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BRIEF COMMUNICATION |
Division of Endocrinology, Diabetes and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Dr. R. Latif, Department of Medicine, Box 1055, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029-6574. E-mail: rauf.latif{at}mssm.edu.
Abstract
The TSH receptor (TSHR) is a prototypic G protein-coupled receptor with a large extracellular domain. We have previously demonstrated homophilic interactions of TSHRs and their existence as constitutive oligomers. However, we have also shown that TSH itself promotes the formation of receptor monomers. We hypothesized, therefore, that TSHR monomers induced by TSH ligand may move into lipid rafts before effective TSH-induced signaling by bringing the cognate signaling molecules resident in such rafts together with the TSHRs. Thus, we aimed to determine whether the TSHRs would partition into these lipid rafts. The B subunit of cholera toxin (CTxB) binds to lipid raft-enriched GM1 ganglioside and has been widely exploited to visualize lipid rafts. Using such a method, we demonstrated the presence of these GM1-enriched lipid microdomains in Chinese hamster ovary cells by using CTxB labeled with a red dye (Alexa 594). To provide evidence for the presence of TSHRs in lipid rafts, we stained Chinese hamster ovary cells expressing TSHRGFP with labeled CTxB. Our results demonstrated that the TSHRGFP complexes localized to GM1-enriched lipid raft microdomains as evidenced by colocalization of the green fluorescent protein tag with the labeled CTxB. Hence, we concluded that a significant proportion of TSHRs were constitutively associated with lipid rafts. Furthermore, upon activation of these stained raft-receptor complexes with increasing concentrations of TSH, we observed that the raft-receptor complexes decreased significantly. The relevance of such receptor movement out of the rafts suggested that these may be the receptors critical in the initiation of signal transduction
THE TSH RECEPTOR (TSHR) is a G protein-coupled receptor expressed on the plasma membrane of thyrocytes and a variety of other cells (1, 2) The TSHR undergoes complex posttranslational processing distinct from other glycoprotein hormone receptors. This includes intramolecular cleavage of the receptor into two subunits followed by shedding of the extracellular 
(or A) subunit (3, 4) after reduction of disulfide bonds (5, 6, 7) between the extracellular domain and the membrane anchored ß (or B) subunit, a process that is unique to this receptor. Another important part of the posttranslational processing of TSHR involves multimerization. In thyroid tissue preparations, as well as in transfected cells, we have demonstrated the presence of multimeric TSHRs (8, 9), and, recently, we have shown that these TSHR multimers dissociated into monomeric forms in response to TSH stimulation (10) We, therefore, hypothesized that the purpose of such monomer formation was to move these dissociated receptors into lipid rafts so that receptor signaling could be initiated (11). We now know that the plasma membrane is a highly organized and dynamic structure. Preferential clustering of sphingolipids and cholesterol on the plasma membrane forms ordered domains known as lipid rafts (12, 13). The existence of lipid rafts in cells has been demonstrated by using biochemical approaches such as density gradient ultracentrifugation or by using biophysical methods such as visualization with fluorescent-labeled lipid raft markers including the B subunit of cholera toxin (CTxB), which binds to the ganglioside GM1 (14). Lipid rafts have been implicated in signal transduction, membrane trafficking, internalization, and other functions initiated at the plasma membrane (13, 15, 16, 17, 18, 19) through their ability to concentrate or exclude proteins and lipid mediators. Signaling molecules found in lipid rafts include dually acylated src family tyrosine kinases, heterotrimeric G protein subunits, adaptor proteins, phosphatidylinositol-3,4-bisphosphate, and lipid kinases and phosphates (12, 15, 16). It has also been indicated recently that cell membranes may have a series of raft subtypes on the basis of differential detergent solubility or ganglioside composition (20, 21). Thus, movement of receptors into, or out of, lipid rafts is a critical event in the initiation and propagation of multiple cellular pathways.
