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Endocrinology Vol. 148, No. 7 3164-3175
Copyright © 2007 by The Endocrine Society

Lipid Rafts Are Triage Centers for Multimeric and Monomeric Thyrotropin Receptor Regulation

R. Latif, T. Ando and T. F. Davies

Division of Endocrinology and Metabolism (R.L., T.F.D.), James J. Peters Veterans Affairs Medical Center, Mount Sinai School of Medicine, New York, New York 10468; and University of Nagasaki (T.A.), Nagasaki 852-8523, Japan

Address all correspondence and requests for reprints to: R. Latif, Ph.D., James J. Peters Veterans Affairs Medical Center, 2F-30 Research Building, 130 West Kingsbridge Road, Bronx, New York 10468. E-mail: rauf.latif{at}mssm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The TSH receptor (TSHR), a heptahelical G protein-coupled receptor on the surface of thyrocytes, is a major autoantigen and physiological regulator of the thyroid gland. Unlike other G protein-coupled receptors, the TSHR undergoes posttranslational cleavage of its ectodomain, leading to the existence of several forms of the receptor on the plasma membrane. We previously hypothesized that to achieve high fidelity and specificity of TSH ligand or TSHR autoantibody signaling, the TSHR may compartmentalize into microdomains within the plasma membrane. In support of this hypothesis we have shown previously that TSHRs reside in GM1 ganglioside-enriched lipid rafts in the plasma membrane of TSHR-expressing cells. In this study, we further explored the different forms of TSHRs that reside in lipid rafts. We studied both TSHR-transfected cells and rat thyrocytes, using both nondetergent biochemical analyses and receptor-lipid raft colocalization. Using the biochemical approach, we observed that monomeric receptors existed in both raft and nonraft fractions of the cell surface in the steady state. We also demonstrated that the multimeric forms of the receptor were preferentially partitioned into the lipid microdomains. Different TSHR forms, including multimers, were dynamically regulated both by receptor-specific and postreceptor-specific modulators. TSH ligand and TSHR antibody of the stimulating variety induced a decrease of multimeric forms in the raft fractions. In addition, multimeric and monomeric forms of the receptor were both associated with Gs{alpha} within and without the rafts. Although failure to achieve total lipid raft disruption prevented a conclusion regarding the relative power of TSHR signaling within and without the raft domains, these data showed clearly that not only were a significant proportion of TSHRs residing within lipid microdomains but that constitutive multimerization of TSHRs was actually regulated within the lipid rafts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HUMAN TSH receptor (TSHR), a heptahelical G protein-coupled receptor present on the plasma membrane of thyrocytes is required to carry out many of the specialized functions of the thyroid gland (1, 2, 3). Like other close members of its family such as the LH receptor and FSH receptor, this receptor also consists of a large ectodomain and a membrane anchored B or ß-subunit. The unique 50-amino acid residue within the large ectodomain of the TSHR undergoes proteolysis (cleavage) leading to the two disulfide-linked subunit (A or {alpha} and B or ß) structures (2, 4, 5, 6, 7). Another post-translational hallmark of this receptor is its propensity to form constitutive dimeric and oligomeric forms. Our laboratory showed the existence of such dimeric and multimeric isoforms of these receptors in native thyroid tissue and subsequently in heterologous cells using fluorescence resonance energy transfer (8, 9, 10). We have also shown that these oligomeric forms undergo dissociation with TSH (10, 11). Subsequently others have confirmed our observation on constitutive oligomerization of the TSHR and have extended the role of these TSHR dimers and oligomers in allosteric interactions and negative cooperativity (12). Recently the role of oligomerization in the dominant inheritance of partial TSH resistance has also been outlined (13). Thus, oligomerization of TSHRs may have physiological and pathological roles in thyroid disease and autoimmunity.

There is now an emerging concept to explain signaling specificity in the face of molecular promiscuity of receptors and the signaling cues that they receive from the external environment of the cell. Specificity, in this model, would be regulated by segregating the signaling molecules and their adaptor proteins in specific cellular compartments. The physical segregation of proteins into such microdomains may regulate their accessibility to regulatory and/or effector molecules. One such compartmentalization of molecules on the plasma membrane is known to occur in cholesterol and sphingolipid enriched platforms of protein segregation namely lipid rafts (14, 15, 16, 17, 18). The classification of the different forms of rafts has been difficult because these microdomains on the cell surface are dynamic structures and there is no clear demarcation of their boundaries on the plasma membrane. However, they have been broadly divided into typical rafts or membrane rafts, which are flat and difficult to differentiate from the rest of the plasma membrane and which are enriched with GM1/3 ganglioside, and atypical rafts, namely caveolae, which are flask-shaped invaginations on the plasma membrane and which are coated with the protein caveolin (14, 15, 16, 17, 18). The extent to which typical lipid rafts and caveolae overlap in composition and function is ill defined. Only cells expressing caveolin have caveolae, whereas all mammalian cells express more typical lipid rafts (19). For example, in caveolin knockout mice, lipid rafts can still be found, implying that caveolae have little to do with typical lipid raft biogenesis (20). Caveolae also function during signal transduction, but they are not absolutely required because several cell types that lack caveolin, such as lymphocytes and neurons, can signal through rafts (18). However, both types of lipid rafts have been proposed to function in a large number of activities ranging from protein and lipid sorting post Golgi to endocytosis to regulation of cell signaling and viral entry and budding (21).

