHR in these regions of the cells was confirmed by interference reflection microscopy (20) (Fig. 1B
). Besides immunolabeling at the plasma membrane, staining was also observed inside the cells, probably corresponding to the precursor protein that has been shown to accumulate in the endoplasmic reticulum (17).

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Figure 1. TSHR Is Concentrated at Sites of Close Contact between Cells and the ECM
A and B, Localization of TSHR by immunofluorescence microscopy in transfected L cells stably expressing the receptor. Note the concentration of TSHR at the leading edge of lamellipodia (A) where close contact between cells and ECM is visualized by interference reflection microscopy (B). CE, Confocal microscopy of transfected L cells comparing the cellular distribution of TSHR (C) with that of -actinin (D), a marker of the leading edge of lamellipodia. E, Regions of the cells where TSHR and -actinin localizations overlap. FH, Control experiment with transfected L cells stably expressing the LHR. Confocal microscopy analysis indicates that the LHR does not concentrate at the leading edge of the lamellipodium (F). It does not colocalize with ß-actin (G, H), another marker of lamellipodia. Bar, 10 µm.
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Cell membrane distribution of TSHR was compared with that of several proteins known to be concentrated at the leading edge of lamellipodia (21). Confocal microscopy showed colocalization of TSHR with
-actinin (Fig. 1
, CE), ß-actin, and talin (not shown).
When LH receptor (LHR) distribution was analyzed in a stably transfected, L cell line (22) there was no concentration of receptor at the leading edge of lamellipodia (Fig. 1F
) and no colocalization with ß-actin (Fig. 1
, G and H),
-actinin, or talin (not shown).
We then examined the time course of redistribution of TSHR by plating cells on fibronectin (see further) for various time periods in serum-free medium. After only 15 min, the TSHR was concentrated at the leading edge of lamellipodia, where it colocalized with ß-actin (Fig. 2
, AC).

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Figure 2. Localization of TSHR in Transfected Cells and in Primary Cultures of Human Thyroid Cells
AC, TSHR accumulation at contacts with the ECM occurs very rapidly after cell adhesion. L cells stably expressing the TSHR were plated for 15 min on a fibronectin substratum. The localization of TSHR was analyzed by confocal microscopy and compared with that of ß-actin. Note that at this early stage of cell adhesion and spreading the TSHR is already concentrated at the leading edge of the lamellipodium (A) where it colocalizes with ß-actin marker (B, C). D, TSHR concentration at contacts with the ECM is also observed in thyroid cells. Primary cultures of human thyroid cells were studied by immunofluorescence microscopy. The TSHR was concentrated at the leading edge of lamellipodia of nonconfluent cells. Bar, 10 µm.
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Addition of low concentrations of hormone (0.011 mU/ml) to the cells was without apparent effect on receptor distribution, whereas high concentrations (>10 mU/ml) provoked receptor redistribution and increased cell spreading (Fig. 3
).

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Figure 3. Hormone Effect on Receptor Distribution in Adherent Subconfluent L-TSHR Cells
Receptor distribution in the absence of hormone (A) in the presence of 1 mU/ml (B) or 20 mU/ml (C) of bTSH. Note that the hormone at high concentration provokes receptor redistribution and increased cell spreading. Bar, 10 µm.
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Finally we examined the distribution of TSHR in primary cultures of human thyroid cells. As shown in Fig. 2D
, the receptor was also concentrated at the leading edge of lamellipodia of nonconfluent cells.
