Thyrotropin-Releasing Hormone Receptor Processing: Role of Ubiquitination and Proteasomal Degradation

Laurie B. Cook, Chang-Cheng Zhu and Patricia M. Hinkle

Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642

Address all correspondence and requests for reprints to: Dr. Patricia M. Hinkle, Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642. E-mail: patricia_hinkle{at}urmc.rochester.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
These studies were designed to characterize ubiquitination of the G protein-coupled TRH receptor (TRHR). TRHRs and ubiquitin coprecipitated with antibodies to either receptor or ubiquitin in Chinese hamster ovary or pituitary GHFT cells. Inhibition of the proteasome with MG-132 resulted in an accumulation of total TRHRs and the appearance of a small amount of cytosolic receptor. MG-132 caused an increase in newly synthesized receptors, detected by microscopy using a TRHR coupled to Timer, a DsRed that undergoes a spontaneous time-dependent color change. Misfolded TRHRs were particularly heavily ubiquitinated. These results show that the proteasome participates in TRHR quality control early after receptor synthesis. Under normal circumstances, most ubiquitinated TRHRs were absorbed to wheat germ agglutinin, indicating that they had undergone complex glycosylation in the Golgi apparatus. When cells were treated with tunicamycin to block glycosylation, a ladder of ubiquitinated species was detectable. Cell surface receptors, which were labeled selectively with either radioligand or antibody, showed no detectable ubiquitin modification. To determine if ubiqutination plays a role in TRH-induced receptor endocytosis, the receptor was expressed in Ts20 cells, which have a temperature-sensitive ubiquitin pathway. TRH induced a significant calcium response and rapid and extensive receptor internalization at both the permissive and nonpermissive temperatures, indicating that ligand-dependent ubiquitination of the receptor, or any other protein, is not necessary for TRHR signaling or internalization. These results show that ubiquitin modification targets misfolded receptors for degradation and suggest a possible role for ubiquitination in receptor trafficking.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
G PROTEIN-COUPLED RECEPTORS (GPCRs) are a large superfamily of transmembrane proteins involved in transmitting a wide variety of signals across the plasma membrane. GPCR targeting and signaling are influenced by posttranslational receptor modifications including glycosylation, phosphorylation, and palmitoylation. Recently, ubiquitination of a few GPCRs has also been reported, but the function of receptor ubiquitination remains uncertain. Ubiquitin, a 76-amino acid protein that has been highly evolutionarily conserved, is covalently attached to lysine {epsilon}-amino groups of target proteins as a single unit or as a chain of ubiquitin monomers (1, 2, 3). Classically, ubiquitination was identified as a pathway for degradation of short-lived cytosolic and nuclear proteins. Ubiquitination was subsequently shown to direct the proteasomal degradation of misfolded intralumenal and transmembrane proteins from the endoplasmic reticulum (ER), signal internalization of several plasma membrane receptors, target proteins for lysosomal degradation, and signal transport into multivesicular bodies (4, 5, 6, 7).

It has been proposed that ubiquitination of GPCRs can serve two different functions, regulation of receptor degradation and regulation of receptor internalization. For example, in mammalian cells, regulation of GPCR degradation by ubiquitin has been described for {delta}-opioid receptors and ß2-adrenergic receptors; {delta}-opioid receptors are degraded by the proteasome, but ß2-adrenergic receptors are degraded by the lysosome (5, 8). In yeast cells, a very different function of ubiquitination has been described. The mating factor receptor is ubiquitinated in response to pheromone binding, triggering its internalization and degradation by the vacuole, a yeast equivalent of the lysosome (9, 10). It is not yet known whether ubiquitin functions as an internalization signal for mammalian GPCRs.

The TRH receptor (TRHR) is a mammalian Gq-coupled receptor involved in controlling the secretion of thyrotropin from the anterior pituitary gland. Tight regulation of the TRHR signal is necessary for proper thyroid function. It is well established that the TRHR undergoes ligand-induced phosphorylation and internalization via clathrin-coated pits within minutes after exposure to TRH (11, 12), but TRHRs are not known to be ubiquitinated. In this study, we demonstrate that TRHRs are ubiquitinated and characterize the role of ubiquitination in receptor quality control and trafficking.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ubiquitination of TRHRs
To establish whether TRHRs are ubiquitinated, we expressed Flag-TRHRs and hemagglutinin (HA)-ubiquitin in Chinese hamster ovary (CHO) cells and immunoprecipitated with anti-Flag and anti-HA antibody. Ubiquitinated TRHRs were detectable in both anti-HA immunoprecipitates and anti-Flag immunoprecipitates (Fig. 1Go, A and B), suggesting that the TRHR was ubiquitinated. TRHRs ran as several broad bands. Endoglycosidase digestion showed that these bands represent nonglycosylated monomer, core-glycosylated monomer, and glycosylated receptor monomer and dimer (Ref.13 and data not shown) (Fig. 1AGo, arrowheads, bottom to top). The ubiquitinated TRHR ran primarily as a high molecular weight smear, shown by the brackets, although lower molecular weight bands corresponding to monomeric receptor species were also detected (Fig. 1AGo, lane 4). No HA staining was seen after immunoprecipitation with anti-Flag antibody unless a Flag-tagged TRHR was expressed, and conversely no Flag staining was seen after precipitation with anti-HA antibody unless HA-tagged ubiquitin was expressed [Figs. 1EGo (left lanes) and 2 and data not shown].



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Fig. 1. Coprecipitation of HA-Tagged Ubiquitin and Flag-Tagged TRHRs

CHO cells were transiently transfected with or without Flag-tagged TRHR and HA-ubiquitin (HA-Ub), as indicated. The following day, cell lysates were prepared. A, B, D, and E, Lysates were immunoprecipitated with anti-Flag or anti-HA antibody. A, B, and D, Immunoprecipitation was performed as described in Materials and Methods. C, Lysates were run directly on SDS-PAGE. E, Cells were solubilized in 100 µl RIPA buffer containing 1% SDS and boiled for 2 min, then diluted to 1 ml with buffer lacking SDS and immunoprecipitated. Immunoblotting was performed using: anti-Flag antibody (A and E), anti-HA antibody (B) or antibody to native ubiquitin (Ub) (C and D). The doublet seen near the 85-kDa molecular mass marker in panel D and several other gels appears to be nonspecific. Molecular mass markers are shown on the sides of gels and IgG denotes immunoglobulin heavy chain. Arrowheads show major TRHR species and brackets ubiquitinated receptors.

 
To rule out the possibility that TRHR ubiquitination was the result of ubiquitin overexpression, we repeated the experiment but performed Western blots with antibodies to native ubiquitin. A high molecular weight smear, representing ubiquitinated TRHR, was observed in cells expressing receptor alone as well as those coexpressing HA-ubiquitin (Fig. 1DGo). We also probed cell lysates with antibody against native ubiquitin (Fig. 1CGo) and found that the overall intensity of ubiquitinated protein bands was only slightly higher in cells that had been transfected with HA-ubiquitin, based on densitometry. Because of this evidence that transfection with HA-ubiquitin causes only modest overexpression, most experiments were performed using HA-ubiquitin transfection so that ubiquitinated TRHRs could be readily immunoprecipitated.

