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J. Biol. Chem., Vol. 282, Issue 17, 12893-12906, April 27, 2007

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Phosphorylation of the Endogenous Thyrotropin-releasing Hormone Receptor in Pituitary GH3 Cells and Pituitary Tissue Revealed by Phosphosite-specific Antibodies^*

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

Received for publication, November 24, 2006 , and in revised form, February 12, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr³⁶⁵ in the receptor tail; distal sites were more phosphorylated in some heterologous models. -Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nM TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The type I thyrotropin-releasing hormone (TRH)² receptor is a seven-transmembrane G protein-coupled receptor (GPCR) that is expressed in the anterior pituitary, where it responds to TRH secreted from the hypothalamus, leading to the release of thyroid-stimulating hormone (TSH) from thyrotrophs, which in turn causes release of T₃ and T₄ from the thyroid. Thyroid hormones exert negative feedback at the level of the hypothalamus and pituitary in order to ensure that hormone levels are tightly regulated. Lactotrophs in the anterior pituitary also express TRH receptors and release prolactin after exposure to TRH. In addition to the anterior pituitary, the TRH receptor is expressed in various areas of the central nervous system. The TRH receptor signals via G_q/11, which activates phospholipase C and leads to the generation of inositol 1,4,5-trisphosphate and diacylglycerol, the release of calcium from the endoplasmic reticulum, and the activation of protein kinase C (PKC).

GPCRs can be phosphorylated by second messenger-activated kinases, such as PKC, or by GPCR kinases (GRKs), which preferentially recognize the agonist-occupied receptor conformation (1, 2). Like other GPCRs, the TRH receptor is phosphorylated after binding to agonist (3, 4), which allows the recruitment of -arrestins from the cytosol to the receptor (5). By binding to -arrestin, the receptor is uncoupled from G proteins and desensitized (6). The -arrestin-receptor complex is then internalized in a dynamin- and clathrin-dependent manner (7). In order to respond to a subsequent pulse of agonist, internalized GPCRs must be recycled to the plasma membrane (8). For some GPCRs, such as the ₂-adrenergic receptor, recycling and resensitization are believed to require receptor dephosphorylation (9).

Despite playing key roles in regulating desensitization, internalization, and resensitization for the majority of GPCRs, receptor phosphorylation and especially dephosphorylation are incompletely understood. In a small number of cases, it has been possible to detect phosphorylation of receptors in native cells or in tissues (10-13), but most information about phosphorylation and dephosphorylation comes from studies using overexpressed receptors in heterologous cells. Several factors contribute to the difficulties in studying receptor phosphorylation. Good antibodies against GPCRs are rare, so epitope-tagged receptors are often used; GPCRs are typically expressed at low concentrations, so isolating sufficient protein for mass spectrometry or analysis of ³²P-labeled peptides is not always possible; many GPCRs have multiple potential phosphorylation sites, and consensus sequences for GRKs are poorly defined (1, 2); and GRKs readily phosphorylate at a different site if their preferred site has been mutated, complicating interpretation of mutagenesis data. Recently, the use of phosphosite-specific antibodies has provided a solution to many of these challenges while avoiding the problems associated with phosphosite mutagenesis (10, 11, 14-16).

We previously showed that heterologously expressed TRH receptors are phosphorylated quite rapidly and that they can be dephosphorylated without internalization (6). Sites of TRH receptor phosphorylation have never been directly identified, however, and phosphorylation of endogenous receptors has not been characterized. The kinetics of phosphorylation and dephosphorylation in pituitary cells are particularly important for the TRH receptor, because TRH is believed to be released into the hypothalamic-hypophyseal portal circulation in a pulsatile manner in vivo.