TSHR Interacts with ECM: Role of Fibronectin
The presence of TSHR in the close contacts between cells and ECM suggested that the receptor might be interacting with the ECM through its ectodomain in both thyroid cells and transfected cells. Because the ectodomain may be experimentaly separated from the transmembrane ß-subunit by thiol-reducing agents (16) we performed the following experiment. L cells expressing the TSHR were cultured for three days on tissue culture plates, treated with ß-mercaptoethanol and detached as previously described (23). The ECM from which the cells have been detached was then probed for the presence of TSHR
-subunit (ectodomain) by antibody TSHR317. As shown in Fig. 4
, a strong reaction was observed. Conversely, no reaction was observed using antibody TSHR365, which recognizes receptor intracellular domain. These data effectively demonstrated that only the
-subunit was present within the ECM and eliminated the possibility of the presence of intact receptor which could have remained attached to membrane fragments or originated from cell lysis. If the cells were not treated by ß-mercaptoethanol before their detachment, a significantly lower amount of TSHR
-subunit was retained in the ECM.

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Figure 4. TSHR Ectodomain Is Bound to the ECM
L-TSHR cells (containing 30,000 receptor molecules/cell) were treated with ß-mercaptoethanol (ß-me) to separate the -subunit (ectodomain) from the membrane spanning ß-subunit. The ECM was exposed by removing the cells and was probed for the presence of the TSHR ectodomain with TSHR317 monoclonal antibody. The strong reaction observed indicates the retention of TSHR ectodomain in the ECM. In contrast, no reaction was observed with the TSHR365 monoclonal antibody that recognizes the intracellular domain of the receptor. Note the low amount of receptor ectodomain retained in ECM in the absence of ß-mercaptoethanol treatment before removal of the cells. Note also that in similar conditions the LHR (used here as a control) was not retained by the ECM of L-LHR cells (containing 120,000 receptor molecules/cell). The affinity for their respective receptors of TSHR317 antibody and LHR38 antibody was similar. Results are given as the mean ± SD of six experiments.
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The LHR has a high degree of homology with the TSHR (13, 24). However, it is not cleaved (25, 26) and thus the ectodomain cannot be detached from cell membranes. We cultured a similar L cell line expressing LHR in the same conditions, did or did not submit them to ß-mercaptoethanol and then detached the cells. As shown in Fig. 4
, the extracellular domain of the LHR could not be detected in the ECM.
We also examined the location of the TSHR
-subunit in the ECM by immunofluorescence after ß-mercaptoethanol treatment and detachment of the cells. The fluorescence followed a pattern similar to that of cell contours with an enhancement corresponding to the location of cell lamellipodia (not shown).
We then determined whether we could demonstrate such a binding in acellular conditions. ECM was deposited on plastic by culturing during the 3 d L cells not expressing TSHR and detaching them from the wells. Immunoaffinity purified cellular TSHR was incubated with the ECM. Binding to the ECM was confirmed using a polyclonal antibody raised against the extracellular region (amino acids 19389) of the TSHR (not shown). Interestingly, antifibronectin antibodies blocked approximately 80% of cellular TSHR binding to ECM. No such effect was observed with antibodies raised against other major components of the thyroid basal membrane such as laminin, collagen I, or heparan-sulfate proteoglycans (not shown). Direct binding of cellular TSHR to fibronectin was also observed (see Fig. 5B
).
To further define the region of interaction, fragments of receptor ectodomain (amino acids 19389, 19243 and 246389) expressed in Escherichia coli (16) were incubated with fibronectin (Fig. 5A
). The existence of an ubiquitin moiety in all recombinant receptor fragments (see Materials and Methods) allowed the use of an antiubiquitin monoclonal antibody to detect receptor binding to fibronectin. Although the interaction involved mainly the segment of the receptor lying between amino acids 246 and 389 there was a weak binding of the 19243 fragment. Thus, definite conclusions on the site of interaction are difficult because the fragments we used may not represent conformations corresponding to the wild-type receptor. As a control, we also included the intracellular domain of TSHR (amino acids 604764), which exhibited a negligible binding to fibronectin.