To eliminate the possibility that TRHRs were not themselves ubiquitinated but were bound to a ubiquitinated protein, we lysed cells in buffer containing 1% sodium dodecyl sulfate (SDS) and boiled before immunoprecipitation (Fig. 1EGo). After these strongly denaturing conditions, ubiquitin-labeled protein was still found in TRHR immunoprecipitates, suggesting that ubiquitin is covalently attached to the receptor.

Ubiquitination of TRHRs in Pituitary Cells
It was not possible to study ubiquitination of endogenous TRHRs in pituitary cells because of the lack of sufficiently specific antibodies. Instead, we transfected Flag-TRHRs and HA-ubiquitin into pituitary GHFT cells, prelactotrophs that have been immortalized by the targeted expression of T antigen (14). Although the overall expression level of TRHRs was very low in these transient transfection experiments, less than 0.05 pmol/mg protein, approximately 10% of the receptor coprecipitated with antibodies to HA-ubiquitin (Fig. 2Go).



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Fig. 2. Ubiquitination of TRHRs in Pituitary Cells

GHFT pituitary cells were transiently transfected with Flag-TRHR, HA-Ub, or both. After 48 h, cell lysates were immunoprecipitated with anti-Flag or anti-HA antibody and immunoprecipitates were run on SDS-PAGE and analyzed by immunoblotting with either anti-Flag (A) or anti-HA (B) antibody. Arrowheads denote major TRHR species and the brackets the major ubiquitinated receptor forms.

 
Effect of Proteasome Inhibition
The importance of proteasomal degradation was assessed by treating CHO cells stably expressing HA-tagged TRHRs with or without the proteasome inhibitor MG132 for 24 h. This stable cell line expressed between 0.5 and 1.5 pmol TRHR/mg protein, similar to the concentration reported for the endogenous receptor in pituitary cells (15). Total receptor concentration in cell lysates was determined by Western blotting and plasma membrane receptor concentration was quantified by binding [3H]MeTRH to intact cells at 0 C.

MG132 caused a slight decrease in [3H]MeTRH binding (Fig. 3AGo), but an increase in total receptor (Fig. 3BGo). To determine where ubiquitinated receptors accumulate, cells were treated with or without 50 µM MG132 for 3 h and fractionated to separate membrane and cytosolic fractions. Proteins were solubilized with detergent and immunoprecipitated with antibody to receptor, then run on SDS-PAGE and immunoblotted with antibody to either HA, for total receptor (Fig. 3DGo, left panel), or with antibody to native ubiquitin, for ubiquitinated receptor (Fig. 3DGo, right panel). Fractions were also probed with antibodies against markers for the ER [calnexin, inositol (1, 4, 5) trisphosphate (IP3) receptor], Golgi apparatus (ß-COP), and plasma membrane (plasma membrane calcium ATPase) (Fig. 3CGo). Most receptor was found in the 10,000 x g membrane pellet, which includes plasma membrane and some ER. MG132 again caused an accumulation of total receptor protein, increasing both the receptor monomer and dimer bands. In MG132-treated cells, ubiquitinated receptor accumulated particularly in the 100,000 x g microsomal pellet, which contains ER and Golgi membranes (Fig. 3DGo, right panel). In the presence of the proteasome inhibitor, some immature receptor was detectable in the cytosolic fraction, and this receptor was ubiquitinated (Fig. 3DGo, asterisks). No cytosolic receptor was isolated in the absence of MG132.



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Fig. 3. Effect of Proteasome Inhibition on TRHR Content

CHO cells stably expressing HA-TRHRs were treated as follows. A, Dishes were incubated for 2 h with 50 µM MG132 or vehicle; 5 nM [3H]MeTRH, with or without excess unlabeled TRH, was then added in the continued presence of MG132. Incubation was continued for 1 h and specific binding of [3H]MeTRH and total protein per dish were measured. Mean ± SE of triplicates is shown. B–D, Cells were incubated with or without 50 µM MG132 or vehicle for 3 h and then harvested and fractionated as described in Materials and Methods. B, Cell lysates were run on SDS-PAGE and blotted with anti-HA antibody. C, Fractions of untreated cultures were run on SDS-PAGE and blotted for ER (IP3 receptor, calnexin), Golgi (ß-COP), or plasma membrane plasma membrane calcium ATPase (PMCA) markers. D, Fractions were immunoprecipitated with anti-HA antibody and immunoblotted with antibody to HA or native ubiquitin. Arrowheads show receptor monomer, dimer, and two faster migrating bands. The asterisk denotes cytosolic receptor and the bracket higher molecular weight ubiquitinated receptor species.

 
Localization of a Timer-TRHR
We took advantage of the DsRed1-E5 "Timer" protein to determine where newly synthesized and older receptors accumulate after proteasome inhibition. DsRed1-E5 (16) fluoresces in the green region shortly after synthesis but over the course of 12 h undergoes a rearrangement such that the reporter fluoresces in the red region. The DsRed1-E5 protein was fused to the C terminus of the TRHR and the fusion, TRHR-Timer, was stably expressed in HEK293 cells. TRHR-Timer was well localized to the plasma membrane (Fig. 4Go) and expressed at 1–2 pmol/mg protein based on radioligand binding (data not shown). When cells were treated with a high dose of MG-132 (50 µM) for 2 h, there was increased accumulation of green fluorescence inside the cells, representing newly synthesized protein. This is consistent with the ER and cytoplasmic localization suggested by the biochemical fractionation studies. After an overnight treatment with 10 µg/ml cycloheximide, a protein synthesis inhibitor, [3H]Leu incorporation into TCA-insoluble material was inhibited by over 95% (data not shown), and there was an increase in the proportion of red fluorescence, as expected.



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Fig. 4. Effect of Proteasome Inhibition on TRHR Localization

HEK293 cells were stably transfected with TRHRs tagged at the C terminus with DsRed1-E5, a fluorescent timer protein (TRHR-Timer). Cells were treated with or without 50 µM MG-132 for 2 h or with 10 µg/ml cycloheximide overnight before fluorescence microscopy. Left panels, Green fluorescence (representing newly synthesized protein) measured with a standard 480/510 nm FITC filter. Right panels, Red fluorescence (representing older protein) measured with a standard 530/585 nm TRITC filter. Green and red fluorescence were measured using the same settings and exposure times in each pair. Exposure times were shorter for MG132-treated cells (middle panels) because otherwise the green fluorescence was too bright. MG-132 caused some increase in green fluorescence in control HEK293 cells but no red fluorescence. This fluorescence due to MG132 was much less than that seen in cells expressing TRHR-Timer, and much of the green fluorescence in MG132-treated cells expressing TRHR-Timer changed to red over time.

