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Endocrinology Vol. 145, No. 2 1003-1010
Copyright © 2004 by The Endocrine Society

Upon Thyrotropin Binding the Thyrotropin Receptor Is Internalized and Localized to Endosome

Surya P. Singh, David McDonald, Thomas J. Hope and Bellur S. Prabhakar

Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Bellur S. Prabhakar, Department of Microbiology and Immunology (M/C 790), Room E-705, 835 South Wolcott Avenue, Chicago, Illinois 60612. E-mail: bprabhak{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To study the fate of TSH receptor (TSHR) on TSH binding, we constructed a chimeric cDNA that encodes a yellow fluorescent protein (YFP) fused to the carboxyl terminus of human TSHR. The protein expression in transfected cells was confirmed using flow cytometry. The functionality of the chimeric protein was determined by its ability to transduce signal leading to activation of cAMP in a TSH dose-dependent manner. The levels of cAMP produced by these cells were comparable with the levels seen in cells transfected with unfused TSHR without the YFP. Using deconvolution microscopy, we observed that the receptor is largely expressed on the cell surface, but on addition of TSH, some of the receptors were rapidly internalized. This conclusion was supported by several independent observations involving different cells expressing either native or recombinant TSHR. On TSH treatment, we observed internalization of human TSHR-YFP and human TSHR, expressed on 293 and CHO cells, respectively. This was further substantiated when we observed colocalization of rhodamine-labeled TSH with TSHR-YFP within the cell and by the uptake of radiolabeled TSH. Furthermore, shortly after ligand binding, there was a profound change in the morphology of the cells and some of the receptors accumulated in the perinuclear region of the cell. The TSHR-YFP was colocalized with RhoB-cyan fluorescent protein, indicating that it accumulated within the endosomes. These results indicate that the receptor internalization might in part be responsible for TSHR desensitization on TSH binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HOMEOSTASIS IS maintained by the mutual regulation of thyroid hormone produced in the thyroid, TSH produced in the anterior pituitary and the TRH produced in the hypothalamus through a classical feedback loop. In the thyroid, TSH binds to the TSH receptor (TSHR) and facilitates heterotrimeric complex of G proteins to exchange GDP for GTP, which activates the G protein. The GTP bound Gs{alpha} subunit dissociates from the Gsß{gamma} and activates adenylyl cyclase causing enhanced production of cAMP. After a short time, the Gs{alpha} subunit, due to its intrinsic GTPase activity, converts GTP to GDP and becomes inactivated and reassociates with the other two subunits. The cAMP in turn activates further downstream signaling leading eventually to the production of thyroid hormone. Increasing thyroid hormone level results in a negative feedback signal resulting in downmodulation of TRH and TSH production. This feedback loop helps maintain the euthyroid status in healthy individuals.

In contrast, our understanding of events as they relate to the fate of the TSHR on TSH binding is less well understood. Moreover, there are conflicting reports on TSHR transcription on ligand binding, with some studies showing up-regulation and others showing downmodulation of TSHR transcripts (1, 2). In many receptor systems, one of the important events following ligand binding is internalization of the receptor-ligand complex (3, 4, 5), which is implicated in termination of cAMP signaling (6, 7), initiation of mitogenic activity (8), dephosphorylation (9), and resensitization or downmodulation of the receptor (10, 11, 12). Although there is abundant information on internalization of receptors that contain a single transmembrane domain (13), there is paucity of data on endocytosis of glycoprotein hormone receptors that contain seven-membrane spanning domains. This is particularly true with regard to the fate of the TSHR protein on TSH binding. Earlier studies (14, 15, 16, 17, 18) on TSHR internalization, with the exception of a very recent study, have used uptake of either [125I] TSH or fluorochrome-TSH without employing any of the direct visualization or biochemical techniques.

A more recent study by Baratti-Elbaz et al. reported TSHR expression on the plasma membrane and clathrin-coated pits, with a proportion of the receptor constitutively endocytosed (19). On TSH addition, the endocytosis of the receptor was increased by 3-fold, with TSH undergoing degradation in lysosomes and 90% of the internalized TSHR recycling to the cell surface, which could be inhibited by monensin.