We took advantage of these observations to analyze the affinity of TSHR for fibronectin (Fig. 5B
) or ECM (not shown). Increasing amounts of full-length immunopurified cellular receptor were incubated with fibronectin in the presence or absence of an excess of receptor ectodomain fragment (amino acids 246389). The binding of the full-length receptor was assessed by using an antibody recognizing the intracellular domain of the protein (this antibody does not recognize the competing extracellular fragment). The binding in the presence of excess extracellular fragment (nonsaturable, nonspecific binding) was subtracted from total binding to yield the specific binding (Fig. 5B
). Half-maximal saturation of specific binding was achieved at a concentration of receptor of approximately 16 nM. Thus, the interaction between the TSHR and fibronectin is saturable and may occur even at low physiological concentrations of the receptor confirming the results observed in whole cell studies. Similar results were obtained (not shown) when fibronectin was replaced by ECM deposited by TSHR devoid L cells. Furthermore, we analyzed the binding of a fixed amount of TSHR to varying concentrations of fibronectin. The half-maximal binding was observed at a concentration of 2.17 pmol/well (not shown).
TSHR Interaction with Fibronectin Modifies Its Functional Properties
We initially studied the stimulation of cAMP synthesis by TSH. Preliminary experiments had shown that cell attachment did not change receptor concentration, or receptor affinity for the hormone (Table 1
). However, optimal stimulation of adenylate cyclase by the hormone was observed only in cells attached to fibronectin as compared with cells in suspension (Fig. 6A
). No such differences were observed in adherent vs. nonadherent L and human embryonic kidney (HEK) 293 cells permanently expressing the LHR (data not shown). Finally, this enhanced cAMP synthesis in the presence of hormone could also be evidenced in FRTL-5 thyroid cells.

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Figure 6. Stimulation of Adenylate Cyclase by TSH in Adherent and Nonadherent Cells
A, Cells expressing the TSHR (thyroid FRTL-5 and transfected L and HEK293) attached to fibronectin or in suspension were stimulated by TSH. Note a 2- to 3-fold enhancement in cAMP synthesis in adherent ( ) vs. nonadherent ( ) cells. B, Effect of substratum. L cells expressing TSHR were attached to fibronectin (Fn), laminin (La), collagen I (Col I) or polylysine (p-Lys). They were stimulated by 1 mU/ml of bTSH. Note that the optimal stimulation by hormone is obtained with fibronectin-attached cells.
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Control experiments with forskolin and cholera toxin produced a similar stimulation of adenylate cyclase in adherent and nonadherent cells (not shown). These observations thus suggest that the mechanism of enhanced stimulation of adenylate cyclase resides at the level of the receptor and not of Gs protein or adenylate cyclase. The role of the substratum was also studied. L-TSHR cells were attached either to fibronectin, laminin, collagen I or polylysine. Optimal TSH stimulation was observed in fibronectin-attached cells (Fig. 6B
).
The TSHR is known to be responsible, in contrast to gonadotropin receptors, for a constitutive activation of adenylate cyclase (27, 28). The mechanism of this activation has raised great interest but remains not understood. During our study of adenylate cyclase activation by TSH in attached and nonattached cells, we observed a constitutive activity of the receptor in the former (Fig. 7
). In TSHR-transfected L and HEK293 cells in suspension, the level of cAMP in the absence of hormone was similar to that observed in nontransfected cells. In attached TSHR-expressing cells, this level was markedly increased, whereas in nontransfected cells there was no difference between attached and nonattached cells. A similar effect of attachment was also seen in FRTL-5 cells.

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Figure 7. Effect of TSHR-Fibronectin Interaction on the Constitutive Activity of the TSHR
cAMP was measured in cells that have not been incubated with hormone. Closed symbols, Adherent cells. Open symbols, Nonadherent cells. Note that 1) in cells permanently expressing the TSHR, the level of cAMP is higher in attached cells than in nonattached cells; 2) in nontransfected cells, there is no difference between adherent and nonadherent cells; and 3) in nonattached transfected cells, the basal cAMP is similar to that observed in nontransfected cells.
