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Endocrinology, doi:10.1210/en.2005-0687
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Endocrinology Vol. 147, No. 6 3114-3122
Copyright © 2006 by The Endocrine Society

The Human Thyrotropin Receptor Is Predominantly Internalized by ß-Arrestin 2

Romy Frenzel1, Carsten Voigt1 and Ralf Paschke

III Medical Department, University of Leipzig, 04103 Leipzig, Germany

Address all correspondence and requests for reprints to: R. Paschke, M.D., III Medical Department, University of Leipzig, Philipp-Rosenthal- Strasse 27, D-04103 Leipzig, Germany. E-mail: pasr{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ß-arrestin-dependent endocytosis of the ß2-adrenergic receptor (ß2AR) has been demonstrated by confocal fluorescence microscopy. Furthermore, a constitutively activated ß2AR is also constitutively desensitized and down-regulated. To clarify the function of ß-arrestin 1 or 2 for TSH receptor (TSHR) desensitization and examine whether constitutively activated TSHR mutants are internalized in a different way, we investigated the TSHR trafficking in association with ß-arrestins in cotransfection experiments in HEK 293 cells using confocal laser-scanning microscopy. We found that both ß-arrestins are able to internalize the TSHR in HEK 293 cells. However, whereas the ß-arrestin 1-mediated TSHR internalization reached its maximum 20 min after TSH stimulation, the ß-arrestin 2-mediated TSHR internalization already reached its maximum 5 min after TSH stimulation. Furthermore, an increased basal desensitization and internalization of constitutively activated TSHR mutants N670S, S505N, and F631L cotransfected with ß-arrestin 2 could not be found. After TSH stimulation the constitutively activated mutants showed the same time course for internalization as the wild-type-TSHR. In summary, contrary to data obtained for the ß2AR, the constitutive activation of the TSHR does not influence the desensitization and time course for internalization of the receptor, and in agreement with findings for the FSH and LH receptors, these results characterize the TSH receptor as a member of the class A of G protein-coupled receptors, which have a higher affinity to ß-arrestin 2 than ß-arrestin 1 and do not colocalize with ß-arrestins in endosomes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BINDING OF ß-ARRESTINS to receptors phosphorylated by G protein-coupled receptor kinases quenches the activation of G proteins and targets the receptors to clathrin-coated pits for internalization (reviewed in Refs.1 and 2). ß-Arrestins therefore play an important role in agonist-dependent desensitization of G protein-coupled receptors (GPCRs). ß-Arrestin 1 mRNA and protein expression and its function as a negative regulator of TSH receptor (TSHR) stimulated cAMP has been reported for FRTL 5 cells (3, 4). We previously demonstrated that both ß-arrestin 1 and 2 are expressed in the human thyroid and that ß-arrestin 1 and 2 are capable of desensitizing the TSHR after stimulation (5). Moreover, we found an increased ß-arrestin 2 expression in toxic thyroid nodules (5).

ß-Arrestins possess different affinities for different receptors (6). Moreover, two classes of GPCRs based on their differential binding affinities for ß-arrestin 1 and 2 could be identified (7). The class A receptors, which interact with ß-arrestin 2 with a higher affinity than with ß-arrestin 1, include the ß2-adrenergic, µ-opioid, endothelin type A, dopamine type 1, and ß1b-adrenergic receptors. The class B receptors, which do not discriminate between ß-arrestin 1 and 2 and show a similar affinity for both proteins, include the angiotensin type 1, vasopressin type 2, neurotensin, TRH, and neurokinin type 1 receptors (7). For the lutropin/choriogonadotropin (LH/CG) or follitropin (FSH) receptors, which are very homologous to the TSHR, the following interactions with ß-arrestins are described: the FSHR undergoes a ß-arrestin 1 and 2-dependent desensitization (8) and shows an enhanced internalization via ß-arrestin 2 (9). The LH receptor shows a ß-arrestin 1 and 2-dependent down-regulation (10) with an enhanced internalization via ß-arrestin 2 (11). Investigations with stably transfected L cells expressing the TSHR showed little endocytosis (30% of receptor molecules) and 90% recycling of the receptor molecules to the cell membrane after TSH stimulation (12). Agonist-dependent translocation of ß-arrestins to various GPCRs (e.g. ß2-adrenergic, m-acetylcholine, angiotensin, dopaminergic, neurotensin, and vasopressin receptors) and the ß-arrestin-dependent endocytosis of these receptors has been demonstrated using confocal fluorescence microscopy (7). Furthermore, the TSHR internalization is mediated by clathrin-coated vesicles (12). However, the role of ß-arrestins in TSHR internalization has not been investigated. Moreover, a ß2-adrenergic receptor constitutively activated by mutations in the third intracellular loop is also constitutively desensitized and down-regulated (1, 13).