 
Role of the TRHR Carboxyl Terminus
Ubiquitin is added to the cytoplasmic carboxyl terminus of several membrane receptors, and this region of the TRHR contains multiple lysine residues that could serve as ubiquitination sites. To test the importance of the carboxyl terminus for receptor ubiquitination, we coexpressed either full-length Flag-TRHR, Flag-TRHRs truncated at L334 ({Delta}335–412), which lack seven of nine lysine residues in the cytoplasmic tail, or Flag-TRHRs truncated at M323 ({Delta}324–412), which lack all nine lysines in the cytoplasmic tail, together with HA-ubiquitin. Cell lysates were immunoprecipitated with anti-Flag or anti-HA antibody and immunoblotted with anti-Flag antibody. Cell lysates contained nonglycosylated monomers and dimers as well as other high molecular weight species that probably represent glycosylated forms and higher oligomers (Fig. 5BGo). All three receptors coimmunoprecipitated with the HA antibody, indicating that they are ubiquitinated. In fact, the {Delta}324–412 receptor was more highly ubiquitinated than {Delta}335–412 and full-length receptors (Fig. 5AGo).



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Fig. 5. Ubiquitination of C-Terminally Truncated TRHRs

CHO cells were transiently transfected with HA-Ub together with full-length Flag-TRHR, {Delta}335–412Flag-TRHR or {Delta}324–412Flag-TRHR. A, Cell lysates were immunoprecipitated with anti-Flag or anti-HA antibody. Left, Equal fractions of the anti-Flag immunoprecipitates (5 µl) were analyzed via Western blot with anti-Flag antibody to verify receptor expression. Right, After immunoprecipitation with antibody to HA-ubiquitin, 20 µl samples were immunoblotted with anti-Flag antibody. B, Cell lysates were run directly and immunoblotted. C, Specific binding of 5 nM [3H]MeTRH to intact cells transfected with the different receptor constructs was measured at 37 C for 1 h. The mean ± range of duplicate dishes is shown. Cultures expressing full-length receptors bound 16048 ± 476 cpm.

 
Cells expressing the {Delta}335–412 TRHR bound 40% as much [3H]MeTRH as the full-length receptor, but those expressing the {Delta}324–412 receptor bound less than 1% as much (Fig. 5CGo). To determine whether the lack of [3H]MeTRH binding to the {Delta}324–412 receptor was due to an inability of the receptor to bind ligand or a loss of surface localization, cells expressing full-length, {Delta}335–412 or {Delta}324–412 Flag-tagged TRHRs were immunostained with anti-Flag antibody both in the presence and absence of detergent. When nonpermeabilized cells were immunostained, full-length and {Delta}335–412 receptors were visible at the plasma membrane, but {Delta}324–412 receptors were not detectable (Fig. 6Go, left panels). In permeabilized cells, all three of the receptors were visible intracellularly. Cells were cotransfected with tagged TRHRs together with markers for either the Golgi apparatus or ER and then permeabilized and immunostained (Fig. 6Go). The wild-type receptor can be seen both at the plasma membrane and colocalizing with ER and Golgi markers, consistent with receptor being synthesized and processed after transient transfection. The {Delta}335–412 TRHR was found primarily in ER and Golgi, and the fully truncated {Delta}324–412 TRHR was only visible intracellularly in the ER. After solubilization with N-dodecylmaltoside, {Delta}324–412 TRHRs showed no measurable [3H]MeTRH binding (data not shown), indicating that this receptor is nonfunctional as well as mislocalized.



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Fig. 6. Localization of C-Terminally Truncated TRHRs

CHO cells were transiently transfected with full length, {Delta}335–412Flag-TRHR or {Delta}324–412Flag-TRHR. Some cultures were cotransfected with DsRed-GRASP, a Golgi marker, or with YC4er, and ER marker. Cells were either not permeabilized (left panels only) or permeabilized, and then immunostained for receptors using anti-Flag primary antibody and either fluorescein-labeled secondary antibody, in panels showing nonpermeabilized cells and cells cotransfected with ds-Red Grasp, or rhodamine-labeled secondary antibody, in panels showing cells cotransfected with YC4er, a green fluorescent protein-labeled protein containing a calreticulin signal peptide and C-terminal KDEL. The panels labeled "Marker" show staining of either the red Golgi marker (G) or the greenYC4er ER marker (ER).

 
In other experiments, which are summarized in Table 1Go, we found that ubiquitination was not prevented by replacing Lys residues in the first and second intracellular loops with Arg or deleting the entire third intracellular loop, suggesting that ubiquitination can take place at multiple sites on the receptor. These results do not prove that ubiquitination takes place at multiple sites on the wild-type receptor, however, because deletion of either the third intracellular loop or the cytoplasmic tail resulted in an inactive, and likely misfolded protein. The finding that misfolded and mislocalized receptors are highly ubiquitinated is consistent with the idea that ubiquitin-mediated degradation plays a role in receptor quality control in the ER.


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Table 1. Ubiquitination of Mutant TRH Receptors

 
Glycosylated TRHRs Are Ubiquitinated
TRHRs contain two putative glycosylation sites in the N terminus and are normally glycosylated (15). To learn whether the ubiquitinated form of the TRHR is glycosylated, we immunoprecipitated Flag-tagged receptors, eluted them with Flag-peptide, and then incubated with wheat germ agglutinin (WGA)-agarose, which binds terminal N-acetyl-glucosamine and N-acetyl-neuraminic acid residues. Receptors retained in the ER should have only core carbohydrates and not bind WGA, but receptors that had undergone processing in the Golgi apparatus would be expected to bind to the lectin. The fraction absorbed to WGA-agarose contained glycosylated receptor monomer and dimer, and also included high molecular weight forms of ubiquitinated receptor (Fig. 7AGo). Several control experiments confirmed that little nonglycosylated receptor bound to WGA-agarose under the conditions used. First, receptors from tunicamycin-treated cells were not absorbed. Second, several misfolded receptors that do not appear to contain mature carbohydrate on SDS-PAGE, including the {Delta}324–412 mutant and a mutant lacking a signal peptide, were not retained by WGA-agarose (data not shown). Based on densitometry, 50% of total receptor in anti-Flag immunoprecipitates and 52% of receptor in anti-HA ubiquitin immunoprecipitates was absorbed to WGA-agarose. Treatment with tunicamycin, which inhibits glycosylation, caused TRHRs to migrate in tighter, faster-migrating monomer and dimer bands (Fig. 7BGo). Antiubiquitin immunoprecipitates from tunicamycin-treated cells contained apparent receptor monomer and a ladder of receptor species; the faster migrating species are separated by approximately 8 kDa, as expected for ubiquitin modifications (Fig. 7BGo, dots). These experiments suggest that ubiquitinated TRHRs contain complex carbohydrate and must have been modified in the Golgi apparatus.



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Fig. 7. Glycosylation of Ubiquitinated TRHRs

CHO cells were transiently transfected with TRHR-Flag and HA-Ub. A, Receptors were immunoprecipitated with anti-Flag antibody and then eluted with Flag peptide. Eluted receptors were incubated overnight with WGA-agarose to bind complex-glycosylated receptors and then run on SDS-PAGE and immunoblotted. The bracket denotes high molecular weight ubiquitinated receptors. B, Cells were incubated overnight with 10 µg/ml tunicamycin or vehicle before immunoprecipitation with either anti-Flag or anti-HA antibody. The blot shown in the right panel was exposed longer. Overnight treatment with tunicamycin did not affect radioligand binding to intact cells. Circles show the various species of ubiquitinated receptor evident on the blot with apparent sizes of approximately 37, 46, 55, 64, and 76 kDa.