The role of caveolae in protein trafficking and internalization is well established (22, 23, 24). Whatever the nature of these microdomains, it has been observed that rafts spatially and temporally segregate signaling, trafficking, and cytoskeletal molecules (16, 17, 18, 25). The role of rafts in signaling has been especially well documented in the case of T and B cell receptors and tyrosine kinase receptors (26, 27, 28). Recent studies have also shown that some G protein-coupled receptors (GPCRs) exist in these lipid microdomains (29, 30, 31, 32). For example, it has been established that the LHR localizes to these membrane-type lipid microdomains on ligand binding (33, 34, 35, 36), whereas the GnRH receptor is constitutively localized to these lipid microdomains (37). Thus, enrichment of GPCRs and its signaling components in lipid rafts or caveolae may be a universal mechanism for increasing the effective concentration of these proteins by restricting their movement and favoring interactions of components in the signal transduction pathways (38).

We have previously shown by confocal microscopy that TSHRs also reside in lipid rafts and exit from the rafts on TSH ligand binding (39). However, the imaging methodology was unable to distinguish the different forms of the receptors that resided in the raft and nonraft fractions. Because the TSHR has subsequently been reported not to be found in caveolae (40), by isolating detergent insoluble membrane fractions, we presumed that our observations had detected TSHRs only in the more typical flat rafts containing ganglioside GM1.

Lipid rafts and raft-associated proteins have usually been isolated by their ability to remain insoluble in detergent-treated cells under low temperatures. Because of the higher saturation of the hydrocarbon chains in the sphingolipids and phospholipids of lipid rafts, these membrane compartments are more tightly packed and have a higher degree of order than the surrounding matrix (16, 25, 41). Recently it has been shown that this detergent methodology, when used for the isolation of rafts, may lead to aggregation, giving rise to false raft formations or even disruption of the existing rafts (18, 42). Therefore, raft isolation using nondetergent biochemical methods and biophysical approaches such as lipid labeling of live cells and protein-protein interaction methods using fluorescence resonance energy transfer and fluorescence lifetime imaging are now the methodologies of choice to study these dynamic structures on the plasma membranes (17, 42). No one method is considered as the gold standard, so a combination of approaches is preferred when studying these specialized membrane microdomains.

The purpose of the present study was: 1) to explore the compartmentalization of TSHRs in rafts prepared from TSHR-transfected Chinese hamster ovary (CHO) cells and rat thyrocytes using a native biochemical approach, 2) to study the forms of the TSHR to be found in raft and nonraft compartments of the cell, 3) to observe the effect of TSH and thyroid-stimulating antibodies on raft-associated TSHR forms, and 4) to examine the association of G proteins with receptors in raft and nonraft compartments and their dynamic changes on contact with TSH ligand.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Opti-Prep density gradient medium was obtained from Sigma Aldrich Chemical Co. (St. Louis, MO; catalog no. D1556). Total cholesterol E assay kits were from Wako (catalog no. 439-17501; Richmond, VA). The flotillin-2 rabbit polyclonal antibody (catalog no. SC25507), the ß-adaptin goat polyclonal (catalog no. SC645), and G{alpha}s rabbit polyclonal (catalog no. SC823) antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The caveolin-1 rabbit polyclonal antibody was obtained from Abcam Inc. (Cambridge, MA; catalog no. Ab2910). The TSHR monoclonal antibody (M1/RSR1) was a kind gift from Dr. B. Rees Smith (RSR Ltd., Cardiff, UK). The epitope that M1 recognizes is 381–385 amino acids and has been shown not to be inhibited by TSH (11). MS-1, a hamster-derived potent stimulating antibody binding to the ectodomain of TSHR, was generated in our laboratory (43). All-labeled secondary antibody was obtained from Jackson ImmunoResearch (West Grove, PA). Bovine TSH was purchased from Sigma Aldrich (catalog no. T8931). The lipid raft staining used the Vybrant Alexa Fluor 555 labeling kit from Molecular Probes (Eugene, OR; catalog no. V-34404). cAMP was measured using the cAMP Biotrak Direct enzyme immunoassay (EIA) system from Amersham Biosciences (catalog no. RPN225; Piscataway, NJ). Methyl-ß-cyclodextrin (MCD) was from Sigma Aldrich (catalog no. C4555).

Cell culture
Stable lines of CHO-TSHR and glycophosphotidyl linked (GPI)-TSHR-transfected cells used in this study were generated by transfecting N terminus HA tagged TSHR construct (kindly provided by Dr. M. W. Szkudlinski, Trophogen, Rockville, MD) GPI-TSHR ectodomain (kindly provided by Dr. A. P. Johnstone, St. George’s Hospital Medical School, London, UK) into CHO cells. The receptor expression and functionality of these cells has been published in our previous studies (11). These cells were maintained in Ham F12 with 10% fetal bovine serum and 100 IU penicillin/streptomycin. Rat-derived thyrocytes and FRTL5 cells were obtained from the laboratory of Dr. Nancy Carraso (Albert Einstein College of Medicine, Bronx, NY). FRLT5 cells were grown in F12 medium with 10% calf serum, 100 µg/ml penicillin/streptomycin, and six-hormone medium containing 1 mU bovine TSH.