We therefore asked the following questions: are ß-arrestins involved in the TSHR internalization? Is there a difference between the ß-arrestin 1- and ß-arrestin 2-induced TSHR internalization? Is the TSHR colocalized with ß-arrestins in endosomes after internalization? To which class of GPCR-ß-arrestin interaction does the TSHR belong? Is there a constitutive desensitization and internalization of constitutively activated TSHR mutants?


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of the green fluorescent protein (GFP)-ß-arrestin 2 fusion protein
The GFP-ß-arrestin 2 fusion protein was generated as follows. The ApaI/HindIII insert released from pcDNA3 containing the coding region for wild-type (wt) ß-arrestin 2 (kindly provided by Dr. J. Benovic, Jefferson Medical College, Philadelphia, PA) was subcloned into the ApaI/HindIII site at the C terminus of the GFP within the pEGFP-C2 vector (CLONTECH, Palo Alto, CA). The resulting open reading frame represents the coding sequence of GFP-ß-arrestin 2. The cDNA clones were sequenced to verify the correct insertion of the ß-arrestin 2 sequence in the pEGFP-C2 vector.

Cell culture and transfection
HEK 293 cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco BRL, Karlsruhe, Germany) at 37 C in a humidified 5% CO2 incubator. HEK MSR 293 GripTite cells were grown in DMEM supplemented with 10% fetal bovine serum, 1% nonessential amino acids and 100 µg/ml geneticin (Gibco) at 37 C in a humidified 5% CO2 incubator. The cells were transfected using the FuGENE 6 (Roche, Mannheim, Germany) transfection reagent according to the manufacturer’s instructions. In brief, for fluorescence-activated cell sorter (FACS) analysis and ELISA 1 x 105 HEK 293 cells per well were seeded into 12-well plates, and for confocal laser-scanning microscopy (CLSM) 2.5 x 105 HEK 293 cells per well were seeded on coverslips into six-well plates. HEK MSR 293 GripTite cells were seeded into 48-well plates (0.25 x 105/well) for cAMP accumulation assay. The cells were incubated 24 h before cotransfection with plasmid constructs (1 µg DNA/1 x 105 HEK 293 cells) containing the coding sequence of the TSHR and ß-arrestin 1, ß-arrestin 2, dominant-negative ß-arrestin 1 mutant (V53D), dominant-negative ß-arrestin 2 mutant (V54D), or TSHR and GFP-ß-arrestin 1, GFP-ß-arrestin 2, the empty pcDNA-vector. In a second experiment, cells were cotransfected with plasmid constructs (1 µg DNA/1 x 105 HEK 293 cells) containing the coding sequence of ß-arrestin 1, ß-arrestin 2, the dominant-negative ß-arrestin 1 mutant (V53D), the dominant-negative ß-arrestin 2 mutant (V54D), the empty pcDNA-vector, and the TSHR or the constitutive active mutants S505N, F631L, N670S. FACS analysis, cAMP accumulation assay, ELISA, and CLSM were performed 48 h after transfection and repeated three times. The chosen transfection relation of 5:1 for TSHR and ß-arrestin constructs was determined by preceding experiments using different transfection relations.

Receptor internalization
Internalization of 125I-bovine (b) TSH was measured by a modification of the acid-wash method of Hazum et al. (14). Forty-eight hours after transfection, HEK MSR 293 GripTite cells were washed once with ice-cold Hanks’ solution [5.36 mM KCl, 0.44 mM KH2PO4, 0.405 mM MgSO4 x 7H2O, 0.334 mM Na2HPO4 x 12H2O, 5.55 mM glucose, 1.3 mM CaCl2, 280 mM sucrose, 0.2% BSA, 2.5% low-fat milk (pH 7.4)] and subsequently incubated at 0 C (on ice) in Hanks’ solution with 150,000 cpm/ml 125I-bTSH for 4 h. Cells were then warmed to 37 C for 40 min, and radioligand internalization was then stopped by placing the cells at 0 C and rapidly washing twice with ice-cold Hanks’ solution. Acid-sensitive bound radioligand, representing cell surface bound label, was removed by the addition of 0.5 ml ice-cold acid solution [150 mM NaCl, 50 mM acetic acid (pH 2.8)] for 10 min. After removal of the acid wash, cells were solubilized with 0.5 ml of 1 N NaOH to determine acid-resistant (internalized) radioligand. Nonspecific binding, consistently found to be less than 10% of total binding, was determined using HEK MSR 293 GripTite cells cotransfected with the empty pSVL-vector and the empty pcDNA-vector. Radioligand internalization was expressed as percent of total cell-associated label (cell surface bound plus acid resistant).