 
Ligand Binding by Ubiquitinated TRHRs
To determine whether ubiquitinated TRHRs are capable of binding ligand, we lysed cells expressing Flag-tagged TRHRs and HA-tagged ubiquitin in buffer containing N-dodecylmaltoside, a detergent that preserves the ligand-binding pocket of the receptor. We then immunoprecipitated either TRHRs or ubiquitinated proteins and measured binding of [3H]MeTRH to the resultant immunoprecipitates. [3H]MeTRH bound specifically to both anti-Flag and anti-HA immunoprecipitates. Receptors precipitated with antibodies to HA-ubiquitin bound about 10% as much radioligand as anti-Flag immunoprecipitates, which contained the total receptor pool (Fig. 8AGo). Either ubiquitinated receptors are competent to bind hormone or they are complexed with functional, nonubiquitinated receptors.



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Fig. 8. Lack of Ubiquitination of Surface TRHRs

A and B, CHO cells were transiently transfected with HA-Ub and Flag-TRHR. A, Cells were lysed in buffer containing 0.1% N-dodecylmaltoside and proteins were immunoprecipitated with anti-HA antibody, anti-Flag antibody, or neither. The immunoprecipitates were incubated with 10 nM [3H]MeTRH for 1 h at 4 C, washed, and counted. B, Cells were incubated with 10 nM [3H]MeTRH on ice for 1 h to label surface receptors. Cells were washed to remove unbound ligand, lysed in buffer containing 1% Triton X-100, and the lysates were immunoprecipitated with anti-Flag antibody, anti-HA antibody, or neither. The immunoprecipitates were then counted. A and B, Bars show mean ± SE of three to four determinations. Expression and immunoprecipitation of total and ubiquitinated receptors were confirmed on Western blots (data not shown). C and D, CHO cells were transiently transfected with HA-TRHRs or empty vector, and a hexahistidine-ubiquitin-c-Myc (6His-Ub-Myc) construct. C, Lysates were immunoprecipitated with anti-HA antibody to precipitate TRHRs or a mixture of anti-6His, anti-myc, and antiubiquitin antibodies to precipitate ubiquitinated proteins. D, Cells were treated with or without TRH for 30 min at 37 C as indicated. To isolate surface receptors, intact cells were incubated with 1:1000 anti-HA antibody on ice for 1 h, washed extensively, lysed, and immunoprecipitated. Western blots were performed with anti-HA antibody to detect TRHRs or a mixture of antiubiquitin, anti-c-Myc, and anti-6His antibodies to detect ubiquitinated proteins.

 
We next asked whether ubiquitinated TRHRs are found at the plasma membrane. Intact cells expressing the Flag-TRHR with or without HA-ubiquitin were incubated with [3H]MeTRH at 0 C. [3H]MeTRH is unable to cross the plasma membrane and labels only surface TRHRs under these conditions. The cells were washed thoroughly and lysed under conditions where rebinding of any dissociated [3H]MeTRH would not occur (15). Lysates were immunoprecipitated with antibody to the HA or Flag epitopes and the amount of radioligand in the washed immunoprecipitates was measured (Fig. 8BGo). Anti-Flag immunoprecipitates, containing all receptors, bound 19566 ± 160 cpm of [3H]MeTRH specifically, but anti-HA immunoprecipitates, containing ubiquitinated proteins, bound less than 1% as much, 179 ± 28 cpm. This result suggests that ubiquitinated receptors are either not localized on the plasma membrane or not able to bind hormone when they are on the cell surface.

To rule out the possibility that surface receptors are ubiquitinated, but cannot bind ligand, we expressed N-terminal HA-tagged TRHRs and epitope-tagged ubiquitin in all cells and then surface-labeled the cells with anti-HA antibody on ice for 1 h. Under these conditions, the high-affinity antibody only binds to cell surface receptors. Surface receptors were then immunoprecipitated and Western blots performed to detect ubiquitin. No ubiquitinated TRHRs were detectable in surface receptor immunoprecipitates (Fig. 8DGo). As a control, we also immunoprecipitated total receptors and ubiquitinated TRHRs from lysates; when all receptors were analyzed in this manner, ubiquitinated receptors were easily detectable (Fig. 8CGo). As previously reported, surface receptors from TRH-treated cells ran more slowly on SDS-PAGE due to receptor phosphorylation (13).

Effect of Ligand on TRHR Ubiquitination
Because ligand binding stimulates ubiquitination of some plasma membrane receptors, including the yeast G protein-coupled receptor Ste2p, we tested the effect of TRH on receptor ubiquitination by exposing cells expressing Flag-tagged TRHRs and HA-ubiquitin to 1 µM TRH for times from 5 sec up to 30 h before cell lysis and immunoprecipitation (Fig. 9Go). The TRH concentration used was 100 times the dissociation constant to ensure a maximal response. TRH did not alter the fraction of total receptors ubiquitinated or the mobility of ubiquitinated receptors on SDS-PAGE, making it unlikely that the ligand promotes receptor ubiquitination. There was a steady increase in the total amount of TRHR immunoprecipitated over time, but the ratio of total receptor to ubiquitinated receptor remained unchanged. Although most ubiquitinated receptor ran at high molecular weight, some monomer was evident, suggesting that ubiquitinated receptors were either coprecipitated with nonubiquitinated receptors or that some deubiquitination took place.



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Fig. 9. Ligand Independence of TRHR Ubiquitination

TRH (1 µM) was added to CHO cells transiently expressing HA-ubiquitin and Flag-TRHRs at intervals up to 30 h before cells were harvested. Lysates were immunoprecipitated with either (A) anti-Flag or (B) anti-HA antibody and immunoblotted with anti-Flag antibodies. Monomeric and oligomeric receptors are denoted by arrowheads and brackets, respectively. IgG bands are not visible.

 
TRHR Signaling and Internalization in Ts20 Cells
To assess the impact of the ubiquitin-proteasome pathway on internalization of the TRHR, we used Ts20 cells, which have a temperature-sensitive E1 ubiquitin-activating enzyme (17). When Ts20 cells are grown at 30 C, this enzyme is fully functional; at 42 C, ubiquitin cannot be covalently attached to proteins. We transiently transfected Ts20 cells with an HA-tagged TRHR, waited for 48 h to permit receptor expression, and incubated the cells with or without TRH for 30 min at the permissive (30 C) or nonpermissive (42 C) temperatures to determine whether the hormone would cause receptor internalization, as it normally does. Analysis of total ubiquitinated proteins showed that levels of ubiquitinated proteins declined rapidly after the switch to the nonpermissive temperature (Fig. 10AGo). As shown in Fig. 10BGo, TRHRs were localized to the plasma membrane of untreated Ts20 cells at 30 C (left panel) or 42 C (not shown). When cells were incubated with TRH on ice, to occupy surface receptor, and then warmed to the permissive temperature for 30 min, a punctate vesicular TRHR-staining pattern was evident (middle panel), indicative of receptor endocytosis. If instead TRH-treated cells were warmed to the nonpermissive temperature, 42 C, plasma membrane staining was lost and endocytic vesicles were still observed, indicating that internalization of the TRHR occurred normally when ubiquitination was blocked (right panel). In fact, receptor endocytic vesicles were larger after incubation at 42 C.