Preparation of detergent-free lipid rafts
The procedure described by Macdonald and Pike (44) was followed with minor modifications. Briefly, four 150-mm dishes of CHO-TSHR cells were cultured in Ham’s F12 medium with 10% fetal bovine serum and 100 µU/ml penicillin and streptomycin until the cells were 90% confluent. Treatment of these cells with TSH or TSHR-stimulating antibody was performed at 37 C for the stipulated amount of time. The cells were washed immediately with cold base buffer [20 mM Tris-Hcl (pH 7.8), 250 mM sucrose] to which 1 mM CaCl2 and 1 mM MgCl2 were added. Protease inhibitor cocktail tablets were added to the base buffer just before its use. The washed cells were gently scraped from the dish into the base buffer with protease inhibitor and were pelleted by centrifugation at 1200 rpm for 5 min at 4 C. The cell pellet was then resuspended in base buffer and was gently homogenized (20 strokes) using a handheld Dounce homogenizer and then aspirated into a 22-g needle and lysed by repeated passage through the needle (20 times). The resulting post nuclear supernatant was then collected into separate tube. The above step was repeated once more with 1 ml of buffer, and the two postnuclear supernatants were combined together. Equal volumes (2 ml) of base buffer containing 50% OptiPrep were combined with the postnuclear supernatant and placed in the bottom of a 12-ml centrifuge tube. Eight milliliters of 5–20% gradient were then poured on top and centrifuged for 90 min at 52,000 x g using the SW41 rotor in a Beckman ultracentrifuge. Gradients were fractionated into 0.7-ml fractions obtained from the top. Fifty microliters of the fraction were combined with 5x sample buffer with and without dithiothreitol as indicated and held at 37 C for 45 min before resolving it on 12% SDS-PAGE.

Protein and cholesterol estimation in the raft fraction
Total protein in each fraction was determined by the Bradford method (45). Total cholesterol was determined using the Wako E cholesterol assay kit briefly; lipid was isolated from 0.5 ml each of the fractions by extracting it with chloroform-methanol mixture [2:1 (vol/vol)]. The chloroform layer was taken out and evaporated to dryness in a water bath and dissolved with isopropyl alcohol containing 10% ether. The pellet was then reconstituted in the buffer, and 300 µl of the color reagent were added to these reconstituted fractions and incubated for 5 min at 37 C followed by spectrophotometric analysis. The end product of this chemical reaction was a blue pigment and the absorbance was measured at 600 nm. The amount of cholesterol in each fraction was read off a standard curve.

Staining of CHO-TSHR cells with labeled cholera toxin B subunit (CtxB)
As described previously (39), 20,000 cells were seeded in 8-well chamber slides (Lab-Tek Nun, Napier, IL) and incubated overnight in 1 ml of Ham’s F12 (Mediatech Inc., Herndon, VA) complete medium supplemented with 10% fetal bovine serum and 100 µU/ml penicillin/streptomycin at 37 C with 5% CO2. Before staining, the cells were chilled on ice for 10 min and washed twice with ice-cold PBS and incubated with 1 µg/ml CtxB Alexa 555 for 10 min. The cells were then washed 5 times with PBS and further incubated with anti-CTxB for 15 min to aggregate the raft molecules. The raft-stained cells were then incubated further with TSHR antibody (biotin labeled M1) and stained further with strepavidin fluorescein isothiocyanate. The images were collected using a 60x oil Plan Apo with 1.4NA objective and Photometric CoolSnapES CCD camera on a TE2000 epifluorescent microscope (Morrell Instruments Co., Melville, NY) using QED software. The colocalization measurements on the cells were carried out using Image Pro plus.

MCD treatment and cAMP measurement
Before treatment with MCD, CHO-TSHR cells were incubated at 37 C for 1 h in a serum-free medium followed by treatment with and without MCD (10 mM) concentrations for 30 min at 37 C. Control cells were in serum-free medium for 1.5 h. Rafts were isolated as described above from untreated and treated cells. For cAMP measurement, 0.5 x106 cells/well CHO-TSHR cells cultured in 24-well plates were first rinsed with serum-free medium and preincubated for 1 h in serum-free medium to deplete any endogenous cholesterol synthesis. The cells were then washed with medium and incubated for 30 min with and without MCD (10 mM) at 37 C. Then the medium was removed and cells were stimulated with bovine TSH (10, 102, 103 µU/ml) in serum-free conditions. At the end of incubation, the cells were lysed by addition of lysis buffer and lysate was measured for cAMP using the EIA kit.

Coimmunoprecipitation with G{alpha}s
Rafts fractions (no. 1) and nonraft fractions (no. 11) prepared from CHO-TSHR cells was immunoprecipitated with anti G{alpha}s (rabbit polyclonal). Briefly, fractions were precleared with protein A agarose beads for 30 min at 4 C. The precleaned supernatant was collected into fresh tubes and precipitated by the addition of 2 µg/ml of rabbit polyclonal G{alpha}s antibody for 2 h at 4 C. This was followed by a pull-down of the immune complex with protein A beads. The beads were reduced by treating them with 5x sample buffer containing 100 mM of dithiothreitol for 45 min at 50 C. The immunoprecipitates were resolved on 12% SDS-PAGE and electroblotted onto polyvinyl difluoride membranes. Membranes were blocked with 5% dried skimmed milk in Tris-buffered saline with 0.05% Tween 20 and then probed with 2 µg/ml monoclonal M1 antibody (TSHR antibody recognizing epitope residues 381–385) for 1 h at room temperature. Washed membranes were then incubated with 1:5000 of secondary antibody (antimouse horseradish peroxidase) for 1 h at room temperature. After final washing, bound secondary antibodies were visualized using enhanced chemiluminescence (Super Signal ECL; Pierce, Rockford, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Initial characterization of lipid rafts
We used the detergent-free method of Macdonald and Pike (44) for our biochemical approach, which is now known to be efficient and simple for the isolation of lipid rafts. The protein and cholesterol profiles of the various fractions prepared from CHO-TSHR cells are shown in Fig. 1AGo. The isolated fractions showed low protein in low-density fractions (fractions 1–4) and higher protein concentrations in high-density fractions (fractions 8–12). The cholesterol profile (Fig. 1Go, bar graph) showed a reversed pattern, indicating that fractions 1–4 were high in lipid and fractions 9–12 were lipid poor, suggesting that the high-density fractions contained abundant membrane-enriched cytoskeleton proteins. The distribution of raft and nonraft markers in these fractions and all subsequent analysis of these fractions was carried out using unconcentrated 50-µl volumes of low- and high-density fractions resolved in 12% SDS-PAGE. For marker distribution, blots were probed with raft markers (flotillin-2 and caveolin-1) and nonraft markers (ß-adaptin). Flotillin-2, as predicted, was restricted to the low-density fractions 1 and 2, whereas caveolin-1, a protein specific to caveolae, was found distributed across the gradient, suggesting that caveolin rafts were not well isolated by this nondetergent method (46). Yet the nonraft marker, ß-adaptin, was appropriately restricted to high-density fractions only (Fig. 1BGo). Thus, the initial characterization of these fractions showed clearly that the nondetergent isolation procedure was capable of separating low-density typical raft fractions and high-density nonraft fractions. Hence, fraction 1, with low density, was subsequently referred to as the raft fraction, and the high-density fraction 12 was referred to as the nonraft fraction. Based on the characterization by raft and nonraft markers, we restricted further analyses to these fractions.