CLSM
Forty-eight hours after transfection, the cells were stimulated with bTSH (100 mU/ml; Sigma Chemical Co., St. Louis, MO) for different time periods as indicated in the figures. Coverslips were rinsed two times with ice-cold PBS and fixed with 2% paraformaldehyde containing 0.1% Triton X-100 (for permeabilization) for 30 min at 4 C. After two 5-min wash steps with cold PBS, the cells were incubated with the primary antibody for 1 h at 4 C. The TSHR was detected using the antihuman TSHR antibody (2C11; Serotec Ltd., Oxford, UK; 1:500 in PBS). Endosomes were detected using the rabbit anti-EEA1 antibody (Affinity BioReagents, Golden, CO). The cells were washed two times for 5 min with cold PBS, and the primary antibody was detected by incubation with an Alexa-Fluor 488 or Alexa-Fluor 546-conjugated goat antimouse secondary antibody (Molecular Probes, Eugene, OR; 1:1000 in PBS) or a Cy5-conjugated goat antirabbit secondary antibody (Dianova, Hamburg, Germany; 1:1000 in PBS) for 1 h at 4 C. After two final 5-min wash steps, the coverslips were mounted onto glass slides. The GFP-ß-arrestin constructs were visualized by the intrinsic fluorescence of the GFP.

Confocal analysis was performed on a confocal laser scanning system (TCS SP2; Leica, Wetzlar, Germany) attached to a microscope (DM IRBE; Leica) with x100 oil immersion lens (PL Fluotar 1.3; Leica). Optical sections (0.45 µm) were taken, and representative sections corresponding to the middle section of the cells were presented. We investigated nearly 200 cells per coverslip, and at least 80% of these cells showed the same level of internalization. After indirect immunofluorescence staining, no specific fluorescence was observed in untransfected HEK 293 cells treated with anti-TSHR antibody and Alexa-Fluor 488-conjugated antibody or in transfected HEK 293 cells treated only with secondary Alexa-Fluor 488-conjugated antibody.

SDS-PAGE followed by Western blotting
Aliquots of cell lysate were mixed with an equal volume of SDS-PAGE sample buffer [4% sodium dodecyl sulfate, 20% glycerol, 100 mmol/liter Tris/HCl (pH 6.8), and 0.002% bromphenolblau], heated to 95 C for 5 min, electrophoresed on a 10% acrylamide gel (SDS-PAGE; Bio-Rad Laboratories Inc., Hercules, CA) at 140 V for 110 min, and blotted onto nitrocellulose (Schleicher & Schuell, Inc., UK Ltd., London, UK) at 12 V for 30 min. The membranes were blocked using 5% low-fat milk in Tris-buffered saline and 0.2% Tween 20, incubated overnight at 4 C with a 1:1000 dilution of horseradish peroxidase (HRP) conjugate anti-GFP (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), and developed by SuperSignal West Pico chemiluminescence reagents (Pierce, Rockford, IL).

Measurement of cAMP
For cAMP assays, HEK MSR 293 GripTite cells were washed in serum-free DMEM, followed by preincubation with the same medium containing 1 mM 3-isobutyl-1-methyl-xanthine (Sigma) for 20 min at 37 C in a humidified 5% CO2 incubator. Subsequently cells were stimulated with bTSH (10 mU/ml; Sigma) for 1 h. Reactions were terminated by aspiration of the medium and addition of 0.2 ml 0.1 N HCl. Supernatants were collected and dried. The cAMP content of the cell extracts was determined with a commercial kit (Amersham Pharmacia Biotech, Braunschweig, Germany) according to the manufacturer’s instructions.

ELISA analysis
Forty-eight hours after transfection, the cells were stimulated for different time periods as indicated in the figures with bTSH (100 mU/ml; Sigma Chemical Co., St. Louis). Cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After three washes with PBS, cells were blocked with DMEM containing 10% fetal calf serum (FCS) for 1 h at room temperature. For the determination of the TSHR cell surface expression, cells were incubated with antihuman TSHR antibody (2C11; Serotec; 1 µg/ml in DMEM containing 10% FCS) for 1 h at 37 C. After three washes with PBS, cells were incubated with the HRP conjugated rabbit antimouse IgG secondary antibody (Cell Signaling, Oxford, UK; 1:2000 dilution in DMEM containing 10% FCS) for 1 h at 37 C. Development was done with 400 µl/well of the o-phenylenediamine as a substrate for HRP, stopped with 200 µl/well 1 M HCL + Na2SO3, and measured at 492/620 nm. The cell surface expression of the TSHR determined by ELISA was confirmed by FACS analysis.