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Fig. 10. TRHR Internalization in Ts20 Cells

A, Ts20 cells were transiently transfected with HA-Ub and incubated for 48 h at 30 C. The temperature was raised to 42 C, and at intervals from 0–6 h dishes were harvested and immunoprecipitated and immunoblotted with anti-HA antibody to detect total ubiquitinated proteins. B and C, Ts20 cells were transiently transfected with HA-TRHRs. B, After 48 h, cells were incubated with 1 µM TRH or vehicle for 30 min at the permissive (30 C) or nonpermissive (42 C) temperature and then fixed. The TRHR was identified by immunocytochemistry using mouse anti-HA antibody and TRITC-conjugated antimouse secondary antibody. Staining of untreated cells was the same when cells were maintained at 30 C, as shown, or at 42 C. C, After 48 h, the rate of internalization of [3H]MeTRH was measured by incubating the cells with 2.5 nM [3H]MeTRH on ice for 1 h and subsequently warming the cultures to 30 C or 42 C and measuring acid/salt resistant (internalized) and sensitive (surface) [3H]MeTRH at intervals. Mean ± range of duplicate samples is shown. D, Ts20 cells were transiently transfected with TRHR-Flag receptors and the YC2 cytoplasmic calcium reporter. Fluorescence resonance energy transfer measurements were carried out as described in Materials and Methods. The mean ± SE of 535/480 fluorescence emission ratio at each time point, relative to the ratio at the start of the experiment, is plotted for cells at 30° (n = 6) or 42 C (n = 9). An increase in the 535/480 fluorescence ratio indicates a rise in intracellular free calcium.

 
To determine whether the kinetics of TRHR internalization depend upon ubiquitination, we again transfected Ts20 cells with HA-TRHRs, incubated with [3H]MeTRH on ice, warmed them to the permissive or nonpermissive temperature, and measured surface and internalized (acid/salt-resistant) [3H]MeTRH binding sites over time (Fig. 10CGo). Internalization of the TRHR occurred rapidly, achieving a plateau at approximately 75–80% internalized within 40 min whether the ubiquitin conjugation system was intact or not. The rates of internalization of the TRHR were the same at the permissive and nonpermissive temperatures. These results for the TRHR are strikingly different from those reported for the GH receptor. The GH receptor normally undergoes internalization and degradation, but when the receptor is expressed in Ts20 cells, endocytosis and receptor degradation are inhibited 3 h after the switch to the nonpermissive temperature (18, 19).

We also asked whether an intact ubiquitination pathway is necessary for signal transduction via the TRHR. Because the transfection efficiency for Ts20 cells was quite low in our experiments, we cotransfected these cells with both the TRHR and the cytoplasmic calcium reporter YC2, one of the cameleon calcium indicators developed by Miyawaki and colleagues (20). This approach allowed us to select only successfully transfected cells for calcium imaging. The YC2 protein contains yellow and cyan variants of green fluorescent protein linked by calmodulin and a calmodulin binding domain, such that fluorescence resonance energy transfer, detected as the 535/480 fluorescence ratio, increases when calcium binds. The transfected cells were maintained at 30 C for 48 h to allow expression of the receptor and the YC2 cameleon and then incubated for 4 h at either 30 or 42 C before imaging, which was performed at either 30 or 42 C. Fluorescence resonance energy transfer was measured in five separate experiments at each temperature, and TRH was tested at a low dose, 1 nM, below the reported dissociation constant of the receptor of 10 nM (15) (Fig. 10DGo). TRH caused a sharp increase in cytoplasmic free calcium ion at both the permissive and nonpermissive temperatures. When all cells expressing YC2 were included in the analysis, TRH increased normalized 535/480 ratios by an average of 15.6 ± 2.1% at 30 C (n = 34) and by 8.9 ± 1.5% at 42 C (n = 39); the fraction of cells responding was 79% at 30° and 41% at 42 C. Although it is not possible to make quantitative comparisons between the results at the two temperatures because calcium pool sizes and YC2 response characteristics at 30 vs. 42 C are not known, we can conclude from these results that TRH can elicit a substantial increase in cytoplasmic calcium under conditions where ubiquitination is blocked.

We tested the effect of MG132 and receptor internalization in HEK293 cells stably expressing the TRHR. As shown in Fig. 11Go, the proteasome inhibitor had no effect on the rate or extent of receptor endocytosis.



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Fig. 11. Effect of Proteasome Inhibition on TRHR Internalization

CHO cells stably expressing HA-TRHRs were incubated for 2 h with 50 µM MG132 or vehicle and then incubated with 2.5 nM [3H]MeTRH on ice for 30 min, washed, and subsequently incubated at 37 C with or without MG132. The fraction of specifically bound [3H]MeTRH that had been internalized was determined at intervals. The mean ± range of duplicates is shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The data reported herein provide compelling evidence that the TRHR can undergo ubiquitination. To our knowledge, this is the first report that the TRHR, or any Gq-coupled receptor, is ubiquitinated. GPCRs have the potential to be modified by addition of both polyubiquitin and monoubiquitin with differing consequences. Polyubiquitin chains usually direct the proteasomal degradation of the proteins to which they are covalently attached, whereas monoubiquitin has been shown to direct the internalization of plasma membrane receptors as well as endosomal sorting. On SDS-PAGE, ubiquitinated TRHRs ran as both high molecular weight smears and sharper monomeric bands. The low molecular weight bands represented receptors that had minimal or no carbohydrate modification. In cells treated with tunicamycin, a traditional ladder of ubiquitinated receptor forms was visible. These species could be due to the presence of branched ubiquitin chains, multiple polyubiquitination sites, or both. Alternatively, the smearing could be due to multiple monoubiquitination, as recently reported for epidermal growth factor receptors (21). TRHRs immunoprecipitated with antibody to ubiquitin after exposure to strongly denaturing conditions, boiling in SDS. Furthermore, truncated receptors precipitated with antibody to ubiquitin ran faster than ubiquitinated full-length receptors on gels. These findings argue that the receptor was itself ubiquitinated and not simply associated with a ubiquitinated protein. Mutation studies suggested that ubiquitin can be added to more than one site on the receptor.