Figure 1
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  		FIG. 1. Biochemical isolation of lipid rafts using a nondetergent method. A, For initial characterization of various density gradient fractions, protein (line graph) and total cholesterol (bar graph) were estimated in fractionation experiments as described in Materials and Methods. B, Raft and nonraft markers in the low- and high-density fractions are indicated. Flotillin-2, a 48-kDa protein, was detected using a 1:200 dilution of rabbit anti-flotillin-2 antibody, whereas caveolin-1, a 22-kDa protein in these fractions, was probed using caveolin-1 antibody (1.5 µg/ml). ß-Adaptin, a nonraft marker, was detected as a 106-kDa protein using a 1:200 dilution of the antibody. Although only representative blots are shown here, these experiments were repeated at least two to three times.

 
TSHRs reside in raft and nonraft fractions of CHO-TSHR cells and rat thyrocytes
Having successfully isolated raft and nonraft fractions by this nondetergent method, we next analyzed the fractions isolated from CHO-TSHR for the presence of TSHR. As indicated in Fig. 2Go, TSHR forms resided in both raft and nonraft fractions. However, the multimeric form of receptors was exclusively found in raft fraction (fraction 1). We did not observe any such multimeric forms of the receptor in the nonraft fraction, even after a 30-min exposure of the immunoblot. Having localized TSHRs in lipid rafts in CHO-TSHR cells by the biochemical method, we next analyzed rat thyrocyte cells for the presence of TSHRs in lipid rafts. Rat thyroid cells (FRTL-5) grown in six-hormone medium (five hormones plus TSH) were deprived of TSH for 48 h and the cells fractionated into raft and nonraft samples. The fractions were probed with TSHR antibody M1 as previously done for CHO-TSHR cells. TSHRs were seen in the control lysate as monomeric and multimeric forms. Because of the low numbers of TSHR in FRTL5 cells, it was difficult to visualize TSHR in the raft and nonraft fractions (Fig. 3Go). However, it was clear that higher order forms were present in the raft but not the nonraft fraction, thus providing a similar pattern to that of CHO-TSHR cells. But only small amounts of the monomer were discernible in the nonraft fractions. The absence of large numbers of oligomeric forms in these cells may have been due to the low numbers of TSHRs on this cell line or due to the presence of other membrane stimulating factors such as insulin and somatostatin in the culture medium. However, a clear TSH holoreceptor band (~85 kDa) was detected in the raft fraction, and small amounts of multimeric forms were observed more in the raft fraction and almost none in the nonraft fraction.


Figure 2
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  		FIG. 2. TSHR forms reside in lipid rafts in CHO-TSHR cells and its regulation by TSH. TSHR multimeric forms (>100 kDa protein bands) and monomeric holoreceptor (100 kDa) in a steady state are indicated by Western blotting (A) and densitometry (B) in raft and nonraft fractions under nonreduced conditions. On TSH treatment (103 µU/ml) for 30 min at 37 C, these forms showed dynamic changes. The multimeric forms had decreased substantially in the raft fraction on exposure to 103 µU/ml of TSH (n = 2). C+, Control lane, total lysate of the cell pellet before and after TSH exposure.

 

Figure 3
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  		FIG. 3. TSHRs in rat thyrocytes resides in rafts. Raft and nonraft fractions obtained from the density gradients of FRTL5 cells grown in 5H medium for 48 h and analyzed under nonreducing condition was probed with TSHR antibody (M1). A major band of receptors corresponding to an expected size of 85 kDa is seen in the lysate and the raft-enriched fraction 1 with bands of higher order forms ranging in size up to 175 kDa. Lane 1 refers to raft fraction and lane 12 refers to the nonraft fraction. C+, Control lane, total lysate of the cell pellet.

 
TSH binding modulates the receptor microenvironment of these multimeric and monomeric forms of TSHRs
We confined our analysis of its regulation to CHO-TSHR cells because of the ability to qualitatively and quantitatively study such changes in these cells. Therefore, to analyze the TSHR forms in the raft and nonraft fractions, we examined them under nonreducing conditions. As indicated in Fig. 2AGo, multimeric TSHRs resided exclusively in the raft fraction in the steady state, but the monomers were distributed in both raft and nonraft preparations. On TSH treatment there was an exit of the raft-associated multimeric receptors with a concomitant increase in monomers in the nonraft fraction (Fig. 2Go, A, lower panels, and B). These multimeric forms in the raft fractions were not observed under reduced conditions, indicating that these are not protein aggregations. However, the monomeric holoreceptor, corresponding to a 100-kDa protein, and cleaved ß-subunits of 37 kDa were both observed in the raft fractions (Fig. 4AGo).