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Activation of adenylate cyclase is the main effect mediated by the TSHR. However, at high concentrations of hormone PLC is also stimulated (15). We thus analyzed PLC activation by TSH in attached and nonattached cells. In contrast to what has been observed for adenylate cyclase, cell attachment did not change PLC stimulation (Fig. 8
). It should be noted that receptor redistribution from cell-ECM contacts has been observed at the concentrations of hormone necessary to observe PLC activation.
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DISCUSSION
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Very few cases of hormone or growth factor receptors interection with the ECM have been described to date: members of orphan receptor tyrosine kinase family serve as nonintegrin receptors activated by collagen (7, 8). The epidermal growth factor (EGF) receptor is activated by the EGF-like repeats of tenascin-C, an antiadhesive ECM component (9). Members of EGF-TM7 family characterized by a chimeric structure in which tandem of adhesion-like modules are coupled to G protein-coupled moieties (reviewed in Ref. 29) behave as functional cell-cell adhesion proteins (10, 11).
The leucine-rich repeats of the extracellular domain of the TSHR have been considered to be responsible for the protein-protein interactions that occur during the binding of the ligand (14, 30, 31). However, many proteins containing the same motifs have been shown to be involved in cell-ECM adhesion [platelet GPIba, platelet GPIX, proteoglycan core, and fibromodulin (32)], whereas drosophila toll and chaoptin proteins have been shown to orient specific cells to adjacent structures (33). It is thus not entirely unexpected that the leucine-rich repeats of the TSHR interact with the ECM. However, it is not clear why this interaction is observed for the TSHR and not for the LHR, which contains similar leucine-rich repeats.
Previous work from our laboratory (16, 17) and from others (34, 35) has shown that the TSHR undergoes a posttranslational cleavage event yielding two subunits: an extracellular
-subunit and a membrane spanning ß-subunit linked together by disulfide bridges. The cleavage of TSHR occurs late in the synthesis process at the cell surface (17). It involves an unknown member of the matrix metalloprotease family (36), enzymes known to be concentrated in cell-ECM contacts and at the leading edges of cells (37). Recent studies from our laboratory (38) suggest that the TSHR cleavage enzyme is related to TACE (TNF
-converting enzyme) but is a different member of the adamalysin family of metzincin metalloproteases. A similar cleavage is not observed for LHR, suggesting a link between matrix metalloprotease activity and receptor-ECM interaction. It has been shown that heparin binding epidermal-like growth factor (HB-EGF) is cleaved by a metalloprotease and sheds a soluble HB-EGF only after cell adhesion and spreading (39). However, when L-TSHR cells were cultured in suspension or after adhesion for 4 h, there was no difference in the extent of TSHR cleavage (data not shown). Unfortunately, this experiment was not conclusive due to the slow turnover of the receptor on the cell surface (17). Cells maintained in suspension for longer periods of time were altered and eventually died.
The TSHR ectodomain binds to fibronectin but not to laminin, collagen or heparin sulfate proteoglycans. It is presently not known whether other components of the ECM also play a role in TSHR-ECM interaction.
K562 cells do not bind to the fibronectin substrate (40). We thus transfected permanently these cells with expression vectors encoding the TSHR and LHR and studied their binding to fibronectin. However, the variability in the results of these experiments, especially when using various cell batches of different origin did not allow us to reach clear conclusions.
Hormone-induced cAMP production is approximately 3-fold higher in attached TSHR-expressing cells than in nonattached cells. Receptor clustering may be necessary for their optimal activation. Indeed, platelet-derived growth factor, insulin, EGF, and vascular endothelial growth factor receptors have been shown to be optimally activated by their respective ligands only under appropriate cell attachment conditions (5). It has been observed in Dictyocelium discoidum that G protein signaling events were activated at the leading edge of chemotactic cells (41). PRL induction of transcription of milk protein genes is only observed in attached mammary cells (42, 43).