We generated phosphosite-specific polyclonal antibodies against all potential phospho-Ser and -Thr residues in the conserved region of the TRH receptor cytoplasmic tail and used these to study phosphorylation and dephosphorylation of the endogenous TRH receptor in rat pituitary GH3 cells and in rat pituitary tissue. For the first time, we characterize phosphorylation and dephosphorylation of the endogenous receptor and demonstrate that changes occur rapidly, suggesting tight control of receptor signaling. We also show that receptor phosphorylation provides a novel marker of the activity of a GPCR in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—GH3, CHO, and HEK293 cells were from ATCC (Manassas, VA). GH-Y cells have been described previously (17). Embryo fibroblasts (MEFs) from mice lacking -arrestins 1 and 2 (18) were from Dr. Robert Lefkowitz (Duke University, Durham, NC). Plasmids encoding wild type and dominant negative GRK2 were from Dr. Jeffrey Benovic (Thomas Jefferson University, Philadelphia, PA); plasmids encoding bovine -arrestins 1 and 2 were provided by Dr. Vsevolod Gurevich (Vanderbilt University, Nashville, TN); GRK3, -5, and -6 plasmids were from Dr. Jonathan Willets (University of Leicester, Leicester, UK). Plasmid encoding an N-terminal hemagglutinin (HA)-tagged TRH receptor has been described (3), and mutations were prepared using QuikChange reagents from Stratagene and confirmed by sequencing. Anti-GRK antibodies against GRK2 (C-15), GRK3 (C-15), GRK5 (C-20), and GRK6 (C-20) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other sources were as follows: BD Falcon multiwell plates (BD Biosciences, Bedford, MA); Lipofectamine, Lipofectamine 2000, DNase I, Moloney murine leukemia virus reverse transcriptase, TaqDNA polymerase, and media (Invitrogen); RNeasy mini kit (Qiagen, Valencia, CA); TRH (Bachem, King of Prussia, PA); chlordiazepoxide (ICN Pharmaceuticals, Costa Mesa, CA); bisindolylmaleimide I, ionomycin, cantharidin, microcystin LF, alkaline phosphatase, staurosporine (Calbio-chem); BAPTA/AM, ProLong Gold with DAPI, and Alexa Fluor 546 goat anti-rabbit antibody (Molecular Probes, Inc., Eugene, OR); phorbol 12-myristate 13-acetate (LC Labs, Woburn, MA); horseradish peroxidase-linked goat anti-mouse and anti-rabbit antibodies (Bio-Rad); -phosphatase (New England Biolabs, Gardner, MA); PAGEr Gold precast SDS-polyacrylamide gels (Cambrex, Rockland, ME); 17-estradiol (Sigma); midazolam (Abbott); polyclonal anti-rat prolactin and anti-rat TSH antibodies (National Hormone and Pituitary Program, National Institutes of Health, Bethesda, MD); polyclonal anti-cyclophilin B antibody (Abcam, Cambridge, MA). Okadaic acid from Sigma and LC Laboratories and calyculin A from Calbio-chem and LC Laboratories were all tested.

Cell Culture and Transfection—GH3 and GH-Y cells were grown in Dulbecco's modified Eagle's medium/F-12 medium, 10% newborn calf serum, 2.5 µg/ml amphotericin B and passaged with trypsin/EDTA. Unless noted, medium was supplemented with 10 nM 17-estradiol for 24-48 h prior to experimentation. GH3 cells were transfected by plating 2 ml of cells at a 1:5 dilution in 6-well plates, immediately adding the DNA mix (0.1 ml of Dulbecco's modified Eagle's medium/F-12 medium, 4-8 µg of plasmid DNA, 25 µl of Lipofectamine 2000), and growing cells 48 h. All other cell lines were grown in Dulbecco's modified Eagle's medium/F-12 medium, 5% fetal bovine serum and transfected as reported (6).

Generation of Phosphosite-specific TRH Receptor Antibodies—Phosphopeptides for immunization and rabbit antisera were prepared by New England Peptides (Gardner, MA). Peptides corresponding to the cytoplasmic tail of the rat TRH receptor containing three or four phosphorylated Ser and Thr residues and an N-terminal acetylated Cys addition were coupled through the Cys to keyhole limpet hemocyanin and used separately as antigens in rabbits, as summarized in Fig. 2. The peptide used for Ab5209 had two phosphorylated residues and a C-terminal Cys addition. Sera were screened by ELISA against the phosphopeptides and had a titer between 1:100,000 and 1:650,000, except Ab5209, which had a titer of 1:25,000. Antibodies were affinity-purified over columns with the immunizing peptide coupled to an insoluble support using the SulfoLink kit (Pierce).