Figure 4
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  		FIG. 4. The cleaved form of the TSHR resides in lipid rafts as seen under reduced conditions. A, In the reduced steady state, the raft fraction 1 showed the 100-kDa holoreceptor seen in CHO-TSHR cells and a 37-kDa cleaved ß-subunit. No multimeric forms were seen in the steady state under reduced conditions, suggesting that the multimers are not just protein aggregates. After TSH stimulation only a small amount of the cleaved ß-subunit was still observed in the raft fraction. B, Data derived from nonreduced gels (not illustrated) as mean of duplicate densitometric data of multimeric form derived from time- and dose-course experiments illustrating the responses of TSHR multimers in raft fraction 1. These are representative data of two experiments.

 
The decrease of multimers from the rafts was observed within 10 min of treatment, and this dramatic decrease was observed even with 100 µU/ml of TSH as shown by the densitometric data in Fig. 4BGo from time- and dose-course experiments derived from immunoblot experiments not illustrated here (data not shown). Thus, the decrease of multimers and monomeric TSHRs in the raft fraction indicated dynamic receptor movement on TSH binding as previously shown by our fluorescence recovery after photo bleaching studies (11).

Effect of TSHR stimulating antibody (MS-1) on TSHR receptor oligomers
Using 5 µg/ml of MS-1 (approximately equal in activity to 30 µU/ml of TSH) for 2 h at 37 C, the multimers decreased in the raft fractions as previously observed with TSH (Fig. 5Go). Interestingly, a fraction of the receptors were still held in dimeric forms (molecular mass corresponds to 200 kDa) in the nonraft fraction, which was not seen by TSH.


Figure 5
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  		FIG. 5. Effect of thyroid stimulating antibody (MS-1) on raft residing TSHR multimers. Multimeric TSHRs again existed in rafts in the steady state (left panel), but only minimal evidence for them was found in the nonraft fractions (right panel) when examined under nonreducing conditions. B, On treatment with MS-1 (~5 µg/ml) at 37 C for 2 h, the multimers, and many monomeric forms, were no longer seen in the raft fraction. Some dimeric forms (~200 kDa) of the TSHR remained intact in the nonraft fraction after antibody exposure. This is a representative blot of two experiments.

 
TSHRs reside within GM1-enriched lipid rafts
Having confirmed the presence of multimeric and monomeric TSHR forms within lipid rafts in CHO-TSHR cells using a biochemical method, we further examined the presence of TSHRs using fluorescent probes. Labeled CTxB Alexa555, which binds to GM1 rafts, and anti-TSHR biotin (biotinylated M1 antibody) for the TSHR detection were used. The GM1 distribution was ascertained by binding the labeled cholera toxin B subunit to the same cells and cross-linking with anticholera toxin to yield aggregated structures of raft, which could be resolved using the fluorescent microscope. The colocalization (Fig. 6AGo, panel 3) indicated that the TSHRs resided constitutively in lipid rafts. The calculated positive correlation coefficient of the double-labeled cells strongly suggested that TSHRs resided in the GM1-enriched lipid rafts. The GM1 raft staining was further confirmed by using GPI-TSHR ectodomain-expressing cells stained by this same method (Fig. 6BGo, panel 3). This showed a more extensive and pronounced double labeling due to the fact that GPI proteins are targeted and enriched in lipid microdomains (47, 48).


Figure 6
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  		FIG. 6. Localization of wild-type TSHRs and GPI-TSHR ectodomain constructs in GM1-enriched lipid rafts. A, Lipid rafts in CHO-TSHR cells were labeled using CtxB Alexa 555 and TSHRs were labeled using biotinylated anti-TSHR (M1, 1 µg/ml) antibody and detected using streptavidin fluorescein isothiocyanate. The nucleus was stained by 4',6'-diamidino-2-phenylindole (DAPI). Panel 1, Raft staining using CtxB Alexa 555 followed by anti-CtxB (1:100) treatment (arrow). Panel 2, TSHR staining using M1 (arrowhead). Panel 3, Colocalized signal of rafts and receptors together. Panel 4, Colocalized red-green signal quantitated using Image Pro plus software (Rr = 0.556). Panels 5 and 6, As controls, colocalization was measured just on the background (Rr = 9999.00) (panel 5) and from a cell showing no colocalization on panel 3 (Rr = 0.082) (panel 6). B, CHO cells stably expressing the GPI-linked TSHR ectodomain were labeled using CtxB Alexa 555 followed by anti-CtxB treatment, and TSHRs were labeled using biotinylated TSHR-specific antibody M1 as described above. Panel 1, Raft staining using CtxB Alexa 555. Panel 2, TSHR staining by M1. Panel 3, Colocalized signal for rafts and receptors together. Panel 4, Measurement of colocalization signal from a randomly selected cell (marked by asterisk in panel 3) (Rr = 0.614).

 
Association of Gs{alpha} protein with receptor-raft fractions
Because the study of Costa et al. (40) demonstrated the partitioning of Gs{alpha} in both raft and nonraft fractions and Gs{alpha} is a major effector of TSHR signaling, we also analyzed the presence of Gs{alpha} in the raft fractions by immunoblotting. As indicated on the Western blot of the raft and nonraft fractions (Fig. 7AGo), we observed the predicted 45-kDa Gs{alpha} protein in both the raft fractions and nonraft fractions, although in much smaller amounts in the raft fraction. Furthermore, to ascertain whether the Gs{alpha} was complexed with receptor in the raft and nonraft fractions or merely existing in these compartments, we immunoprecipitated both fractions with anti-Gs{alpha}, and the immunoblot was probed with anti-TSHR (M1) antibody under reduced conditions (Fig. 7BGo). TSHRs were observed in both raft and nonraft fractions. Hence, Gs{alpha} was associated with the TSHR throughout the membrane environment in the steady state. However, after TSH stimulation, there was no precipitation of receptors in the raft fraction, whereas the 100-kDa holoreceptor was precipitated from the nonraft fraction, again suggesting exit/decrease from the rafts on ligand exposure.