Studies using LH/TSHR chimeras showed that the extracellular domain determined the degree of internalization, whereas the transmembrane and the cytoplasmic domains determined whether the protein was degraded or recycled. Based on these studies, the authors speculated that differences in the fate of the receptor might be important for the differential effects of the hormones (19). The degradation of LH/chorionic gonadotropin (CG) receptor might explain its negative regulation (20), whereas recycling of the TSHR might be responsible for maintenance of the thyroid stimulatory antibody activity and development of hyperthyroidism (19). Developing insights into these issues is critical not only for understanding the normal function of the TSHR but also its role in the pathogenesis of Graves’ disease and other autoantibody mediated thyroid disorders.

Therefore, in the present study, we studied the interaction of TSHR, fused to a fluorescence protein, with TSH employing Delta Vision wide-field restoration microscopy, which has allowed us to study dynamic interactions in a living cell. In addition, we used biochemical approaches to demonstrate TSHR internalization on TSH binding and its accumulation in endosomes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
CHO cells and 293 cells (ATCC, Manassas, VA) were grown in Ham’s F-12 and DMEM-F12 (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and 2 mM (0.29 mg/ml) L-glutamine (Life Technologies, Inc., Invitrogen Corp., Grand Island, NY). FRTL-5 cells were obtained from ATCC and cultured in 6H medium; 5 d before the experiment, medium was replaced with 5H medium that was devoid of TSH (21).

Cloning of human TSHR (hTSHR) cDNA into pEYFP-N1 vector
TSHR-specific PCR primers, containing EcoR1 and BamH1 sites, and hTSHR cDNA as a template, were used to amplify hTSHR cDNA devoid of a stop codon. The amplified cDNA was then digested with the above-mentioned enzymes and cloned in frame upstream of the yellow fluorescent protein (YFP) using the same restriction sites within the multiple cloning site of the pEYFP-N1 vector (Clontech, Palo Alto, CA). Cloning was confirmed by restriction analysis using EcoR1 and BamH1 as well as by sequencing. Additionally, we prepared another construct containing a linker (sequence: ccGGATCTGC AGGGAGCTTC TAGACCGGAT CC) between the TSHR and YFP, which was used only to obtain results shown in Fig. 2Go, A and C.



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  		FIG. 2. A, Surface expression of TSHR-YFP. 293 cells transfected with a TSHR-YFP were stained with an anti-TSHR antibody followed by an antirabbit Cy3 antibody. Cells were fixed with 4% paraformaldehyde and the nuclei were stained with Hoechst. A-i, TSHR expressed as YFP. A-ii, Cy3 staining of TSHR. A-iii, A merged image of the two. B, Rhodamine-TSH binding to TSHR-YFP. 293 cells stably expressing TSHR-YFP were incubated with rhodamine-labeled TSH (1 x 10-8 M) at 4 C for 15 min, and the unbound TSH was removed by washing with PBS twice. Cells were fixed, mounted, and visualized. B-i, TSHR-YFP. B-ii, Binding of rhodamine TSH. B-iii, A merged image of the two. C, Internalization of TSHR-YFP. Rhodamine-labeled TSH (1 x 10-8 M) was added to the TSHR-YFP-expressing cells at 4 C for 15 min, washed with PBS, incubated at 37 C for 5 min, fixed, and mounted using a gel mount. C-i, TSHR-YFP. C-ii, Binding of rhodamine TSH. C-iii, Merged image of the two with TSH and TSHR colocalized inside the cell.

 
Expression of TSHR-YFP
The hTSHR-pEYFP-N1 plasmid was used to transfect 293 and CHO cells using the superfect reagent (Qiagen Inc., Valencia, CA) following the manufacturer’s protocol. Upon transfection, cells were cultured in medium containing G418 (1 mg/ml) for 2 wk to obtain stable transfectants. After selection, cells were sorted by flow cytometry to select for cells with varying levels of expression of YFP.