Fixed Cell ELISA—To measure phosphorylated TRH receptor, a modified ELISA protocol was used in which antibody was added to fixed cells. Specifically, cells were rinsed on ice with PBS, fixed for 5 min with 1:1 MeOH/acetone (stored at -20 °C), air-dried, washed with PBS for 5 min, blocked for 20 min with radioimmune precipitation (RIPA)/milk buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, 10 mM NaF, 100 nM sodium orthovanadate, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, pH 8.0, 5% nonfat dried milk), incubated with 1:500-1:000 affinity-purified antibody for 90 min, washed three times for 5 min with PBS, incubated with 1:5000-7500 horseradish peroxidase-linked anti-rabbit secondary antibody for 45 min, washed three times for 5 min with PBS, and incubated for 5-20 min with BM Blue POD Substrate (Roche Applied Science). The reaction was terminated with 5% sulfuric acid, 300 µl were transferred to a 96-well plate, and the absorbance was read at 450 nm on a Benchmark Plus microplate spectrophotometer (Bio-Rad).

To optimize detection of phosphorylated TRH receptors, different fixation and permeabilization procedures were tested. The ELISA signal to background ratio was higher when cells were permeabilized with RIPA buffer than with Nonidet P-40 and higher with MeOH/acetone than with paraformaldehyde fixation (supplemental Fig. 1). Combinations using two other blocking agents, goat serum or bovine serum albumin, and two other detergents, Tween or Triton X-100, were tested, but none was as effective as RIPA buffer with milk (data not shown). The pattern of phosphorylation over time was the same regardless of fixative, buffer, or detergent.

To block phosphosite-specific antibody binding, immunizing peptides were incubated with antibody for at least 20 min at high concentration and then diluted to 1-10 µg/ml in blocking buffer before use. In order to dephosphorylate receptors, fixed cells were incubated with -phosphatase in reaction buffer (50 mM Tris-HCl, 100 mM NaCl, 2 mM dithiothreitol, 0.1 mM EGTA, 0.01% Brij 35, pH 7.5), or 100 units/ml alkaline phosphatase in 15 mM Tris, pH 9.5, for 1-2 h at 37 °C. Phosphatase was removed with three quick washes with PBS.

To measure surface TRH receptors, cells in multiwell plates expressing receptors with N-terminal HA epitopes were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, blocked in PBS containing 5% goat serum, and incubated with mouse anti-HA antibody (Covance) at 1:5000 followed by goat horseradish peroxidase-anti-mouse antibody (Bio-Rad) at 1:7500. Washes and color development were the same as described above.

Receptor Internalization—An antibody feeding protocol proved to be the most sensitive assay for internalization in CHO cells. In this method, cells expressing receptor with an N-terminal HA tag were incubated with anti-HA antibody at 1:1000 in labeling buffer (F-12 medium, 20 mM HEPES, pH 7.4, 5% goat serum) for 2 h at room temperature to label surface receptors. They were then washed twice for 5 min at room temperature in labeling buffer and incubated with or without TRH at 37 °C for the indicated times. Plates were placed on ice and washed, and the cells were fixed for 20 min with 3% paraformaldehyde in PBS without detergents and blocked in PBS with 5% goat serum. The amount of surface antibody was measured by ELISA using anti-HA antibody. The loss of surface receptor was the difference in ELISA signal between TRH-treated and naive cells. Background obtained without antibody or in untransfected cells was normally less than 10% of total signal and has been subtracted.

Immunoprecipitation and Western Blotting—Immunoprecipitation and Western blotting were performed essentially as described (3), except that crude serum for immunoprecipitation was used at 1:100 dilution, samples were rotated with 30 µl of Protein A/G beads at 4 °C overnight, and the pellet was not washed. Western blots of cell lysates were incubated with a 1:1000 dilution of antibodies against GRK2, -3, and -5 or a 1:500 dilution of anti-GRK6. Polyclonal anti-cyclophilin B was used at 1:5000.

RNA Extraction and cDNA Synthesis—Total RNA was extracted from GH3 cells using the RNeasy mini kit and treated with DNase I to digest contaminating DNA, and 1 µg was reverse transcribed into cDNA using oligo(dT) and MML reverse transcriptase, all according to the manufacturer's instructions. Reverse transcriptase was omitted as a negative control.