Figure 7
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  		FIG. 7. Association of G proteins with raft-residing TSHRs. A, Raft and nonraft fractions were analyzed by Western blotting for the presence of Gs{alpha} using 1:1000 dilution of anti Gs{alpha} antibody. The 45-kDa Gs{alpha} protein was found in both raft and nonraft fractions. A nonspecific band of 70 kDa was also observed in the nonraft fractions. B, Coimmunoprecipitation was then performed to detect TSHRs in raft and nonraft fractions, which were bound with Gs{alpha}. TSH (103 µU/ml) and non-TSH-treated fractions were first precipitated using anti-Gs{alpha} (2 µg/ml), and then the immunoprecipitate was probed for TSHR using M1 antibody (2 µg/ml), which was detected using antimouse conjugated to horseradish peroxidase (1:5000) followed by enhanced chemiluminescence. Again TSH holoreceptors were seen in both raft and nonraft fractions, providing evidence of previous Gs{alpha} binding, and these receptor forms exited the rafts on exposure to TSH as previously shown. Because this was a reduced gel, the TSHR ß-subunit was prominent in the raft fraction before TSH exposure but was less prominent in the nonraft fractions for unclear reasons. The TSHR ß-subunit was much reduced after TSH exposure probably due to endodomain degradation. C+, Negative control lane of CHO cell lysate with identical treatment for immunoprecipitation.

 
Effect of postreceptor modification on multimeric and monomeric TSHRs
Although TSH ligand modulated the distribution of TSHR forms in the cell membrane, it was unclear whether this was due to ligand binding itself or secondary to signal transduction as observed in our earlier report (10). Therefore, the postreceptor perturbation of the cell was examined in two ways.

1) Effect of forskolin on receptor multimers and monomers.
To ascertain whether the receptor movement from lipid rafts was influenced by postreceptor activation of the cells and changes in the cellular matrix rather than ligand binding, we stimulated CHO-TSHR cells with 10 µM of forskolin for 30 min at 37 C and then isolated the rafts. As indicated in Fig. 8Go, we observed that the TSHR multimers moved into the nonraft fraction after postreceptor signaling evidenced by cAMP generation. Although the multimers exited the rafts, they remained intact multimeric in the nonraft fractions, suggesting that they were incapable of dissociating into monomeric forms by this postreceptor stimulation unlike as seen with TSH or stimulating antibody. These data suggested that a structural change induced by the ligand was necessary for monomerization, and the raft environment supported such changes.


Figure 8
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  		FIG. 8. Effect of forskolin on raft-residing TSHR forms. CHO-TSHR cells were incubated with 10 mM forskolin in complete medium for 30 min at 37 C and washed twice with ice-cold base buffer and raft fractions prepared as described in Materials and Methods. A, Raft and nonraft fractions prepared from untreated cells under nonreduced conditions, illustrating multimeric and monomeric TSHR forms with the multimeric variety confined to the raft fraction. B, In contrast, after exposure to forskolin, there was an absence of multimeric TSHR forms in the raft fractions.

 
2) Effect of cholesterol depletion on TSHR forms.
MCD is known to alter the cytoskeleton structure of the cell by removing cholesterol and thus effective in disrupting lipid rafts. However, an earlier report suggested that disrupting rafts by this chemical method did not lead to loss of TSH-induced cAMP signaling in dog thyrocytes (40). To examine the effect of MCD on TSHR forms and their regulation, we treated cells for 30 min with 10 mM MCD in serum-free medium. Disruption at this concentration and for this length of time failed to remove all of the monomeric forms of TSHR from the lipid rafts (Fig. 9Go). However, the multimeric forms of the TSHR moved out into the nonraft fraction due to the perturbation of the cytoskeleton structure, but monomeric forms of the receptor remained in the rafts. To ascertain whether cholesterol removal by MCD and the pertuburation of receptors as observed above affected signaling, cAMP was measured in cells pretreated with 10 mM MCD for 30 min at 37 C. As seen in Fig. 10AGo, there was no significant difference in cAMP production between the MCD-treated cells and the untreated cells. This was true even with increasing doses of MCD up to 30 mM (Fig. 10BGo). These data suggested that TSHRs associated with lipid rafts were not altered sufficiently by MCD treatment to disrupt their signaling.


Figure 9
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  		FIG. 9. Effect of MCD on TSHR multimers. In an attempt to disrupt lipid rafts, CHO-TSHR cells were incubated with 10 mM MCD in complete medium for 30 min at 37 C, and rafts were prepared as described previously. A, Raft and nonraft fractions in untreated cells illustrating the TSHR forms seen earlier. B, After the raft and nonraft fractions were prepared from cells treated with MCD, there was a loss of multimeric TSHR forms in the raft fractions.