The wild-type TSHR displays readily measurable constitutive activity upon transfection in various adherent cell types (27, 28, 44). To explain this ligand-independent activity, it has been hypothesized that two forms of the unliganded receptor coexist in equilibrium: a closed or inactive conformation stabilized by putative interactions between the extracellular and the serpentine domains, and an open or active conformation where these interactions are released (45, 46). The concentration of the active form of TSHR would be responsible for its constitutive activity. TSHR ectodomain interaction with fibronectin may contribute to the stabilization of the serpentine portion of the receptor in the active (open) conformation. Indeed, we detected a constitutive activity of the TSHR only in adherent cells. In contrast, the inactive (closed) conformation may predominate in cells maintained in suspension. It should be noted that the LHR, which is randomly distributed on the membrane, does not display constitutive activation. It should also be emphasized that aggregation of growth factor receptors has been shown to result in their partial activation in the absence of ligand (5, 47). This activation is sufficient to prevent apoptosis, which occurs when the cells are detached from the ECM (47).
Attachment to the ECM or fibronectin enhances the TSH effect on adenylate cyclase but not on PLC. This may be related to the fact that high concentrations of TSH are necessary to activate the latter and at these concentrations redistribution of receptor is observed. It is also possible that the G protein-mediated coupling mechanisms differ in each case. It has been shown that mammary cells respond differentially to signaling ligands (insulin, EGF, interferon
) according to cell attachment conditions. For instance, activation of insulin receptor substrate and PI3K was restricted to cells contacting basement membrane, whereas phosphorylation of Erk occured equally in cells contacting basement membrane or collagen (48).
Human thyroid carcinoma tissues are known to be relatively insensitive to TSH (49). Furthermore, two human thyroid carcinoma cells lines have been shown to be unresponsive to TSH. However, these cell lines contain normal TSHR and Gs protein and their nonresponsiveness is related to an abnormality of TSHR-G protein coupling (50). We have observed that basolateral localization of TSHR is altered in biopsies of human thyroid carcinomas (Milgrom, E., unpublished data).
The observations reported here highlight a new aspect of TSHR signaling. Because the TSHR is concentrated at the close contacts between cells and ECM it should now be possible to determine: 1) which cytoplasmic and/or membrane proteins interact with the clustered TSHR and whether these interactions are modified by hormone binding and 2) whether the TSHR-ECM interaction triggers signal transduction through mechanism(s) differing from the classical receptor/G protein/effector cascade. It is interesting to note that the TSHR cytoplasmic domain contains PTB, SH2 and PDZ motifs. These motifs have been shown in other systems to function as scaffolds for the assembly of signaling complexes (51).
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MATERIALS AND METHODS
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Cells
Mouse L and HEK293 cell lines expressing either the TSHR or the LHR were prepared and grown as described (17, 22, 52). Primary human thyrocytes and FRTL-5 (ATCC, Manassas, VA) were cultured as previously described (19, 53).
Isolation and Purification of TSHR
Native TSHR was solubilized from membranes of L cells expressing the TSHR (17) and immunopurified as described (16).
Fragments of TSHR cDNA encoding amino acids 19389 (the putative signal peptide extends to amino acid 21), 19243, 246359, and 604764 were cloned into the polylinker of the pNMHUB (54) vector at the 3'-end of the gene encoding human ubiquitin. The constructions were tailed at the 3'-end with a sequence encoding 6 His. Expression in E. coli and purification by nickel-agarose chromatography were performed as described (16).
Immunofluorescence
Cells (at
60% confluence) were fixed in 3% paraformaldehyde, permeabilized in 0.1% Triton X-100, stained for indirect immunofluorescence, and examined by confocal microscopy. The following primary antibodies were used: rabbit antibodies against the TSHR ectodomain [TSHR 19389 (18)] and against actin (Sigma, St. Louis, MO), mouse monoclonal antibodies against the human TSHR ectodomain [TSHR51 (16)], the pig LHR [LHR38 (55)],
-actinin, ß-actin, and talin (Sigma). All these primary antibodies were visualized using Alexa 546 goat antirabbit IgG and Alexa 488 goat antimouse IgG (Molecular Probes, Inc., Eugene, OR).
