| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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 INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-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 
-opioid receptors and ß2-adrenergic receptors; -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 |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
, 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. 1A, 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. 1A, 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. 1E (left lanes) and 2 and data not shown].
|
). We also probed cell lysates with antibody against native ubiquitin (Fig. 1C) 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. 1E). 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. 2).
|
MG132 caused a slight decrease in [3H]MeTRH binding (Fig. 3A), but an increase in total receptor (Fig. 3B). 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. 3D, left panel), or with antibody to native ubiquitin, for ubiquitinated receptor (Fig. 3D, 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. 3C). 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. 3D, right panel). In the presence of the proteasome inhibitor, some immature receptor was detectable in the cytosolic fraction, and this receptor was ubiquitinated (Fig. 3D, asterisks). No cytosolic receptor was isolated in the absence of MG132.
|
) 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.
|
335–412), which lack seven of nine lysine residues in the cytoplasmic tail, or Flag-TRHRs truncated at M323 (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. 5B). All three receptors coimmunoprecipitated with the HA antibody, indicating that they are ubiquitinated. In fact, the 324–412 receptor was more highly ubiquitinated than 335–412 and full-length receptors (Fig. 5A).
|
335–412 TRHR bound 40% as much [3H]MeTRH as the full-length receptor, but those expressing the 324–412 receptor bound less than 1% as much (Fig. 5C). To determine whether the lack of [3H]MeTRH binding to the 324–412 receptor was due to an inability of the receptor to bind ligand or a loss of surface localization, cells expressing full-length, 335–412 or 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 335–412 receptors were visible at the plasma membrane, but 324–412 receptors were not detectable (Fig. 6, 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. 6). 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 335–412 TRHR was found primarily in ER and Golgi, and the fully truncated 324–412 TRHR was only visible intracellularly in the ER. After solubilization with N-dodecylmaltoside, 324–412 TRHRs showed no measurable [3H]MeTRH binding (data not shown), indicating that this receptor is nonfunctional as well as mislocalized.
|
, 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.
|
). 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 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. 7B). 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. 7B, dots). These experiments suggest that ubiquitinated TRHRs contain complex carbohydrate and must have been modified in the Golgi apparatus.
|
). Either ubiquitinated receptors are competent to bind hormone or they are complexed with functional, nonubiquitinated receptors.
|
). 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. 8D). 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. 8C). 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. 9). 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.
|
). As shown in Fig. 10B, 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.
|
). 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. 10D). 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. 11, the proteasome inhibitor had no effect on the rate or extent of receptor endocytosis.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
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 -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, , and 
-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 (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 
-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 |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
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 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'). 324–412 was amplified using short-tail and pcDNA3-UP. 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 |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome. J Biol Chem 276:4416–4423 opioid receptors. J Biol Chem 276:12345–12355-opioid receptor: internalization is required for down-regulation. Mol Pharmacol 58:795–801-subunit. J Biol Chem 276:18384–18391This article has been cited by other articles:
 |
|
 |
|
  B. W. Jones and P. M. Hinkle Subcellular Trafficking of the TRH Receptor: Effect of Phosphorylation Mol. Endocrinol., September 1, 2009; 23(9): 1466 - 1478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Torres, M. J. Lee, F. Ding, C. Purbeck, B. Kuhlman, N. V. Dokholyan, and H. G. Dohlman G Protein Mono-ubiquitination by the Rsp5 Ubiquitin Ligase J. Biol. Chem., March 27, 2009; 284(13): 8940 - 8950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Mines, J. S. Goodwin, L. E. Limbird, F.-F. Cui, and G.-H. Fan Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation J. Biol. Chem., February 27, 2009; 284(9): 5742 - 5752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Rondou, G. Haegeman, P. Vanhoenacker, and K. Van Craenenbroeck BTB Protein KLHL12 Targets the Dopamine D4 Receptor for Ubiquitination by a Cul3-based E3 Ligase J. Biol. Chem., April 25, 2008; 283(17): 11083 - 11096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Shirai, A. Matsuyama, Y. Yashiroda, A. Hashimoto, Y. Kawamura, R. Arai, Y. Komatsu, S. Horinouchi, and M. Yoshida Global Analysis of Gel Mobility of Proteins and Its Use in Target Identification J. Biol. Chem., April 18, 2008; 283(16): 10745 - 10752. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-G. Li, D. S. Haines, and L.-Y. Liu-Chen Agonist-Promoted Lys63-Linked Polyubiquitination of the Human {kappa}-Opioid Receptor Is Involved in Receptor Down-Regulation Mol. Pharmacol., April 1, 2008; 73(4): 1319 - 1330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.-T. Lin, Y.-J. Lai, N. Makarova, G. Tigyi, and W.-C. Lin The Lysophosphatidic Acid 2 Receptor Mediates Down-regulation of Siva-1 to Promote Cell Survival J. Biol. Chem., December 28, 2007; 282(52): 37759 - 37769. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Conn, A. Ulloa-Aguirre, J. Ito, and J. A. Janovick G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo Pharmacol. Rev., September 1, 2007; 59(3): 225 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Shenoy Seven-Transmembrane Receptors and Ubiquitination Circ. Res., April 27, 2007; 100(8): 1142 - 1154. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Huang, J.-i. Niwa, G. Sobue, and G. E. Breitwieser Calcium-sensing Receptor Ubiquitination and Degradation Mediated by the E3 Ubiquitin Ligase Dorfin J. Biol. Chem., April 28, 2006; 281(17): 11610 - 11617. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. W. Jones and P. M. Hinkle {beta}-Arrestin Mediates Desensitization and Internalization but Does Not Affect Dephosphorylation of the Thyrotropin-releasing Hormone Receptor J. Biol. Chem., November 18, 2005; 280(46): 38346 - 38354. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-C. Lu, T. M. Piazza, and L. A. Schuler Proteasomes Mediate Prolactin-induced Receptor Down-regulation and Fragment Generation in Breast Cancer Cells J. Biol. Chem., October 7, 2005; 280(40): 33909 - 33916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Pietila, J. T. Tuusa, P. M. Apaja, J. T. Aatsinki, A. E. Hakalahti, H. J. Rajaniemi, and U. E. Petaja-Repo Inefficient Maturation of the Rat Luteinizing Hormone Receptor: A PUTATIVE WAY TO REGULATE RECEPTOR NUMBERS AT THE CELL SURFACE J. Biol. Chem., July 15, 2005; 280(28): 26622 - 26629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Xia, M. Roundtree, A. Merikhi, X. Lu, S. Shentu, and G. LeSage Degradation of the Apical Sodium-dependent Bile Acid Transporter by the Ubiquitin-Proteasome Pathway in Cholangiocytes J. Biol. Chem., October 22, 2004; 279(43): 44931 - 44937. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. B. Cook and P. M. Hinkle Fate of Internalized Thyrotropin-Releasing Hormone Receptors Monitored with a Timer Fusion Protein Endocrinology, July 1, 2004; 145(7): 3095 - 3100. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