Some intrinsic membrane proteins, such as the cystic fibrosis transmembrane regulator, have been shown to be ubiquitinated cotranslationally in the ER (22). Other membrane receptors, including the GPCR Ste2p and several receptor tyrosine kinases, are ubiquitinated at the plasma membrane. It has been postulated that most GPCRs are degraded in the ER and never reach the plasma membrane. ß-Adrenergic receptors (23) and {delta}-opioid receptors (8) are largely degraded before they exit the ER, and ubiquitinated, partially deglycosylated opioid receptors can be detected in the cytosol if proteasome inhibitors are present. We showed that newly synthesized TRHRs accumulated when proteasomes were inhibited as detected both by Western blot and fluorescence microscopy. These results imply that ubiquitination and proteasomal degradation of receptors occurs normally during receptor synthesis, probably as a quality control mechanism to dispose of misfolded receptors. This idea is supported by the finding that several mutant receptors that were unable to bind hormone, undergo N-glycosylation, or exit the ER were more heavily ubiquitinated than wild-type receptors.

Proteasomal degradation of intrinsic membrane proteins is thought to involve their retrograde transfer into the cytoplasm followed by deglycosylation, ubiquitination, and finally degradation (1, 3, 24, 25). In the presence of MG-132, TRHRs became detectable in cytosolic fractions, suggesting that this receptor is degraded by the proteasome in a similar way. The Sec61p translocon is proposed to play a role in ER-associated degradation (26), and may be responsible for the cytosolic translocation of TRHRs. Several diseases arise from the aggregation of proteins destined for ER-associated degradation by the proteasome. For example, certain mutant forms of rhodopsin aggregate in cells and cannot be degraded properly by the proteasome causing an autosomal dominant form of retinitis pigmentosa (27). Also, mutant cystic fibrosis transmembrane regulator protein can accumulate in aggresomes when misfolded (28). Proteasomal degradation of misfolded TRHRs appears to be quite efficient, because there is little accumulation of ubiquitinated receptor forms unless proteasome inhibitors are present.

ER-associated degradation may not be the only function for ubiquitination of the TRHR. In cells expressing wild-type receptor, most of the ubiquitinated receptor had undergone complex glycosylation, indicating that it had been modified in the Golgi apparatus. The data do not allow us to distinguish whether: 1) receptors were ubiquitinated in the ER, escaped proteasomal degradation and were transported to the Golgi apparatus where they underwent further glycosylation; 2) receptors were ubiquitinated after exit from the ER, possibly in the Golgi apparatus; or 3) complex-glycosylated receptors were ubiquitinated in the ER after retrograde transport from the Golgi apparatus. There is precedent for ubiquitination taking place in the Golgi apparatus. A recently described transmembrane ubiquitin ligase, Tul1, is found in the Golgi and necessary for sorting membrane proteins into multivesicular bodies in yeast (7). Another E3 ubiquitin-ligase, SCFFbx targets proteins with N-linked glycans, and a related enzyme could be responsible for ubiquitination of glycosylated TRHRs (29). Experiments using brefeldin A to differentiate among these possibilities were uninformative (Cook, L. B., and P. M. Hinkle, unpublished data).

The down-regulation of many plasma membrane receptors is regulated by ubiquitin. Yeast pheromone receptors are modified by the addition of a ubiquitin residue on their C-terminal tails after ligand binding, triggering internalization (9, 30). Receptor ubiquitination is also involved in the endocytosis of the met tyrosine kinase (31). Ligand binding promotes ubiquitination of the platelet-derived growth factor receptor (32), and ligand-independent, cell detachment-induced receptor ubiquitination has been reported to cause its degradation (31). Proteasome inhibitors reduce ligand-induced down-regulation of the µ, {delta}, and {kappa}-opioid receptors (33, 34). We investigated the possibility that TRHR internalization and signaling were affected by ubiquitin by a variety of approaches. The following pieces of evidence suggest that TRHR ubiquitination is not regulating internalization of the TRHR. 1) Although the TRHR is phosphorylated and over 80% of surface receptor is rapidly internalized in response to ligand binding, TRH did not affect the extent of receptor ubiquitination over the time course of endocytosis. 2) A C-terminally truncated TRHR ({Delta}335–412) was ubiquitinated, but cannot internalize (35). 3) TRHRs internalized normally in the absence of a functional ubiquitin pathway in Ts20 cells at the nonpermissive temperature. 4) Proteasome inhibitors had no effect on TRHR internalization. 5) Ubiquitinated TRHRs were not detected at the cell surface. From these results, we conclude that TRHR internalization can proceed without transient ubiquitination of the receptor itself or other proteins. TRHR internalization is clathrin-mediated and involves tethering of the receptor C terminus to ß-arrestin. Lefkowitz and co-workers (5) found that a loss of ß-arrestin ubiquitination resulted in the inhibition of ß2-adrenergic receptor internalization and that MG-132 treatment blocked receptor endocytosis. We were not able to show any effect of the ubiquitin-proteasome pathway on TRHR internalization in CHO cells or Ts20 cells, a CHO cell derivative. Furthermore, ubiquitin modification is not required for receptor signaling, because TRH was able to generate a strong calcium signal in the absence of a functional ubiquitination system.

Ubiquitinated TRHRs are not detectable at the plasma membrane. One possibility is that ubiquitinated TRHRs do reach the plasma membrane but are degraded or deubiquitinated so quickly that they are not detectable. Deubiquitinating enzymes are found throughout the cell, including the plasma membrane, and have been reported to remove single, or even poly-ubiquitin groups (36, 37). Another possibility is that the ubiquitinated TRHR in the Golgi apparatus is targeted for lysosomal degradation. Addition of ubiquitin to proteins in the Golgi apparatus by Tul1 results in their transport into the endosomal-vacuole pathway (7). A homologous ubiquitin-ligase may perform a similar function in mammalian cells, serving as a secondary checkpoint to inhibit misfolded transmembrane proteins from reaching the plasma membrane. An additional possibility is that ubiquitin functions to prevent forward trafficking of TRHRs out of the Golgi apparatus. This model is fashioned after a recent study by Keller and co-workers (38), who showed that ubiquitin functions to regulate post-Golgi trafficking of the nicotinic acetylcholine receptor. Ubiquitin masks signals required for transport of the {alpha}-subunit of the nicotinic receptor to the plasma membrane, resulting in retention of the receptor in the Golgi apparatus. If this is the case, ubiquitinated receptors could serve as a reserve and deubiquitination could make them available for trafficking to the plasma membrane.

In summary, we have shown that the TRHR undergoes posttranslational modification by ubiquitin addition. The function of ubiquitination includes degradation of misfolded newly synthesized receptors in the ER, and post-ER quality control that may include regulation of receptor trafficking. The ubiquitin-proteasome pathway is not, however, necessary for TRHR internalization or calcium signaling. Future experiments will explore the regulation and quality control of receptor trafficking to and from the plasma membrane by ubiquitin and other proteins.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
CHO cells were maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS). Cell lines expressing TRHRs were created by transfecting cells with the relevant plasmid using Lipofectamine (Invitrogen Life Technologies, Carlsbad, CA). CHO cells expressing a rat TRHR tagged with two HA epitopes at the N terminus (13) were selected with 850 µg/ml G-418 and individual clones were picked. HEK293 cells expressing a rat TRHR with a C-terminal Timer (CLONTECH, Palo Alto, CA) construct were selected with 600 µg/ml G-418 and a pool was used for experiments. GHFT cells were provided by Dr. Richard N. Day (University of Virginia Medical School, Charlottesville, VA) and grown in DMEM/F12 medium supplemented with 10% FBS. Cells were grown as monolayer cultures in a humidified 95% air and 5% CO2 environment at 37 C. Ts20 cells were obtained from Dr. Anthony Bianco (Harvard Medical School, Boston, MA). They were maintained as monolayer cultures in DMEM supplemented with 10% FBS at 30 C in a humidified 95% air and 5% CO2 environment. All cells were passaged when 80–90% confluent.