PCR—cDNA was amplified using primers corresponding to rat GRK2, -3, -5, and -6 or TRH receptor sequences. Primers were synthesized by Invitrogen and designed as follows: GRK2 forward, 5'-GATGAGGAGACACAAAAGGAATC-3'; GRK2 reverse, 5'-TCAGAGGCCGTTGGCACTGCCACGCTG-3'; GRK3 forward, 5'-AATTGAGGCCAGGAAGAAGGCTA-3'; GRK3 reverse, 5'-TCAGAGGCCGCTGCTATTTCTGTGACA-3'; GRK5 forward, 5'-GAACCACCAAAGAAAGGGCTG-3'; GRK5 reverse, 5'-CTAGCTGCTTCCAGTGGAG-3'; GRK6 forward, 5'-TTTGGGCTGGATGGGTCTGTTC-3'; GRK6 reverse, 5'-CGCTGCAGTTCCCACAGCAATC-3'. All PCRs were performed using TaqDNA polymerase in 50-µl volumes containing 2 µl of cDNA. For GRK cDNA amplification, 35 cycles of denaturation (94 °C for 60 s), annealing (55 °C for 60 s), and extension (72 °C for 60 s) were conducted with an automated thermal cycler. Product sizes were as expected: 606, 463, 144, and 123 bp for GRK2, -3, -5, and -6, respectively. Plasmids encoding GRKs were used as positive controls.

Synthesis and Transfection of Small Interfering RNAs (siRNA)—siRNA duplexes targeting rat GRK2 were synthesized by Dharmacon (Lafayette, CO). The sense siRNA sequences targeting rat GRK2 were 5'-GCAAGUGUCUCCUGCUUAAUU-3' (positions 1993-2011) and 5'-GAGCAAGGUGCCACUGAUUUU-3' (position 2168-2186). siRNA targeting cyclophilin B (sense sequence 5'-GGAAAGACUGUUCCAAAAA-3') was used as a control. A total of 0.2 ml of Opti-MEM containing 100 pmol of siRNA and 3 µl of Lipofectamine 2000 was added to 0.8 ml of suspended cells in 10% newborn calf serum in Dulbecco's modified Eagle's medium/F-12 medium, and cells were plated in 12-well plates.

Immunofluorescence Microscopy—To image phosphorylated receptor, GH3 cells were grown on untreated coverslips or BD Biocoat human fibronectin-coated coverslips (BD Biosciences, Bedford, MA). Fixing, blocking, and incubating with antibodies was performed exactly as in the fixed cell ELISA protocol, except that affinity-purified primary antibody was used at 1:100 and Alexa Fluor 546 goat anti-rabbit secondary antibody was used at 1:500. To image HA-tagged TRH receptor, CHO cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, blocked in PBS containing 5% goat serum and 0.2% Nonidet P-40, and then incubated for 90 min with mouse monoclonal anti-HA antibody at 1:1000, washed, and incubated for 45 min with goat Alexa 546-labeled anti-mouse antibody at 1:1000. Coverslips were mounted using ProLong Gold with DAPI and imaged as described (19).

Immunofluorescence in Rat Pituitary Tissue—The present study was approved by the University of Rochester Committee on Animal Resources and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Lactating Sprague-Dawley rats were anesthetized with chloral hydrate/sodium pentobarbital, injected intraperitoneally with saline or 0.1 mg/kg TRH, and sacrificed after 5 min by cardiac perfusion with 100 ml of perfusion buffer (150 mM NaPO₄ (pH 7.2), 145 mM NaNO₂, 1000 units/ml heparin) followed by fixation with 200 ml of low pH buffer (100 mM sodium acetate, 4% paraformaldehyde, pH 6.5) and 200 ml of high pH buffer (100 mM sodium borate, 4% paraformaldehyde, pH 9.5). Harvested pituitaries were stored in high pH buffer at 4 °C overnight and embedded in paraffin and sectioned by the University of Rochester Pathology Core. Sections were deparaffinized with three 5-min washes in xylenes and rehydrated with 10-min washes in 100, 95, 80, and 50% ethanol in PBS and two 10-min washes in PBS. Following rehydration, sections were stained for phosphorylated TRH receptor, prolactin, or TSH using the procedures described above for GH3 cells, except that primary antibody was applied overnight at room temperature and secondary antibody was applied for 3 h. Ab6959 was used at 1:20, anti-prolactin was used at 1:500, and anti-TSH was used at 1:500.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Ab6959 specifically recognizes phosphorylated TRH receptor in pituitary cells. A, GH3 cells were treated without or with 30 µM chlordiazepoxide (CDE) or 30 µM midazolam (MID) for 30 min. TRH (30 nM) was then added or not for 2 min. Cells were fixed and blocked, Ab6959 was added without or with 4pp6959, and phospho-TRH receptor was detected by ELISA. Shown are mean ± range of duplicate determinations representative of two independent experiments. **, p < 0.01 versus naive vehicle-treated cells. B and C, GH3 cells were grown without or with 10 nM 17-estradiol (E2) for 48 h. B, receptor concentration was determined by incubating cells with 5 nM [3H]MeTRH. C, cells were incubated with or without 100 nM TRH for 1 min, and phospho-TRH receptor was measured by ELISA with Ab6959. D, GH3 and GH-Y cells were treated or not with 30 nM TRH for 2 min and fixed and incubated without or with Ab6959 to quantify phospho-TRH receptor by ELISA. E, GH3 cells were treated with 1 µM TRH for 1 min, fixed, and incubated with 0-20,000 units/ml -phosphatase as described under "Experimental Procedures" before measuring phospho-TRH receptor by ELISA with Ab6959. Shown are means ± range of duplicate determinations representative of two experiments. B-D, **, p < 0.01.