Fluorescence-activated cell sorter (FACS) analysis to detect hTSHR-YFP expression
Cells were analyzed for TSHR-YFP expression with and without further staining using a rabbit anti-TSHR antibody. First, 1 x 106 cells were incubated for 15 min with a rabbit anti-TSHR antibody (1:100) (22) in PBS containing 2% FBS followed by fluorescein isothiocyanate (FITC)-labeled secondary antibody. Samples were acquired and analyzed on a Becton Dickinson FACS calibur instrument using Cell Quest software (Becton Dickinson Co., San Jose, CA). All assays included cells incubated with second antibody alone and normal mouse serum as controls (data not shown).

cAMP assay
Cells were grown to full confluence in a 96-well plate and washed twice with prewarmed Hanks’ balanced salt solution (HBSS). HBSS containing 0.5 mM 3-isobutyl-1-methylxanthine along with TSH (10-8, 10-9, 10-10 M) was added. Cells were incubated at 37 C for 3 h. Supernatants were collected and the levels of cAMP determined using a RIA kit (NEN Life Science Products, Boston, MA).

Visualization of receptor internalization
293-YFP-TSHR cells (4 x 104) were plated in {Delta}-T black dishes (Bioptech, Butler, PA) 1 d before the experiment, and the next day the medium was replaced with fresh medium. Live cell images showing receptor expression were acquired before and after TSH stimulation employing a temperature-controlled stage and objective.

Binding and internalization of [125I] TSH
Control, TSHR, or TSHR-YFP-expressing CHO cells (5 x 104 cells) were plated into 24-well plates. After overnight incubation, cells were washed with PBS twice and incubated with [125I] TSH (27,000 cpm) at 4 C for 15 min. Unbound TSH was removed by washing cells with PBS twice, and cells were incubated at 37 C for 5 min, washed once with PBS, and then treated with isotonic buffer with pH 3 to remove TSH that was not internalized. Subsequently, cells were solubilized using NaOH before counting in a {gamma}-counter (23).

Immunostaining of TSHR using anti-TSHR antibody
CHO cells (105/chamber) expressing hTSHR were seeded into two chamber slides (Falcon/Becton Dickinson, Franklin Lakes, NJ). The next day the medium was replaced and 10-7 M bovine TSH (Sigma Chemical Co., St. Louis, MO) was added. After 1, 5, and 10 min, the cells were fixed in 4% paraformaldehyde and washed twice with PBS containing 2% FBS and then treated with a rabbit polyclonal anti-TSHR antibody for 15 min (22). Cells were washed twice with PBS followed by the addition of an FITC-labeled antirabbit antibody. Cells were washed again and mounted with Gel/Mount (Biomeda Corp., Foster City, CA). Dried slides were imaged on an IX70 epifluorescent microscope (Olympus, Tokyo, Japan) fitted with a Cooke Sensicam CCD (The Cooke Corp., Auburn Hills, MI). Fluorescent channels were captured individually and merged into a single RGB image using Slidebook imaging software (Intelligent Imaging Innovations, Denver, CO).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of cDNA encoding hTSHR and its cell surface expression
In an effort to develop an appropriate system that could be used to study TSHR trafficking in viable cells, we made a chimeric cDNA construct by cloning a full-length cDNA capable of encoding hTSHR into the EcoR1-BamH1 sites within the multiple cloning site of the pEYFP-N1 plasmid (not shown). We designated this plasmid as TSHR-YFP and was used to transfect 293 and CHO cells to establish stably transfected 293-TSHR-YFP and CHO-TSHR-YFP cell lines. As shown in Fig. 1AGo, transfected cells showed significant YFP expression (green fluorescence seen in FL1), whereas control untransfected (CHO-N) cells were completely negative. The protein expression was further confirmed when we stained viable cells with an antibody specific to the TSHR and a biotinylated second antibody followed by the addition of PE-avidin (Fig. 1BGo, red fluorescence seen in FL2). It is interesting to note that the degree of TSHR-YFP expression seen in both FL1 (direct visualization without staining with anti-TSHR antibody in A) and FL2 (due to PE-avidin used for detection in B) were comparable. Similarly, we were able to detect cell surface expression of TSHR using an anti-TSHR antibody in transfected CHO cells and FRTL-5 cells using an FITC-labeled (Fig. 1CGo) and a PE labeled second antibody (Fig. 1DGo), respectively.