Cell Transfection
A plasmid encoding HA-ubiquitin was donated by Dr. R. K. Rottapel (Ontario Cancer Institute, Toronto, Ontario, Canada), and plasmids encoding HA- and Flag-tagged TRHRs have been described (13). A prolactin signal peptide preceded the Flag epitope tags unless noted. A plasmid encoding the rat TRHR tagged with 6 His at the amino terminus and a double Flag epitope in the C terminus and a plasmid encoding the mouse TRHR (pcDM8mTRHR) were provided by Dr. Marvin C. Gershengorn (National Institutes of Health, Bethesda, MD). The extent of ubiquitination was similar in receptors tagged with either an N- or C-terminal Flag epitope. Receptors tagged at the N terminus are denoted Flag-TRHR or HA-TRHR, and those at the C terminus TRHR-Flag. A larger fraction of receptors tended to be localized to the plasma membrane when the epitope tag was on the C terminus. Plasmid encoding the yellow cameleon YC2 was donated by Dr. Roger Tsien (University of California, San Diego, CA), and pCW-7, a plasmid encoding 6 His and c-myc tagged ubiquitin, was provided by Dr. Ron Kopito (Stanford University, Stanford, CA). A plasmid encoding TRHR-Timer was made by inserting the rat TRHR sequence lacking a stop codon between the KpnI and BamHI sites in pTimer (CLONTECH). The receptor was then cut with BamHI and EcoRI and moved into pcDNA3 (Invitrogen). Cells were transfected with 3, 4, or 10 µg total plasmid DNA in 35-, 60-, or 100-mm dishes, respectively. Lipofectamine transfection reagent was used according to manufacturer’s recommendations. After 4–5 h, cells were rinsed once in serum-free medium and split, if necessary. Cells were incubated 18–48 h in serum-containing medium before experiments, except as noted for Ts20 cells.

TRHR Mutants
All TRHR mutants were constructed from a pcDNA3 plasmid encoding the long form of the rat TRHR containing two Flag epitopes separated by a Gly residue with a sequence encoding a prolactin signal peptide upstream of the Flag epitope tags (13). Primers to pcDNA3 were pcDNA3-Up (5'-ATGTCGTAACAACTCCGCCCCATTG-3') and pcDNA3-Down (5'-GAATGACACCTACTCAGACAATGCGAT-3'). For the K54R mutant, two separate PCR products were constructed. The upstream portion was constructed with pcDNA3-UP and K54-Down (5'-TAGCGGTTCTCATGTGCCTCGTTCTCATCATGACCACC-3'). The downstream portion was constructed using pcDNA3-Down and K54-UP (5'-GGTGGTCATGAGAACGAGGCACATGAGAACCGCTA-3'). These PCR products were annealed and amplified again using pcDNA3-UP and pcDNA3-Down. K132R was constructed similarly with the following TRHR primers: K132R-UP (5'-ATCTGCCACCCCATCAGAGCCCAGTTTCTGTG-3') and K132R-Down (5'-CACAGAAACTGGGCTCTGATGGGGTGGCAGAT-3'). The template for K54/132R was a pcDNA3 vector encoding K54R. K228R was constructed with the following TRHR primers: K228R-Up (5'-CATTCCTTCAGACCCTAGAGAAAACTCTAAGAC-3') and K228R-Down (5'-GTCTTAGAGTTTTCTCTAGGGTCTGAAGGAATG-3'). To construct {Delta}324–412, the primer, short-tail, was made to introduce a stop codon between Met323 and Ser324 with an ApaI sequence at the 3' end (5'-CGCGGGCCCCTTCTGAGATCACATGAGGTTGTA-3'). {Delta}324–412 was amplified using short-tail and pcDNA3-UP. {Delta}210–266 was amplified using the following primers: P2 (5'-CACCACTGCAAGCATCACAGTGGCCAGGAT-3') and P3 (5'-ATCCTGGCCACTGTGATGCTTGCAGTGGTG-3') and pcDNA3-UP and pcDNA3-Down. All final PCR products were digested with KpnI and ApaI and ligated into the parent vector, which was also digested with KpnI and ApaI.

Immunoprecipitation
Cells were washed twice in PBS and lysed in 1 ml ice-cold lysis buffer [150 mM NaCl, 50 mM Tris-base (pH 8.0), 1% (wt/vol) Triton X-100, 1 mM EDTA, 1:200 Protease Inhibitor Cocktail Set III (Calbiochem), and 10 mM iodoacetamide]. In one experiment, cells were lysed in RIPA buffer [150 mM NaCl, 50 mM Tris (pH 8), 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate] containing 1% SDS. Cells were incubated on ice for 5 min, then harvested into chilled microfuge tubes and set on ice for an additional 10 min. The lysate was pelleted by centrifugation for 10 min at 16,000 x g in an Eppendorf microcentrifuge at 4 C. Supernatants were divided in half and brought to 1 ml with lysis buffer. Primary antibody was added at a 1:5000 dilution and incubation continued for 1–18 h at 4 C. In some cases, 1 ml of a 1:1000 dilution of primary antibody was absorbed to protein A/G beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1.5 h at 4 C before the addition of cell lysate. M2 monoclonal antibody to the Flag epitope (4.4 mg/ml) was from Sigma (St. Louis, MO) and monoclonal antibodies to HA (6 mg/ml) and ubiquitin (4 mg/ml) were from Covance (Princeton, NJ). Twenty microliters of protein A/G beads were washed once in buffer [20 mM Tris-base (pH 8.0) and 1 mM EDTA] and then added to each lysate for 1–2 h at 4 C. Beads were collected by centrifugation and washed 4 times in lysis buffer. Beads were resuspended in 75 µl 2x sample buffer and boiled for 2 min. Samples were either used immediately or stored at -20 C. Alternatively, Flag-TRHRs were eluted from beads with two successive 100-µl elutions with 0.15–0.25 mg/ml Flag peptide (Sigma) in 10 mM Tris HCl, 150 mM NaCl (pH 7.4) for 30 min at 4 C.

Antibody Labeling of Surface Receptors
Cells expressing full-length receptors in 100-mm dishes were placed on ice and rinsed twice with ice-cold Hanks’ balanced salt solution (HBSS). They were then incubated on ice for 1 h with 1:1000 anti-HA monoclonal antibody in HBSS, rinsed with ice-cold HBSS three times, and lysed in 1 ml ice-cold lysis buffer. Control experiments, in which the lysate was spiked with solubilized truncated receptor, showed that the truncated receptor was not immunoprecipitated.