In this study, we have used a fluorescent microscopic approach to 1) visualize the presence of lipid rafts in Chinese hamster ovary (CHO) cells, 2) demonstrate the presence and membrane distribution of TSHRs in lipid rafts using cells expressing TSHRGFP, and 3) study the influence of TSH on these raft-receptor complexes by time-lapse imaging of TSHRGFP cells.
Materials and Methods
Staining of CHO cells with labeled CTxB
CHO cells transfected with an empty vector (JPO2, kindly provided by Dr. G. Vassart) were used for examining the presence of lipid rafts in CHO cells. Cells (0.3 x 106) were seeded in a four-well chamber slide (LAB-TEK brand, Nalge Nunc Inc., Naperville, IL) and incubated overnight in 1 ml of Ham’s F-12 (Mediatech, Inc., Herndon, VA) complete medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin at 37 C with 5% CO2. Before staining, the cells were incubated for 10 min on ice and then washed twice with ice-cold PBS (pH 7.4) and incubated for another 5 min on ice with 0.1% BSA-PBS buffer. After gently aspirating the buffer, the cells were incubated with 1 µg/ml of BODIPY-FL (Molecular Probes, Inc., Eugene, OR) in 0.1% BSA-PBS buffer for 20 min at 4 C. BODIPY-FL is a GM1 analog labeled with a green dye. At the end of this incubation, unbound BODIPY-FL was washed out with ice-cold PBS and immediately followed with 2 µg/ml of CTxB Alexa 594 (CTxB conjugated to Alexa 594, Molecular Probes, Inc.) in 0.1% BSA-PBS for another 20 min at 4 C. Finally, after several washings with cold PBS, the cells were mounted on a non-glycerol-based mounting fluid (Slow Fade; Molecular Probes Inc.) and observed under immunofluorescence microscope using fluorescein isothiocyanate filter for BODIPY-FL/green fluorescent protein (GFP) and Texas Red filter for CTxB Alexa 594. Digital images were obtained using a SPOT RT charge-coupled device camera (Diagnostics, Inc., Sterling Heights, MI) attached to a TE 2000 microscope (Nikon, Melville, NY). The individual images obtained in each filter set were overlaid using Adobe Photoshop 5.5. To demonstrate the aggregation of rafts, we used anti-CTxB (Calbiochem, La Jolla, CA) at 1:100 and incubated the cells for for 20 min at room temperature after staining for rafts using CTxB Alexa. The cells were mounted and observed with oil immersion objective (x100).
Live cell microscopy
The effect of TSH on receptor-raft interaction was studied by live cell imaging. These studies were performed using TSHRGFP cells generated by transfecting CHO cells with TSHR-tagged with GFP at its carboxyl terminus as described in our previous report (9). Briefly, 0.5 x 106 cells per dish of CHO TSHRGFP, seeded in Ham’s F12 complete medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and streptomycin, were adhered to Delta T glass dishes (Bioptechs, Butler, PA) by incubating at 37 C overnight. Before treatment with TSH, TSHRGFP cells were stained for lipid rafts with labeled CTxB. Increasing concentrations of bovine TSH (Sigma, St. Louis, MO) were added in 500 µl of medium to the same dish, mixed by gentle pipetting, and time-lapse imaging was continued after 5 min of incubation. Images were sequentially acquired every 60 sec for a period of 3 min. Time-lapse imaging of the treated cells was performed using a laser scanning confocal microscope (Microscopy Core Facility, Mount Sinai School of Medicine, New York, NY) on a temperature-controlled stage with a x63 oil objective with a numerical aperture (NA) of 1.32.