 

Figure 10
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  		FIG. 10. Effect of MCD on TSH-stimulated cAMP levels. A, As described in Materials and Methods, CHO-TSHR cells were preincubated in serum-free medium for 1 h followed by MCD treatment. After this pretreatment, the cells were stimulated with increasing concentrations of TSH (10–103 µU/ml) for 30 min at 37 C. The resulting stimulated cAMP levels were measured in the lysates using a direct cAMP EIA kit. There was no difference observed after MCD treatment. B, Increasing doses of MCD were then used and again followed by TSH (103 µU/ml) and forskolin (10 mM) treatment with cAMP measurement. Again there was no apparent difference in cAMP responses with MCD. F (B), Forskolin. C, This histogram shows the total cellular cholesterol after varying the doses of MCD as shown in B. These data demonstrate a failure to remove all the cholesterol, even with high concentrations of MCD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently oligomerization of GPCRs has been recognized as part of normal GPCR biology (49, 50, 51, 52, 53). The TSHR is no exception to this as we have shown by using biochemical and biophysical approaches (8, 9, 10, 12). Due to the additional complex posttranslational changes involving the TSHR, such as cleavage and oligomerization, the TSHR exists on the cell surface in multiple forms, many with the potential for signaling (2, 12, 54, 55, 56). For such signaling to be effective and specific, these receptor forms, like other related GPCRs, may be compartmentalized into lipid rafts within the plasma membrane. Lipid rafts are microdomains that are lateral heterogeneities in membranes of living cells and are enriched in sphingolipids, cholesterol, and other membrane proteins (16, 17, 18). These structures have been proposed to be responsible for sorting of proteins from the Golgi and acting as sites for the assembly of cytoplasmic signaling complexes in various biological contexts (18, 25, 41, 57).

Previously we have shown in TSHRGFP-transfected cells, using CtxB Alexa 594 and BodipyFL staining under confocal imaging, that TSHRs resided in GM1-rich typical lipid microdomains (39). Recently detergent-based isolation of caveolae-type lipid rafts failed to demonstrate TSHRs in this species of lipid raft (40). However, most investigators do not consider these as typical lipid rafts but rather as a specific subset (17, 18). Nevertheless, these observations prompted us to further analyze typical lipid rafts and to incorporate a detergent-free biochemical approach that has been found to produce more stable, and predictable, raft preparations. The intention of these studies was to further extend our observations to the characterization of the different forms of the receptors, which may colonize lipid rafts to begin to understand their physiology. We also examined the regulation of receptors within these lipid microdomains by TSH ligand and TSHR stimulating monoclonal antibody, MS-1 (43), and their association with Gs{alpha} to reveal the role of lipid rafts in TSHR signaling.

Several studies have recently shown that detergent isolation of lipid rafts may introduce biochemical changes such as complex associations between the membrane skeleton and the lipid bilayer (19, 46, 58). Furthermore, membrane fractions of variable composition can be generated by using a single detergent. Thus, the extraction patterns obtained may be investigator specific (42). Several additional observations have raised concern over extraction of cells with detergent. The procedure may generate clusters of raft lipids and proteins that did not exist in the intact cell. In fact, it has been pointed out that resistance to detergent solubilization might arise from thermodynamic or kinetic factors contributed by the detergent molecules themselves (59). Thus, the localization of all lipid rafts into one major fraction from dog thyrocytes, as shown by Costa et al. (40), may have been due to the clustering effect caused by detergent. In lieu of these complications associated with the detergent method of lipid raft preparation, there have been several recent methods described for isolating rafts from cells in the absence of detergent (16, 44, 60). By following a simplified biochemical method, we successfully isolated low- and high-density fractions and the protein and cholesterol profiles in these fractions followed the pattern of typical raft isolation (44). We were able to classify the fractions as raft and nonraft using antibody probes of raft and nonraft markers (Fig. 1Go).

Flotillin-2 was originally identified as an integral membrane protein resident in caveolin-enriched membrane domains (61). Whether the flotillins are resident in caveolae, noncaveolar lipid rafts, or both has not been determined by immunogold electron microscopy, but there is, however, compelling evidence that flotillin-2 is a resident of both caveolar and membrane lipid raft protein (45, 62). Both in detergent and detergent-free methods, flotillin-1 and -2 cofractionate with caveolin-1. Flotillin-2 has been shown by several investigators to be a specific lipid raft marker and is detected as a 42-kDa protein. In keeping with this, we found flotillin-2 only in the raft fraction 1 and very small amounts in fraction 2. However, we also observed that caveolin-1, which is the marker for caveolae, was present in all the fractions, although most highly enriched in the low-density fractions 1–4 (raft fractions). Such unrestricted distribution of caveolin has also been observed by several other investigators (32). Although the interrelationship between caveolae and rafts is still a source of confusion, caveolae are generally considered a subset of lipid rafts, which may also have other differences from typical rafts (63, 64). Thus, it would seem that due to very similar lipid composition, caveoles are found in the same subcellular fraction as other lipid rafts on density gradients. Our cells certainly expressed both types of rafts, as shown by our present biochemical isolation study and GM1 colocalization (Figs. 2AGo and 6AGo) as previously shown (39).

Having confirmed that TSHRs in these cells resided in typical lipid rafts, we further analyzed the raft and nonraft fractions prepared from cells subjected to TSH treatment. Interestingly in the steady state, we observed that TSHR multimeric forms preferentially localized to the raft fraction, whereas the 100-kDa monomeric form localized to both the raft and nonraft fraction under nonreducing conditions. This suggested, most likely, that either multimerization of the receptor controls its lipid raft partitioning or the lipid raft environment was more capable of supporting multimerization. However, other possibilities include selective trafficking of these multimerized receptors from endoplasmic reticulum to the cell surface via lipid raft targeting vesicles and the fact that the multimeric form may alter the functional palmitoylation state of the receptors and thus may target it to the lipid rafts. As yet there no direct proof to say that the putative palmitoylation site Cys699 on the TSHR (65) is involved in lipid raft targeting, although it known that this site is not crucial for cAMP or phosphoinositide signaling (40, 66). Although these possibilities are open to investigation, it has been shown that the partitioning of urokinase-type plasminogen activator receptor (uPAR/CD87) into lipid rafts is regulated by its dimerization (67). Induced clustering of different membrane proteins has also been shown to enhance their partitioning to lipid rafts (68, 69, 70). Furthermore, it has been suggested that dimerization and oligomerization of membrane proteins may have a fundamental regulatory function in their association with lipid rafts (18). Similarly, in the case of the LH receptor, it was observed that ligand bound oligomerized receptors translocated to lipid rafts (36). The translocation of these receptors into lipid rafts after ligand binding not only was essential for signaling but also the dimerized or oligomerized receptors stabilized in lipid rafts. It has also been shown that GPI-anchored proteins in polarized cells are transported apically and are stabilized in rafts by protein oligomerization, increasing their raft affinity (71). We and others (8, 9, 12) reported the TSHR to be constitutively oligomerized. Therefore, it seems logical that these constitutively oligomerized receptors should also be exclusively localized in lipid rafts. Alternatively the lipid raft environment may regulate the degradation and proteolysis of these multimeric forms.