FACS analysis
Forty-eight hours after transfection the cells were stimulated for 1 h with bTSH (100 mU/ml; Sigma). For the determination of the TSHR cell surface expression, cells were incubated with antihuman TSHR antibody (2C11, Serotec; 10 µg/ml in PBS containing 0.1% BSA). After two washes with PBS (0.1% BSA), cells were incubated with the fluorescein-5-isothiocyanate-conjugated F(ab')2 rabbit antimouse IgG secondary antibody (Star9B, Serotec; 1:100 dilution in PBS containing 0.1% BSA) for 1 h on ice in the dark. The fluorescence of 10,000 cells per tube was assayed by a FACScan cytofluorometer (Becton Dickinson, San Jose, CA).

Statistical methods
For comparison involving three or more groups, data were assessed for normality by Dunnett’s test one-way ANOVA. Comparison of between-group mean values were performed using an unpaired Student’s t test for two-group comparison. Statistical significance was defined as P < 0.05. All values are presented as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TSHR internalization via ß-arrestins
In the experimental protocol used here, radioligand binding to intact cells was allowed to reach equilibrium at 0 C, under which conditions receptor internalization does not occur. After warming of cells to 37 C for 40 min, radioligand receptor-mediated internalization was measured and expressed as percent of total cell-associated ligand (Fig. 1Go).


Figure 1
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  		FIG. 1. Internalization of 125I-bTSH-labeled TSHR. HEK MSR 293 GripTite cells were cotransfected with the TSHR and the pcDNA expression vector encoding various ß-arrestin constructs or the empty pcDNA vector. Internalized radioligand 125I-bTSH is expressed as a percent of total cell-associated radioligand as described in experimental procedures. Data shown are representative of three independent experiments (*, P < 0.05). bArr2, ß-Arrestin 2; bArr1, ß-arrestin 1; V53D, dominant-negative ß-arrestin 1 mutant V53D; V54D, dominant-negative ß-arrestin 2 mutant V54D.

 
By radioligand binding analysis, HEK MSR 293 GripTite cells cotransfected with the TSHR and the empty pcDNA-vector showed 37.6% ± 1.5 TSHR internalization (Fig. 1Go). In HEK MSR 293 GripTite cells cotransfected with the TSHR and ß-arrestin 1 or 2, the TSHR internalization was significantly increased for ß-arrestin 2 [63.9% ± 0.5 (P < 0.05)] and markedly increased for ß-arrestin 1 (45.3 ± 2.3%). The internalization of TSHR after cotransfection of the dominant-negative ß-arrestin 1 mutant V53D (30.3 ± 0.9%) or the dominant-negative ß-arrestin 2 mutant V54D (33 ± 1.7%) was comparable with TSHR cotransfected with empty pcDNA-vector. All cotransfection experiments were reproduced three times. The TSHR transfection efficiency was verified by means of FACS (data not shown). In unstimulated cells with equivalent amount of ß-arrestin 1 and 2 cotransfected with the TSHR, the transfection efficiency and cell surface expression of TSHR was identical.

In confocal laser scanning experiments, HEK 293 cells, cotransfected with the TSHR and empty pcDNA-vector, showed no visually TSHR internalization over a time course of 40 min after TSH stimulation (Fig. 2Go). Cells, cotransfected with the TSHR and ß-arrestin 1, reached the maximum of TSHR internalization 20 min after TSH stimulation (Fig. 2Go). However, HEK 293 cells cotransfected with the TSHR and ß-arrestin 2 already reached the maximum of TSHR internalization 5 min after stimulation (Fig. 2Go). After coexpression of the dominant-negative ß-arrestin 1 mutant V53D or dominant-negative ß-arrestin 2 mutant V54D, there was no detectable TSHR internalization over a time course of 40 min after TSH stimulation (Fig. 2Go).


Figure 2
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  		FIG. 2. Distribution of the TSHR after TSH stimulation. A, HEK 293 cells were transiently cotransfected with the TSHR and the pcDNA expression vector encoding various ß-arrestin constructs or the empty pcDNA vector and stimulated for 0, 2, 5, 10, 20, or 40 min with 100 mU/ml bTSH. B, Nonpermeabilized vs. permeabilized HEK 293 cells transiently cotransfected with the TSHR and ß-arrestin 2, stimulated for 0 and 10 min with 100 mU/ml bTSH. The distribution of the receptor was assessed by CLSM as described in experimental procedures using a TSHR antibody and an Alexa Fluor 488-conjugated secondary antibody. The white arrows in the image indicate the internalized TSHR. Data shown are representative of three independent experiments. V53D, Dominant-negative ß-arrestin 1 mutant V53D; V54D, dominant-negative ß-arrestin 2 mutant V54D.