Results
Lipid rafts in CHO cells
Before examining the presence of TSH receptors in lipid rafts, it was essential to study the localization and distribution of lipid rafts in CHO cells. For this purpose, we made use of the CTxB that binds to the GM1 ganglioside. To further demonstrate that CTxB bound to GMI ganglioside, we used a GM1 analog directly labeled with a green fluorescent dye (BODIPY-FL) labeled with fluorescein isothiocyanate on the acylsphingosine portion of the molecule, which did not interfere with the binding of CTxB to GM1. Therefore, using CTxB Alexa 594 (red dye; Fig. 1B
), we were able to demonstrate the presence of GM1-enriched lipid rafts in the CHO cell plasma membranes. However, coincident staining patterns of CTxB Alexa 594 and BODIPY-FL (Fig. 1C) obtained using BODIPY-FL (Fig. 1A) and CTxB (Fig. 1B) further confirmed the localization of rafts. Figure 1C is a merged image showing a uniform orange to yellow staining on the surface of the cell due to the colocalization of green BODIPY-FL with the red CTxB Alexa 594 stains. After treatment with anti-CTxB (1:100), which would result in the cross-linking of rafts leading to their aggregation on the surface (22), we observed characteristically punctate and random distribution of these microdomains across the plasma membrane (Fig. 1E).
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). Thus, using the fluorescent probe-labeled CTxB on TSHRGFP cells, we were able to demonstrate the presence of tagged TSHRs in rafts by the colocalization observed between the TSHRGFP and lipid rafts stained with CTxB Alexa 594 (Fig. 2C). This indicated constitutive localization of TSHRs to lipid rafts in these cells.
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, in the absence of TSH (Fig. 3A), there was constitutive localization of the receptor molecules to the rafts. However, these raft-receptor complexes disappeared from the surface of the cells after exposure to at least 100 µU/ml of TSH (Fig. 3C), although the cells continued to show the presence of remaining tagged TSHRs, which were non raft-associated residual receptors on the cell surface.
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Previous reports studying the composition of the TSHR have suggested that, besides the major glycoprotein component, solubilized wild-type TSHRs also contained a ganglioside component belonging to the GM1 family, found in lipid rafts, that may be of considerable functional significance (23, 24, 25). These ganglioside components have also been reported to be an integral part of the purified TSHR that interact with the TSHR -subunits (26). In our present study, we demonstrated that TSHRs were actually present in lipid rafts. Having visualized, microscopically, the constitutive raft-receptor complexes in these cells, we also examined their response to TSH ligand. Due to the importance of lipid rafts as a hub for cellular signaling and protein trafficking (12, 13), it is likely that understanding the organization of TSHRs in these membrane microdomains is critical for TSH and TSHR autoantibody-induced intracellular signaling and other posttranslational modifications unique to this receptor.
The coalescence of lipid rafts and entry, or exit, of crucial signaling molecules into these microdomains upon ligand binding suggests that lipid rafts may serve as a site for signaling and other posttranslational events following receptor activation. Our observations using time-lapse imaging of rafts with tagged TSH receptors exposed to TSH indicated the disappearance of raft-receptor complexes from the surface of the cells after exposure to TSH, and this phenomenon requires explanation. First, the data suggested that a large fraction of the raft-receptor complexes were subjected to TSH regulation, and, second, our original hypothesis (11) that TSH would induce monomeric TSHR entry into lipid rafts was proven to be false. At the least we could conclude that, if TSH did indeed induce some monomeric TSHRs (10) to enter lipid rafts, its effects on TSHR exit from these domains was dominant. More likely, it would seem that monomer formation may actually occur within the lipid raft domains allowing their rapid exit.
In summary, our microscopic imaging experiments have shown that TSHRs are constitutively localized to lipid rafts. The role of TSHRs in these rafts awaits further investigation, but, as with other receptors, it is likely to be the lipid raft-associated TSHRs that initiate signal transduction and cell activation.
Acknowledgments
We thank Dr. Scott Henderson for assistance with the confocal laser scanning microscopy, which was performed at the Mount Sinai School of Medicine Microscopy Center with funding sources listed below. We also thank Dr. Reigh-Yi Lin for reviewing the manuscript.
Footnotes
This work was supported in part by NIH Grants DK52464, DK-45011, and AI-24671 (to T.F.D.) and NIH shared instrumentation Grant 1S10RR9145-01 and National Science Foundation major research instrumentation Grant DBI-9724504.
Abbreviations: CHO, Chinese hamster ovary; CTxB, cholera toxin B subunit; GFP, green fluorescent protein; TSHR, TSH receptor.
Received July 24, 2003.
Accepted for publication August 26, 2003.
References
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