Binding of TSHR to ECM
L-TSHR cells were treated with 5% ß-mercaptoethanol for 10 min at room temperature to separate the
-subunit (ectodomain) from the membrane spanning ß-subunit (16). The free thiol groups were blocked with 40 mM iodoacetamide (10 min at room temperature). The ECM was exposed by removing cells with 0.5% Triton X-100/20 mM NH4OH (23) in the presence of a cocktail of protease inhibitors (1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 mM leupeptin, 100 µg/ml bacitracin, and 70 µg/ml aprotinin) in PBS. The exposed ECM was probed for the presence of TSHR ectodomain with TSHR317 monoclonal antibody (5 µg/ml) (16). The latter was detected by peroxidase-conjugated sheep antimouse IgG (1:1,000 dilution) (Amersham Pharmacia Biotech Europe GmbH, Saclay, France). 2,2-Azino-bis-[3-ethylbenzthiazoline-6-sulfonate] (Amersham Pharmacia Biotech) was the substrate. Control experiments involved: 1) L-TSHR cells not treated with ß-mercaptoethanol; 2) replacement of TSHR antibody, which recognizes the ectodomain by TSHR365 monoclonal antibody, which recognizes the intracellular domain (16); and 3) replacement of L-TSHR by L-LHR cells.
Interaction of the TSHR with Fibronectin or with ECM-Coated Wells
TSHR was incubated with either fibronectin (20 µg/ml)-coated wells or with ECM deposited by nontransfected L cells. The latter were cultured in 24-well plates and treated by Triton X-100/NH4OH (23) to detach the cells. Coated wells were then incubated either with the entire recombinant receptor ectodomain (amino acids 19389) expressed in Escherichia coli or with fragments of this ectodomain (amino acids 246389 or 19243) or with the immunoaffinity purified full-length cellular receptor (17). Bacterially expressed intracellular domain of the receptor (amino acids 604674) was used as control. In all cases, the concentration of protein was 1 pmol/well. After washing, wells were incubated for 2 h at room temperature with the primary antibodies. The rabbit polyclonal anti-TSHR ectodomain antibody (1:10,000 dilution) was used in wells incubated with the cellular receptor or with the entire ectodomain expressed in E. coli (amino acids 19389). Antiubiquitin antibodies were used (1:5,000 dilution) in wells incubated with full-length ectodomain or fragments of the receptor expressed in E. coli. Bound immunoglobulins were detected with peroxidase-conjugated donkey antirabbit IgG and sheep antimouse IgG, respectively (Amersham Pharmacia Biotech, 1:1,000 dilution).
The affinity of purified cellular TSHR for fibronectin was determined by incubating fibronectin (20 µg/ml)-coated wells (96-well plates) with increasing concentrations of the immunoaffinity purified full-length cellular receptor in the presence or absence of a 150-fold excess of the 246389 receptor fragment, which has been expressed in E. coli. The latter incubations allowed the measurement of the nonsaturable nonspecific binding. We performed similar experiments by coating wells with 50 µl of various concentrations (from 01 mg/ml) of fibronectin. After saturation with BSA (10 mg/ml) the immunoaffinity purified cellular TSHR was added at a concentration of 10 pmol/well. The cellular receptor bound to fibronectin-coated wells was detected with the TSHR365 antibody (16), which recognizes the cytoplasmic domain of the receptor (and thus binds to the full-length cellular receptor but not to the competing 246389 fragment). The peroxidase-coupled sheep antimouse IgG (Amersham Pharmacia Biotech, 1: 1,000 dilution) was used as secondary antibody. 2,2-Azino-bis-[3-ethylbenzthiazoline-6-sulfonate] was used as the peroxidase substrate.