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  		FIG. 1. Analysis of TSHR and TSHR-YFP expression using FACS. A, Cells were analyzed for TSHR-YFP expression without staining with anti-TSHR antibodies. FACS analysis shows CHO cells expressing TSHR-YFP (open area) relative to untransfected CHO cells (filled area). B and D, TSHR expression was detected using a rabbit anti-TSHR antibody followed by biotinylated antirabbit and phycoerythrin-labeled avidin (FL-2). B, TSHR-YFP expression on CHO cells (open area) relative to untransfected cells. D, TSHR expression on FRTL-5 cells detected by anti-TSHR antibody (open area) relative to FRTL-5 cells stained with normal rabbit serum. C, CHO cells expressing TSHR without YFP were stained with a rabbit anti-TSHR followed by FITC-labeled antirabbit (FL1). Samples were analyzed on a Becton Dickinson FACS calibur. FL-1 and FL-2 represents green and red fluorescence, respectively.

 
We confirmed cell surface expression of TSHR and suitability of this protein for trafficking studies using fluorescent microscopy. As evident from the data shown in Fig. 2AGo, TSHR-YFP was readily detected in transfected cells (Fig. 2AGo-i). To confirm cell surface expression, viable cells were stained with a rabbit anti-TSHR antibody followed by an antirabbit Cy3 antibody (Molecular Probes, Inc., Eugene, OR). This showed that most of the receptors were expressed on the cell surface (Fig. 2AGo-ii). This was further substantiated when we observed almost complete overlap of area stained with YFP and Cy3 (Fig. 2AGo-iii).

Ability of TSHR-YFP to activate cAMP production
The intracellular domain of the TSHR is involved in signaling; therefore, we wanted to ensure that the fusion of YFP to the cytoplasmic tail of TSHR did not affect its ability to transduce intracellular signaling. We tested cells expressing no TSHR, full-length TSHR, or TSHR-YFP for their ability to produce cAMP on addition of TSH. Results from these experiments clearly showed that the cells expressing TSHR-YFP were able to produce cAMP relative to the control cells (Fig. 3Go). Both CHO-TSHR and CHO-TSHR-YFP cells produced comparable levels of cAMP at a higher concentration of TSH (10 nM), but the level of cAMP was higher in CHO-TSHR-YFP when lower amounts of TSH (e.g. 1 nM) were used. Collectively, our data showed that the degree of cAMP production correlated with the levels of TSHR expression and that the TSHR-YFP is expressed on the cell surface and is functional.



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  		FIG. 3. TSH-induced cAMP production. Different cells were grown to confluency in a 96-well plate and washed twice with prewarmed HBSS. HBSS containing 0.5 mM 3-isobutyl-1-methylxanthine along with TSH (10-8,10-9,10-10 M) was added to the cells and incubated at 37 C for 3 h. Supernatants were collected and subjected to cAMP assay using NEN Life Science Products cAMP [125I] RIA kit. These experiments have been repeated three times with very similar results.

 
Binding and internalization of TSH
TSHR-YFP expression appeared to be largely localized to the cell surface area (Fig. 2BGo-i). To further confirm this and evaluate its ability to interact with TSH, we stained viable cells with rhodamine (Pierce Chemical Co., Rockford, IL)-labeled TSH at 4 C and found that the distribution of rhodamine-TSH (Fig. 2BGo-ii) was very similar to the distribution of TSHR-YFP (Fig. 2BGo-i). This was proven to be the case when the two images were overlaid (Fig. 2BGo-iii). These results further confirmed that the receptor was expressed on the cell surface and that the TSH could bind to it. To study the fate of TSHR-bound TSH, we allowed rhodamine-TSH to bind to TSHR-YFP expressed on 293 cells at 4 C for 15 min and then incubated the cells at 37 C for 5 min to allow for receptor trafficking. The cells were fixed and observed using a fluorescence microscope. As expected, a significant amount of TSH was colocalized with TSHR-YFP within the cytoplasm, suggesting that the receptor-ligand complexes were internalized (Fig. 2CGo). To further demonstrate internalization of TSHR, we treated 293 cells expressing TSHR-YFP with TSH at 37 C for 10 min, and images of live cells were captured. As indicated earlier, the TSHR-YFP protein was expressed on the cell surface before addition of TSH (Fig. 4AGo). However, after TSH addition, the TSHR-YFP was redistributed from the cell surface with some of the protein internalized (Fig. 4BGo). Concomitant with redistribution of the TSHR, we also noticed a profound change in the cell morphology, perhaps due to significant rearrangement of cytoskeletal structures (see supplemental data of time-lapse live images). To confirm internalization of the ligand along with the TSHR, we used [125I]-labeled TSH and found that approximately 20% of the receptor bound TSH was internalized along with the receptor (Fig. 5Go).