 
To clarify that this different influence of ß-arrestin 1 and ß-arrestin 2 is not a result of different ß-arrestin protein levels, we investigated the protein expression of GFP-ß-arrestin 1 and GFP-ß-arrestin 2 after cotransfection with TSHR by Western blot (Fig. 3Go). No differences in the intensity of the 82-kDa GFP-ß-arrestin 1 band and the 72-kDa GFP-ß-arrestin 2 band could be found. These data suggest that the differences we observed with ß-arrestin 2 over ß-arrestin 1 were not dose dependent.


Figure 3
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  		FIG. 3. Protein expression levels of GFP-ß-arrestin 1 and ß-arrestin 2. HEK 293 cells were cotransfected with the TSHR and the pcDNA expression vector encoding nothing (lane 1), GFP-ß-arrestin 1 (lane 2), or GFP-ß-arrestin 2 (lane 3). The protein expression levels were assessed by Western blotting as described in experimental procedures using a HRP-conjugated GFP antibody. ßA1-GFP, GFP-ß-arrestin 1; ßA2-GFP, GFP-ß-arrestin 2.

 
TSHR and ß-arrestin distribution in HEK 293 cells
In addition to the time-course experiments, further cotransfection experiments with the TSHR and GFP-ß-arrestin constructs were performed to identify a possible colocalization of the TSHR and GFP-ß-arrestins. After stimulating the cells with TSH, a colocalization of the TSHR and ß-arrestin 1 or ß-arrestin 2 was found only in small areas at the cell membrane but not inside the cell (Fig. 4AGo). To clarify that there is no colocalization of TSHR and ß-arrestins in endosomes, we also stained early endosomes. Besides the colocalization of ß-arrestin 2 and the TSHR near the plasma membrane and the colocalization of early endosomes and the TSHR in the cytoplasma, we could not detect colocalization of ß-arrestin 2 with the stained early endosomes (Fig. 4BGo). There is no colocalization of ß-arrestin linked TSHR in early endosomes (Fig. 4BGo). To quantify this visual result, we added graphs of sections through the cells to show the distribution of red and green fluorescence (Fig. 4BGo). In unstimulated cells, TSHR immunofluorescence (red) was confined to the plasma membrane, whereas the ß-arrestin 2-GFP fluorescence (green) was distributed in the cytoplasm (Fig. 4BGo, 0 min). This is illustrated in the overlay and quantified in the line profile by the peaks of TSHR-Alexa546, in which the line crosses the plasma membrane and the even distribution of ß-arrestin 2-GFP fluorescence throughout the cytosol (Fig. 4BGo, 0 min). In response to agonist activation of the TSHR, ß-arrestin 2-GFP was translocated to the membrane in which it colocalizes with TSHR as visualized by the appearance of yellow spots (Fig. 4BGo, 5 min). The colocalization is also demonstrated in the line profile by the same peak position for the intensity of TSHR-Alexa546 and ß-arrestin 2-GFP fluorescence (Fig. 4BGo, 5 min). Yellow spots and colocalization of fluorescence intensity in the line profile could be observed only at or near the cell membrane. No colocalization of ß-arrestin 2-GFP with TSHR was observed in the TSHR-containing intracellular vesicles in the cytoplasm of the cell (Fig. 4BGo, 5 min). This is also illustrated by the line profile, in which the increase in intracellular TSHR-Alexa546 fluorescence representing internalized TSHR is not colocalized with ß-arrestin 2-GFP (Fig. 4BGo, 5 min).


Figure 4
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  		FIG. 4. Distribution of the TSHR and ß-arrestins. A, HEK 293 cells transiently cotransfected with the TSHR and GFP-ß-arrestin 1 or ß-arrestin 2 unstimulated and stimulated for 40 min with 100 mU/ml TSH. B, HEK 293 cells transiently cotransfected with the TSHR and GFP-ß-arrestin 2 unstimulated and stimulated for 5 min with 100 mU/ml TSH. The distribution of the receptor (red) and the GFP-ß-arrestins (green) was assessed by CLSM as described in experimental procedures. The TSHR was detected with an anti-TSHR antibody and an Alexa Fluor 546-conjugated secondary antibody. The GFP-ß-arrestins were detected by the fluorescence of the GFP. The colocalization of the TSHR and GFP-ß-arrestins near the plasma membrane is shown in the overlay images (yellow). Colocalization of the TSHR and GFP-ß-arrestins in early endosomes were detected with the EEA1 antibody for early endosomes and a Cy5-conjugated secondary antibody (cyan). For a better understanding, we changed the color of the endosomes from cyan to red or green to show colocalization with TSHR (red) or ß-arrestin (green) in yellow. No colocalization of GFP-ß-arrestins (green) and early endosomes (red) could be found in the overlay images. The colocalization of the TSHR (red) and early endosomes (green) in the cytoplasm is shown in the overlay images (yellow). The quantitative fluorescence intensity (in arbitrary units; y-axis) vs. distance (in micrometers; x-axis) profiles were obtained by fluorometric scanning along the straight line shown on confocal images using the Leica LSM software. Plasma membrane (pm) boundaries were indicated by arrows. Data shown are representative of two independent experiments. ß-arr1, GFP-ß-arrestin 1; ß-arr2, GFP-ß-arrestin 2.