Cell Fractionation
Cells from two 100-mm dishes were rinsed once in homogenization buffer (50 mM Tris-Cl, 1 mM EDTA, 0.25 M sucrose, 10 mM iodoacetamide, 1:200 protease inhibitor cocktail) and cells were homogenized on ice with 30 strokes in a Dounce homogenizer. After a low-speed spin to remove nuclei and unbroken cells, the supernatant fraction was centrifuged at 10,000 x g for 30 min. The supernatant fraction was then spun at 100,000 x g for 1 h. The pellets were resuspended in homogenization buffer with 1% Triton X-100 to solubilize proteins and centrifuged at 10,000 x g. The supernatant fractions were all brought to 1% Triton X-100 and immunoprecipitated with anti-HA antibody at 1:5000 dilution. Samples were resuspended in 50 µl 2x sample buffer. Sources and dilutions for antibodies to markers were: monoclonal antibody to type 3 IP3 receptor from Transduction Laboratories (San Diego, CA) (1:1000), antibody to calnexin N terminus (50–68) from Calbiochem (San Diego, CA) (1:5000), mouse monoclonal antibody to ß-COP from Sigma (1:5000) and mouse monoclonal antibody to plasma membrane calcium ATPase from Affinity Bioreagents (Golden, CO) (1:1000).

Lectin Affinity Purification
Eluted TRHR immunoprecipitates were diluted to 1 ml and incubated with 25 µl of WGA agarose (Vector Laboratories Inc., Burlingame, CA) overnight at 4 C with rotation. The following day, the WGA-agarose was pelleted and washed twice with lysis buffer. Pellets were resuspended in 30 µl 2x sample buffer.

Electrophoresis and Immunoblotting
Samples were boiled for 2 min and microfuged for 2 min. Equal amounts of sample (5 to 25 µl) were loaded onto a 10% gel and SDS-PAGE was performed at 100–175 V. In most cases, 5 µl were loaded when immunoblotting was performed with the precipitating antibody and 10–25 µl when immunoblotting was done with a different antibody, and exposure times were longer in the latter cases. Proteins were transferred to a nitrocellulose membrane using a Semi-dry Transblot apparatus (Bio-Rad, Hercules, CA) at 15 V for 1 h. Membranes were then incubated in blocking buffer containing 5% evaporated milk in TBS-T [20 mM Tris-Cl, 0.14 M NaCl, 0.05% Tween 20 (pH 7.6)] for 1 h at room temperature or overnight at 4 C. The membrane was incubated with primary antibodies diluted in blocking buffer for at least 2 h at room temperature or overnight at 4 C. Anti-His-tag monoclonal antibody was obtained from Novagen (Madison, WI) and anti-c-myc 9E10 antibody was from Santa Cruz Biotechnology, Inc. Antiubiquitin and anti-His-tag antibodies were used at 1:1000, anti-myc antibody at 1:500, and all other antibodies at 1:5000. Membranes were then washed in TBS-T and incubated for 45 min at room temperature with horseradish peroxidase-conjugated secondary antibodies from Amersham Life Sciences (Piscataway, NJ) diluted 1:2000 in blocking buffer. Horseradish peroxidase-conjugated proteins were detected using Chemiluminescence Reagent Plus (New England Nuclear Life Science Products, Boston, MA) or SuperSignal (Pierce, Rockford, IL) on Kodak film.

Radioligand Binding Assays
To measure radioligand binding to intact cells, dishes were incubated in serum-free media or HBSS at pH 7.4 containing [3H]MeTRH (Dupont/New England Nuclear, 60–100 Ci/mmol) with or without a 1000-fold molar excess of nonradioactive TRH for the times indicated. Cells were then placed on ice and washed three times in ice-cold saline solution. To measure internalization of receptor-bound [3H]MeTRH, cells were washed for 15–60 sec with ice cold acid/salt buffer [0.2 M acetic acid, 0.5 M NaCl (pH 2.5)] to extract surface ligand, and the cells were solubilized and counted to measure internalized hormone (39).

To measure binding to solubilized receptors, cells were lysed in lysis buffer containing 0.1% N-dodecylmaltoside or 1% Triton X-100, as noted in the text, and centrifuged as described above. Supernatant fluids were incubated with 10 nM [3H]MeTRH with or without excess unlabeled TRH on ice for 2–18 h and receptor-bound [3H]MeTRH was trapped on a glass fiber filter that had been soaked in 0.3% polyethyleneimine. Protein concentrations were determined by the Bradford method with BSA as a standard.

Immunocytochemistry
Cells were plated onto coverslips the day before immunostaining, which was carried out as described (40). Cells on coverslips were rinsed in HBSS, fixed in 1 ml 4% paraformaldehyde in PBS for 20 min and permeabilized in blocking buffer (PBS containing 5% goat serum and 0.2% Nonidet P-40) for 20 min at room temperature. Mouse monoclonal anti-HA antibody was added at a 1:1000 dilution in the same blocking buffer for 1–3 h. Coverslips were washed three times in 2 ml PBS for 5 min each. fluorescein isothiocyanate (FITC)- or tetramethyl rhodamine isothiocyanate (TRITC)-conjugated antimouse secondary antibody (American Qualex, San Clemente, CA) was added at a 1:500 dilution in blocking buffer for 40 min. Coverslips were then washed again as before and mounted. In some experiments, cells were cotransfected with plasmids encoding GRASP-55-DsRed, donated by Dr. Francis Barr (Max Planck Institute of Biochemistry, Martinsried, Germany) or YC4er from Dr. Roger Tsien (University of California at San Diego, San Diego, CA). DsRed was visualized with TRITC filters and YC4er, which encodes a green fluorescent protein, with FITC filters.

Calcium Imaging
Ts20 cells were plated onto coverslips and then transfected with 2 µg of plasmid encoding TRHR with a C-terminal Flag epitope and 1 µg of plasmid encoding YC2. Cells were maintained at 30 C for 48 h and then incubated for a further 4 h at either 30 or 42 C before imaging was begun. Coverslips were washed and incubated in HBSS at either 30 or 42 C. Fluorescence resonance energy transfer was measured by exciting cells with 440 nm light and quantifying fluorescence emission at 535 and 480 nm alternately every 3 sec as previously described in detail (41).


    ACKNOWLEDGMENTS
 
We are grateful to John Puskas and Carrie Perkowski at the University of Rochester (Rochester, NY) for excellent technical assistance.


    FOOTNOTES
 
This work was supported by a grant from NIH (DK19974) (to P.M.H.), a Wilmot Cancer Research Fellowship (to C.-C.Z.) and a Pharmaceutical Manufacturers’ Association Predoctoral Fellowship (to L.B.C.).

Abbreviations: CHO, Chinese hamster ovary; ER, endoplasmic reticulum; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GPCR, G protein-coupled receptor; HA, hemagglutinin; HBSS, Hanks’ balanced salt solution; IP3, inositol (1 4 5 ) trisphosphate; SDS, sodium dodecyl sulfate; TRITC, tetramethylrhodamine isothiocyanate; WGA, wheat germ agglutinin.

Received for publication March 5, 2003. Accepted for publication June 2, 2003.


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 DISCUSSION
 MATERIALS AND METHODS
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