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  		FIG. 4. Live-cell imaging of TSHR-YFP internalization. 293 cells were plated in {Delta} T black disc 24 h before the experiment. Medium was replaced, cells were treated with TSH for 10 min at 37 C, and live cell images were captured using a deconvolution microscope. A and B, Cells before and after TSH treatment, respectively.

 


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  		FIG. 5. TSH binding and internalization. Control and TSHR- or TSHR-YFP-expressing CHO cells (5 x 104) were plated into 24-well plates. After overnight incubation, cells were washed with PBS twice and incubated with125 I -TSH (27,000 cpm) at 4 C for 15 min. Cells were washed twice with PBS to remove unbound TSH and incubated at 37 C for 5min to allow for TSH internalization. Cells were washed once with PBS and then treated with isotonic pH 3 buffer to remove TSH that was not internalized. Internalized TSH was quantitated by solubilizing the cells with NaOH and then counting in a {gamma}-counter. This procedure is identical to that described by Kishi and Ascoli (23 ).

 
Internalization of TSHR in CHO-TSHR cells on TSH treatment
To confirm that the redistribution seen with TSHR-YFP was not due to aberrant behavior of TSHR due to its fusion with YFP, we monitored trafficking of the TSHR, without YFP, using an anti-TSHR antibody. As is apparent from Fig. 6Go, the protein was readily detected in CHO cells expressing TSHR but not in control cells (not shown). The TSHR was expressed throughout the cell (Fig. 6AGo). The receptor was internalized 1 min after addition of TSH (Fig. 6BGo) and had accumulated around the nucleus after 5 min (Fig. 6CGo). However, the receptor reappeared on the cell surface within 10 min of TSH addition (6DGo).



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  		FIG. 6. Internalization of TSHR upon TSH treatment. CHO cells (105/chamber) expressing hTSHR were seeded into two chambered slides (lab-TekII). The next day the medium was replaced and 10-7 M bovine TSH was added. After 1, 5, and 10 min of incubation with TSH, the cells were washed twice with PBS containing 2% FBS, fixed in cold methanol, and then treated with a rabbit polyclonal anti-TSHR antibody for 15 min. Cells were washed twice with PBS followed by the addition of an FITC-labeled antirabbit antibody. Cells were washed again, mounted, and then observed under a deconvolution microscope. A, Untreated cells. B–D, Cells treated with TSH for 1, 5, and 10 min, respectively.

 
Colocalization of TSHR in endosome
Within minutes after addition of TSH, the receptor was internalized and accumulated primarily in the perinuclear region of the cell, suggesting that they might have localized to the endosome. To confirm the location of TSHR within the cell, we transfected TSHR-YFP-expressing CHO cells with a RhoB-cyan fluorescent protein (CFP) construct (BD Biosciences Clontech, Palo Alto, CA), which is commonly used as an endosomal marker. Before the addition of TSH, RhoB protein showed a distinct intracellular distribution (Fig. 7BGo, shown in blue), whereas TSHR appeared to be on the surface (Fig. 7AGo, shown in green) with little or no overlap between the two (Fig. 7CGo). On addition of TSH, a significant amount of the TSHR signal (Fig. 7DGo) overlapped with the RhoB (Fig. 7EGo), indicating that it had accumulated in the endosomes (Fig. 7FGo).