 
Desensitization and internalization of constitutively activated TSHR mutants
Because a ß2-adrenergic receptor constitutively activated by mutations in the third intracellular loop is also constitutively desensitized and down-regulated, we also studied the influence of ß-arrestins on the desensitization and internalization of three different constitutively activating TSHR mutations (Table 1Go). All mutations have been functionally characterized in COS 7 cells [see previous publications (15, 16, 17)]. Cotransfection of various ß-arrestin constructs indicates no response in basal cAMP values of constitutively activated TSHR mutants (Fig. 5Go, filled bars). Therefore, HEK MSR 293 GripeTite cells, cotransfected with the constitutively activated TSHR mutants and ß-arrestin constructs, showed no basal TSHR desensitization in cAMP assays. After stimulation with TSH for 1 h, the investigated mutants showed a similar desensitization with ß-arrestin 1 and 2 in cAMP assays as the wt-TSHR (Fig. 5Go, open bars).


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  		TABLE 1. Functional characteristics of the investigated constitutively activated TSHR mutants as determined in COS 7 cells in previous publications (15 16 17 )

 

Figure 5
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  		FIG. 5. Desensitization of constitutively activated TSHR mutants. Basal (filled bars) and TSH-stimulated (10 mU/ml, 1 h; open bars) cAMP accumulation in HEK MSR 293 GripTite cells cotransfected with the TSHR or constitutively activated TSHR mutants (S505N, F631L, N670S) and the pcDNA expression vector encoding various ß-arrestin constructs or the empty pcDNA vector. Data are expressed relative to expression level of unstimulated cells (set at 100%). Data are given as means ± SEM of three independent experiments, each carried out in duplicate. bA1, ß-Arrestin 1; bA2, ß-arrestin 2; V53D, dominant-negative ß-arrestin 1 mutant V53D; V54D, dominant-negative ß-arrestin 2 mutant V54D.

 
HEK 293 cells, cotransfected with the constitutively activated TSHR mutants (Table 1Go) and ß-arrestin 2, showed no basal TSHR internalization in confocal laser scanning experiments (Fig. 6AGo). Same results for coexpression of ß-arrestin 1 and the dominant-negative ß-arrestin mutants V53D and V54D were found (data not shown). After coexpression of ß-arrestin 2 and stimulation with TSH over a time course of 10 min, the investigated mutants showed the same internalization time course as the wt-TSHR (data shown for N670S, Fig. 6BGo). After coexpression of ß-arrestin 1 and stimulation with TSH over a time course of 30 min, the investigated mutants showed the same internalization time course as the wt-TSHR (data shown for N670S, Fig. 6BGo). After coexpression of the dominant-negative ß-arrestin 1 mutant V53D or the dominant-negative ß-arrestin 2 mutant V54D and stimulation with TSH over a time course of 30 min, the investigated mutants showed no internalization, like the wt-TSHR (data not shown). The same results were achieved by ELISA (Fig. 7Go) and FACS (data not shown).


Figure 6
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  		FIG. 6. Internalization of constitutively activated TSHR mutants. A, HEK 293 cells transiently cotransfected with ß-arrestin 2 and the wt-TSHR or constitutively activated TSHR mutants (S505N, F631L, N670S). B, HEK 293 cells transiently cotransfected with the wt-TSHR or the constitutively activated TSHR mutant N670S and ß-arrestin 2 or ß-arrestin 1 stimulated for various times with 100 mU/ml bTSH. The distribution of the receptor was assessed by CLSM as described in experimental procedures using an anti-TSHR antibody and an Alexa Fluor 488-conjugated secondary antibody. The white arrows in the image indicate the internalized TSHR. Data shown are representative of three independent experiments.