Previous work from several laboratories has shown that the TSHR undergoes posttranslational cleavage, resulting in two subunits: an extracellular {alpha}-subunit and a membrane-spanning ß-subunit held together by disulfide bridges. This cleavage event occurs late in the synthesis process at the cell surface (2, 5, 54, 55). Although metzincin metal proteases, belonging to the adamalysin family of enzymes, have been suggested as involved in this process of cleavage, the exact protease or the mechanism of cleavage remains under investigation (54, 55, 72). Interestingly, we observed that under reduced conditions, the TSHRs, which were localized within the rafts, appeared to undergo ectodomain cleavage, resulting in the detection of 37-kDa ß-subunits (Figs. 4BGo and 7BGo). These data indicated that the protease responsible for cleavage may be not be restricted in its localization, unlike previous suggestions (73).

Cholesterol disruption using various chemical agents has been a standard method of disrupting lipid rafts to study functional loss (18). Lately it has been shown that there are several caveats to this approach because some protein disruption of cholesterol leads to a gain of function rather than a loss (74). Our data on methyl cyclodextrin treatment of CHO-TSHR cells (Fig. 8Go) showed this method to be unable to completely remove all of the monomeric forms of the TSHR from the raft fractions (maximum 50%), although the major fraction of multimers decreased in the rafts. Measurement of cAMP in the MCD-treated cells vs. the non-MCD-treated cells showed no significant disruption of cAMP signaling (Fig. 8Go), as observed by Costa et al. (40) in dog thyrocytes. These experiments suggested that TSHRs localized to lipid rafts were not sufficiently altered enough by MCD treatment to interfere with signaling. Hence, MCD is ineffective in disrupting TSHR-associated rafts and use of other agents like filipin and saponin may be more effective.

Recent reports show that G proteins, because of their acylated nature, have a tendency to localize in lipid rafts (29). It has also been show that protein modification, such as acylation and palmitoylation, further increases their tendency to localize in lipid rafts (18, 21). It is known that one TSHR signaling pathway is via G{alpha}s leading to the production of cAMP and activation of protein kinase A-dependent and -independent signaling. It is also known that a structural change in G{alpha}S occurs in the receptor complex after ligand binding and subsequent signal transduction (75, 76). Although G{alpha}S was found in both the raft and nonraft TSHR fractions by immunoblotting, we wanted to see whether these molecules were indeed associated with the receptor. Our coimmunoprecipitation studies showed that G{alpha}s was associated with the TSHR in both the raft and nonraft fractions but did not discriminate between signaling and nonsignaling structures (Fig. 7Go).

Oligomeric forms of protein are transported in lipid rafts to the plasma membrane (18, 25, 67, 71). A recent report discussed the role of oligomeric forms of mutant TSHR on the entrapment of wild-type TSHR by direct interaction of the two receptor molecules within the endoplasmic reticulum. This may function as a mechanism leading to TSH resistance in congenital hypothyroidism (13) and would suggest that in the wild-type situation, the oligomers formed intracellularly would be transported from the Golgi to fuse with the plasma membrane via lipid rafts. Hence, TSHR oligomers may be involved in cell pathology and their role in normal physiology requires further clarification.

Our current data suggest that oligomeric forms of the TSHR are preferentially localized in typical lipid microdomains on the plasma membrane. According to our current model, we propose (Fig. 11Go) that these oligomeric forms within the rafts are monomerized by TSH as previously shown (11). The monomerized receptors are capable of binding G{alpha}s. It is not yet clear whether these monomerized receptors encased in the rafts initiate the first wave of signal transduction via adenyl cyclase or are involved in other signaling pathways. This pattern may also be true for thyroid stimulating antibodies because these antibodies also move/decrease the receptors out of rafts. These mechanisms of action need further investigation.


Figure 11
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  		FIG. 11. A model of multimeric TSHR regulation at the cell surface. In the steady state, TSHRs first appear as oligomers and monomers in the lipid raft environment. On stimulation by TSH, or thyroid stimulating antibody, the multimers exit/decrease in the rafts. The TSHRs have GS{alpha} associated with them in both the compartments. The TSHR monomers then leave the nonraft domain by internalization for recycling or degradation. In addition, they may remain associated with Gs{alpha} at the cell surface and presumably remain in a desensitized state in the nonraft fraction after ligand exposure.

 


   Acknowledgments
 
We thank Zhong Yao for technical help and Drs. Syed Morshed, Xiaoming Yin, and Marcos Agote-Robertson for critical review of the manuscript. We also thank Dr. Linda J. Pike of Washington University School of Medicine (St. Louis, MO) for her helpful suggestions on lipid rafts.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK069713, DK052464, and VA Merit Award (to T.F.D.).

First Published Online April 5, 2007

Abbreviations: CHO, Chinese hamster ovary; CtxB, cholera toxin B subunit; EIA, enzyme immunoassay; GPCR, G protein-coupled receptor; GPI, glycophosphotidyl linked; MCD, methyl-ß-cyclodextrin; TSHR, TSH receptor.

Received November 27, 2006.

Accepted for publication March 26, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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