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  		FIG. 7. Colocalization of TSHR with RhoB. 293 cells were cotransfected with TSHR-YFP and {rho}B-CFP (Endovector, Clontech) cDNAs. A and B, Localization of TSHR-YFP and RhoB-CFP, respectively. C, Merged image of the two. D and E, Distribution of TSHR-YFP and RhoB-CFP, respectively, after TSH treatment. F, Merged image of D and E and, unlike C, shows colocalization of the two proteins upon TSH addition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Receptor proteins fused to fluorescent proteins have been successfully used for directly visualizing protein expression and following the protein redistribution in real time on stimulation with an appropriate ligand (24, 25, 26, 27). This approach has also been successfully used for studying G protein-coupled receptor expression (transient and stable) like ß2-adrenergic receptor (28, 29) and the cholecystokinin receptor type A (30). Our initial attempts to express a functional TSHR protein fused to either a red or green fluorescent protein resulted in successful production of both chimeric proteins. However, these two proteins showed varying degrees of self-aggregation and thus were found to be less than optimal for TSHR trafficking studies (not shown). However, we prepared and employed a TSHR-YFP chimeric protein, either with or without a linker sequence, to study the fate of the TSHR protein on TSH binding. As evident from our results, we successfully expressed a functional TSHR-YFP chimeric protein on the cell surface.

One of the most important considerations while modifying a protein is to ensure that it maintains its normal ligand binding and signal transduction properties. We tested cells expressing either a hTSHR or a hTSHR-YFP chimeric protein for its ability to respond to TSH. Our results showed that the chimeric receptors could not only bind TSH but also transduce intracellular signals resulting in enhanced production of cAMP. The level of cAMP produced by cells expressing the chimeric protein was comparable with the level produced by cells expressing the hTSHR protein.

The molecular mechanisms that lead to agonist-dependent desensitization of G protein-coupled receptor are not fully defined, but several distinct events occur. In general, the receptor numbers on the cell surface are reduced due to internalization, reduced transcription, and/or degradation. One of the important events following ligand binding to G protein-coupled receptors is internalization of the receptor-ligand complex (3, 4, 5). Concomitantly, changes also occur inside the cell including uncoupling of the receptor from the heterotrimeric G proteins (4, 32, 33).

Although there is abundant information on internalization of receptors that contain a single transmembrane domain (13), there is paucity of data on the fate of glycoprotein hormone receptors that contain seven-membrane spanning domains. This is particularly true with regard to the fate of the TSHR protein on TSH binding. Based on earlier studies (14, 15, 16, 17, 18), the fate of the TSHR on ligand binding can be inferred. However, the present study directly shows that some of the TSHR is internalized, along with TSH, within minutes of TSH addition. The time required for, and the degree of, internalization was different in different cells and suggested that it might be dependent on the density of receptor expression on the cell surface. In addition, we saw a profound change in the morphology of the cell shortly after addition of TSH, perhaps due to cytoskeletal changes (see time-lapse live images provided as supplemental data). These changes are analogous to the dynamic changes that we observed earlier with another seven-transmembrane region containing CCR5 receptor exposed to the ligand regulated upon activation, normal T cell expressed, and secreted (34).

Much of what we know about the fate of the receptors upon ligand binding comes from studies using receptors for transferrin, low-density lipoprotein, and epithelial growth factor, and more recently ß2 adrenergic, LH/CG, and FSH (35, 36, 37, 38, 39, 40, 41). Usually ligand-receptor complexes formed on the cell surface accumulate in the clathrin-coated pits that then form clathrin-coated vesicles (42, 43). These vesicles lose their coat and immediately fuse with early endosomes (44). Once taken up by the endocytic vesicles, the receptor-ligand complexes are dissociated due to mild acidic pH 6.3–6.8 (36), sorted, and delivered to specific compartments in the cell. Freed receptors accumulate in the tubular extensions of the early endosomes and bud off to become recycling vesicles. Either directly or indirectly these vesicles facilitate recycling of the receptor to the plasma membrane. The dissociated ligand accumulates in the vesicular portion of the early endosome, which traverses to the perinuclear region of the cytoplasm and then fuses with the late endosomes and lysosomes. In these vesicles, because of low pH (pH 5) and presence of lysozymes, the ligand undergoes degradation. Thus, early endosomes are involved in dissociation and sorting of receptor-ligand complexes in an environment that is least damaging (45), whereas the late endosomes and lysosomal vesicles are primarily involved in accumulating and digesting both exogenous and endogenous macromolecules (46). It is clear from the perinuclear accumulation of the TSHR and its colocalization with RhoB-CFP that the TSHR, upon TSH binding, internalizes and accumulates in the endosomal vesicles. Furthermore, our studies, similar to an earlier study (19), strongly suggest that the receptor is recycled back to the cell surface. However, additional studies are necessary to determine how exactly the TSHR is internalized and show conclusively whether it is recycled back to the cell surface or degraded in lysosomal vesicles.