 

Figure 7
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  		FIG. 7. Cell surface expression of wt-TSHR and constitutively activated TSHR mutants. A, HEK 293 cells transiently cotransfected with ß-arrestin 1 and the wt-TSHR (lane 1) or constitutively activated TSHR mutants N670S (lane 2), S505N (lane 3), or F631L (lane 4) stimulated for 0 min (white), 2 min (gray), 5 min (dark gray), or 20 min (black) with 100 mU bTSH per milliliter. B, HEK 293 cells transiently cotransfected with ß-arrestin 2 and the wt-TSHR (lane 1) or constitutively activated TSHR mutants N670S (lane 2), S505N (lane 3), or F631L (lane 4) stimulated for 0 min (white), 10 min (gray), 20 min (dark gray), or 40 min (black) with 100 mU bTSH per milliliter. C, HEK 293 cells transiently cotransfected with the dominant-negative ß-arrestin 1 mutant V53D and the wt-TSHR (lane 1) or constitutively activated TSHR mutants N670S (lane 2), S505N (lane 3), or 631L (lane 4) stimulated for 0 min (white), 10 min (gray), 20 min (dark gray), or 40 min (black) with 100 mU bTSH per milliliter. D, HEK 293 cells transiently cotransfected with the dominant-negative ß-arrestin 2 mutant V54D and the wt-TSHR (lane 1) or constitutively activated TSHR mutants N670S (lane 2), S505N (lane 3), or F631L (lane 4) stimulated for 0 min (white), 2 min (gray), 5 min (dark gray), or 20 min (black) with 100 mU bTSH per milliliter. Cell surface expression was assessed by ELISA as described in experimental procedures using a mouse antihuman TSHR antibody. All data are presented as mean ± SEM of two independent experiments, each performed in duplicate (Dunnett’s test: 0 min vs. 2/5/10/20/40 min; *, P < 0.05, **, P < 0.01). bA2, ß-Arrestin 2; bA1, ß-arrestin 1; V53D, dominant-negative ß-arrestin 1 mutant V53D; V54D, dominant-negative ß-arrestin 2 mutant V54D.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The desensitizing effect of ß-arrestins on GPCR signaling has been demonstrated in many studies (2, 7, 8, 9, 10, 18, 19, 20, 21, 22). Furthermore, ß-arrestin binding to ß2-AR not only terminates receptor-G protein coupling followed by a decrease of cAMP, but also it initiates a second wave of signal transduction in which the desensitized receptor acts as a structural component of a mitogenic signaling complex (23).

TSH stimulation of FRTL 5 cells increased the level of cytosolic ß-arrestin 1, and, in turn, the elevated levels of ß-arrestin 1 attenuated TSH-induced cAMP accumulation and increased TSHR desensitization (3). In contrast, Nagayama et al. (4) did not find a TSH-dependent mechanism of ß-arrestin 1 regulation in FRTL 5 cells.

A functional importance of ß-arrestins for TSH receptor signaling is also implied by experiments with the lutropin (LH)/CG or the follitropin (FSH) receptor, which display a high homology to the TSHR. The desensitization of LH/CG receptor-stimulated adenylyl cyclase activity in porcine ovarian follicular membranes is mediated by ß-arrestin 1 (19). Cotransfection of HEK 293 cells with the LH/CG receptor and G protein-coupled receptor kinase-2 or ß-arrestin 1 or ß-arrestin 2 enhances internalization and increases down-regulation of the receptor (9, 10). The FSHR expressed in HEK 293 cells (9, 20) or mouse Ltk cells (8) shows enhanced agonist-induced internalization after cotransfection with ß-arrestin 1 or ß-arrestin 2.

Overexpression of ß-arrestin 1 in COS 7 or FRTL 5 cells decreased TSHR-dependent cAMP production (3, 4), probably by an increased desensitization of the TSHR. We could previously demonstrate the expression of both ß-arrestins in human thyroid tissue and found that both ß-arrestins are able to desensitize the TSHR in vitro in HEK 293 cells (5). In this study TSH-stimulated cAMP accumulation in HEK 293 cells, cotransfected with the TSHR and ß-arrestin 1 or 2, was significantly decreased to 37.2 ± 6.4% (P < 0.05) for ß-arrestin 1 and to 33.9 ± 7.5% (P < 0.05) for ß-arrestin 2, compared with HEK 293 cells transfected only with the TSHR (100%). cAMP was unchanged after stimulation in HEK 293 cells cotransfected with the TSHR and a dominant-negative ß-arrestin 1 mutant (V53D), compared with HEK 293 cells transfected only with the TSHR. Furthermore, we identified an increased ß-arrestin 2 expression in hot thyroid nodules, which suggests ß-arrestin 2 as the predominant cAMP-dependent regulator of TSHR activation (5).

Here we demonstrate by radioligand binding analysis and CLSM the capability of ß-arrestin 1 as well as ß-arrestin 2 to internalize the TSHR. Previous investigations with L cells stably transfected with the TSHR alone showed a 30% internalization of the receptor (12). This is in line with our observation in HEK MSR 293 GripTite cells (see Fig. 1Go).