Hormone Stimulation of Adenylate Cyclase
Cells attached to fibronectin were compared with nonadherent cells (cultured on BSA-coated wells). Similar numbers of adherent and nonadherent cells were incubated for 60 min at 37 C with various concentrations of bovine TSH (bTSH) (0.0110 mU/ml) in 500 µl of Krebs-sucrose buffer containing 0.5 mM isobutylmethylxanthine. Cells incubated with the Krebs-sucrose buffer containing 0.5 mM isobutylmethylxanthine in absence of bTSH were used to determine the constitutive activity of the TSHR. Nontransfected cells were used as control. cAMP levels (extracellular + intracellular) were determined as previously described (15) and expressed as pmol x10-6 cells. Cells from three separate identical wells were trypsinized and counted in a hemocytometer to assess the number of adherent cells. The same number of nonadherent cells was used for TSH stimulation. L cells and HEK293 cells expressing the LHR were used as additional controls.
To study the effect of various substrata, L-TSHR cells and nontransfected L cells were attached for 4 h at 37 C in 12-well culture plates either to fibronectin (20 µg/ml) or to collagen I (20 µg/ml), laminin (20 µg/ml) and polylysine (100 µg/ml). The cells were then treated as above except that a single hormone concentration (1 mU/ml) was used.
Hormone Stimulation of PLC
Myo-[3H]-inositol (specific activity 115 Ci/mmol; Amersham Pharmacia Biotech) was added (5 µCi/ml) to the culture medium 24 h before the experiment. At the start of the experiment, the cells were rinsed three times with Krebs-sucrose buffer and then incubated with hormone as described above, except that the medium contained 20 mM LiCl. After 20 min of incubation with various concentrations of bTSH (from 1 to 100 mU/ml), formic acid was added (3 mM final concentration). After neutralization with ammonium hydroxide, the [3H]-labeled inositol phosphates were isolated by stepwise chromatography on an AG1-X8 resin column (15).
Binding of [125I]-TSH to Adherent Cells and to Cells in Suspension
Cells cultured as described were incubated for 60 min at 4 C with 2.8 x 104 cpm of [125I]-bTSH (ERIA Diagnostics, specific activity 70 µCi/µg) in PBS containing 1% BSA in presence of increasing concentrations (from 10-11 to 10-6 M) of unlabeled bTSH (Calbiochem). At the end of the incubation, the cells were rinsed three times with ice-cold PBS containing 1% BSA and were solubilized in 0.5 ml 1 N NaOH and radioactivity was measured in a
-counter. Nonspecific binding was measured in the presence of 10-6 M of unlabeled bTSH and was subtracted from the total binding. The KD and Bmax were calculated by Scatchard analysis.
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ACKNOWLEDGMENTS
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We thank Mrs. M. T. Groyer-Picard for excellent technical assistance. We gratefully acknowledge the gift of the following material: L-LHR cells (M. T. Vu Hai-Luu Thi), HEK293-LHR cells (N. de Roux), HEK293-TSHR cells (M. Atger and M. Misrahi), L-TSHR cells and purified cellular TSHR (M. Misrahi and S. Sar), various purified TSHR fragments expresed in E. coli (H. Loosfelt), anti-LHR monoclonal antibodies (M. T. Vu Hai-Luu Thi and K. Echasserieau), anti-TSHR monoclonal and polyclonal antibodies (H. Loosfelt, A. Jolivet, and C. Pichon), and antihuman ubiquitin monoclonal antibody (M. Rauch). We thank P. Leclerc for help with confocal microscopy and A. D. Dakhlia for secretarial assistance. We are grateful to D. Louvard and J. P. Thiery for very helpful discussions.
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FOOTNOTES
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This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Faculté de Médecine Paris-Sud, and the Association pour la Recherche sur le Cancer.
Abbreviations: Bmax, Number of cell surface-associated TSHR molecules/cell; bTSH, bovine TSH; ECM, extracellular matrix; EGF, epidermal growth factor; HB, heparin binding; HEK, human embryonic kidney; KD, dissociation constant; LHR, LH receptor; TACE, TNF
-converting enzyme; TSHR, TSH receptor.
Received for publication November 20, 2001.
Accepted for publication January 3, 2002.
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REFERENCES
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