Like other G protein-coupled receptors, binding of hormone to either FSH receptor or LH receptor that are structurally very similar to TSHR initiates endocytosis of the receptor-hormone complex involving clathrin-coated pits/vesicles (37, 38, 39, 40, 41). It has been shown that the LH receptor-LH complexes are eventually transported to lysosomes in which both are degraded (39). However, the fate of FSH receptor is not as well understood, although the FSH itself is targeted for degradation in the lysosome (38). Studies involving muscarinic acetylcholine receptor (47) and ß-adrenergic receptor (48) showed that these receptors were internalized via caveolae and suggested that all G protein-coupled receptors might follow the same pathway. However, other studies clearly demonstrated that LH/CG receptor (49) and a number of other receptors (50, 51, 52, 53, 54, 55, 56) are internalized using the clathrin-coated vesicles. Some of these receptors were recycled to the cell surface (e.g. receptors for GnRH, TRH, ß2 adrenergic, and angiotensin II) (57, 58, 59, 60, 61), whereas some others (e.g. receptors for LH/CG, thrombin, and yeast pheromone) were degraded in the lysosomes (49, 51, 53, 62). Studies on cholecystokinin receptor, using electron microscopy, have shown that this receptor uses two different pathways (63): one involving clathrin-coated vesicles that target the protein to lysosomes for degradation and the other involving caveolae, which facilitates recycling of the receptor to the plasma membrane (63).

A recent study by Baratti-Elbaz et al. (19) showed that the TSHR is internalized upon TSH addition, and most of the internalized TSHR is recycled to the cell surface. Results of the present study are consistent with these earlier observations. In contrast, another study by Latif et al. (64) showed TSHR-green fluorescent protein capping upon TSH binding. Subsequently, Latif et al. demonstrated that the receptor can undergo oligomerization that is dependent on receptor cleavage into {alpha}- and ß-subunits. In another study, receptor-receptor interaction was further demonstrated using fluorescence resonance energy transfer analysis (65). However, upon TSH binding there was almost complete lack of FRET-positive cells, indicating a reduction in receptor oligomerization. It was concluded that the constitutive activation of the receptor is inhibited by the formation of higher-order complexes, which is prevented by TSH binding, resulting in receptor activation. Although these studies are very interesting and informative, they did not fully resolve the mechanism of receptor desensitization upon TSH binding. It is interesting to note that TSH was internalized and colocalized with the TSHR-YFP; this suggested that {alpha}- and ß-subunits were not separated upon ligand binding. An earlier study (19) and the present study, using several different cells expressing the TSHR, clearly show that the TSHR is internalized after the addition of TSH. Developing further insights on TSHR trafficking is critical not only for understanding its normal function but also might shed new light on its role, if any, in the pathogenesis of Graves’ disease.


   Acknowledgments
 
The authors thank Osvaldo Martinez and Chentamarakshan Vasu for their technical help and thoughtful suggestions.


   Footnotes
 
This work was supported by the NIH Grants DK47417, DK57938, and DK44972.

Abbreviations: CFP, Cyan fluorescent protein; CG, chorionic gonadotropin; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HBSS, Hanks’ balanced salt solution; hTSHR, human TSHR; TSHR, TSH receptor; YFP, yellow fluorescent protein.

Received September 12, 2003.

Accepted for publication October 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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