There are two classes of GPCRs based on their differential binding affinities for ß-arrestin 1 and 2 (7). The class A receptors (e.g. FSHR, LH receptor), which internalize through a ß-arrestin 2-dependent mechanism, show a transient ß-arrestin-GPCR interaction, and ß-arrestin does not colocalize with these receptors in endosomes. The class B receptors (e.g. angiotensin type 1 receptor), which have no preference for ß-arrestin 2 over ß-arrestin 1, show a more stable ß-arrestin-GPCR interaction and the receptor and ß-arrestins are colocalized in endosomes (7, 24). Our results demonstrate, that both ß-arrestins are able to internalize the TSHR: the ß-arrestin 1-mediated TSHR internalization reaches its maximum 20 min after TSH stimulation, whereas the ß-arrestin 2-mediated TSHR internalization already reaches its maximum 5 min after TSH stimulation. In addition to this faster action of ß-arrestin 2, compared with ß-arrestin 1, we observed a stronger effect of ß-arrestin 2 for TSHR internalization (see Fig. 1Go). In conclusion, ß-arrestin 2 has a predominant role over ß-arrestin 1 for the TSHR internalization. This conclusion is in line with the previously described predominant role of ß-arrestin 2 in FSHR and LHR internalization (9, 11).

In accordance with Singh et al. (25), we see the TSHR localized to endosomes after internalization. However, we demonstrate a lack of colocalization of the TSHR together with ß-arrestins in endosomes and a colocalization of ß-arrestins and the TSHR only near the plasma membrane. Therefore, the earlier ß-arrestin 2-dependent TSHR internalization, the more pronounced internalization of the TSHR in radioligand-binding analysis of stimulated HEK MSR 293 GripTite cells cotransfected with ß-arrestin 2, compared with cells cotransfected with ß-arrestin 1, and the lack of colocalization of the TSHR with ß-arrestins in endosomes characterize the TSHR as a member of the class A receptors together with FSHR and LH receptor, which are very homologous to the TSHR.

Furthermore, we investigated the internalization of constitutively activated TSHR mutants (Table 1Go) for two reasons: first because our data demonstrate that the TSHR belongs to the class A of GPCRs, like the ß2-adrenergic receptor, and because it was previously shown that a constitutively activated ß2-adrenergic receptor mutant is constitutively desensitized and down-regulated (13); and second because an N-terminally truncated TSHR mutant (deletion of amino acids 22–313) is constitutively activated and shows an increased basal internalization (26).

Our investigation shows that, contrary to the ß2-adrenergic receptor, constitutively activated TSHR mutants are not constitutively desensitized (Fig. 5Go). As shown in Fig. 5Go, there is no difference in the basal cAMP levels after coexpression of the TSHR mutants with various ß-arrestin constructs; therefore, there is no spontaneous desensitization of the constitutively active TSHR mutants. After stimulation with TSH, the constitutively activated TSHR mutants cotransfected with ß-arrestin 1 or 2 in HEK 293 cells show the same time course for internalization in ELISA and CLSM analysis as the wt-TSHR (Figs. 6Go and 7Go).

In conclusion we found that both ß-arrestins are able to internalize the TSHR. However, based on the earlier and more prominent internalization, a predominant role of ß-arrestin 2 for the TSHR internalization was shown. We could thus classify the TSHR as a member of the class A of GPCRs. In addition, we did not find a constitutive desensitization for constitutively active TSHR mutants as has previously been demonstrated for a constitutively active ß2-adrenergic receptor.


   Acknowledgments
 
We thank Dr. J. Benovic (Jefferson Medical College, Philadelphia, PA) for the ß-arrestin 1 and ß-arrestin 2 cDNA and the ß-arrestin 1 (V53D) mutant cDNA; Dr. A. Hanyaloglu (Western Australian Institute for Medical Research Inc., Perth, Australia) for the GFP-ß-arrestin 1 cDNA; and Dr. G. E. Breitwieser (Weis Center for Research, Geisinger Clinic, Danville, PA) for the ß-arrestin 2 (V54D) mutant cDNA.


   Footnotes
 
This work was supported by grants from the Interdisciplinary Centre for Clinical Research at the University of Leipzig (project B19) and the Deutsche Forschungsgemeinschaft Pa 423/12-1.

Romy Frenzel, Carsten Voigt, and Ralf Paschke have nothing to declare.

First Published Online March 2, 2006

1 R.F. and C.V. contributed equally to this work. Back

Abbreviations: b, Bovine; CG, choriogonadotropin; CLSM, confocal laser-scanning microscopy; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HRP, horseradish peroxidase; TSHR, TSH receptor; wt, wild type.

Received June 8, 2005.

Accepted for publication February 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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