Statistical Analysis—Data are shown as mean ± S.E. of triplicate determinations representative of at least three independent experiments unless otherwise noted. Where not visible, error bars fell within symbol size. Data were statistically analyzed, as appropriate, with one-way analysis of variance and post hoc Tukey's test or Student's unpaired t test, and differences were considered significant at p < 0.05 or 0.01, as indicated.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Phosphorylation of Endogenous TRH Receptors in Pituitary Cells—Previous studies of Ala-substituted and truncated TRH receptors suggested that potential phosphosites between residues 350 and 380 of the cytoplasmic tail are important for internalization and desensitization (20, 21). We prepared a rabbit antibody against a peptide representing residues 351-370 with all four Ser and Thr residues phosphorylated. This antibody was tested further to determine whether it could be used to analyze phosphorylation of endogenous TRH receptors.

Phosphorylation was measured by a modified ELISA procedure in which rat pituitary GH3 cells, which endogenously express TRH receptor (22), were incubated with or without TRH, and cells were fixed in culture dishes and then incubated with affinity-purified antibody (Ab6959). A small ELISA signal was obtained with Ab6959 in unstimulated GH3 cells, but the signal increased dramatically (5-20-fold) when cells were incubated with TRH (Fig. 1A). Chlordiazepoxide and midazolam, both inverse agonists at the TRH receptor, did nothing to the basal absorbance but blocked the TRH-induced signal. The immunizing phosphopeptide, 4pp6959, completely blocked the TRH-induced signal but did not affect the absorbance in naive cells.

TRH receptor expression is reportedly up-regulated by 17-estradiol in GH3 cells (23). Consistent with this, overnight incubation with 17-estradiol increased both [³H]MeTRH binding and TRH receptor phosphorylation by about 75% (Fig. 1, B and C). There was a linear correlation between [³H]MeTRH binding and the phosphoreceptor ELISA signal when receptor concentration was manipulated by plating cells at different densities or by growing cells in dexamethasone (a glucocorticoid that increases TRH receptors), ICI 182,780 (an estrogen receptor antagonist that blocks the action of estrogens from serum and decreases TRH receptor density), or 17-estradiol (data not shown).

To determine the specificity of Ab6959 for phosphorylated TRH receptors, we compared the ELISA signal in GH3 cells to that of GH-Y cells, a rat pituitary cell line that does not express TRH receptors (17). There was virtually no ELISA signal in either cell type when primary antibody was omitted, and the addition of antibody caused the same small signal in both cell types (Fig. 1D). GH-Y cells showed the same low level of absorbance as unstimulated GH3 cells due to nonspecific antibody binding.

To confirm that Ab6959 binding was due to receptor phosphorylation and not to other agonist-induced changes in receptor conformation, fixed cells were incubated with phosphatase prior to the addition of primary antibody. The phosphoreceptor signal was reduced to near background levels by -phosphatase (Fig. 1E) and by alkaline phosphatase at 100 units/ml (data not shown). Phosphatase treatment did not decrease the ELISA signal in cells that had not been exposed to TRH. As an additional control, when a polyclonal antibody against a nonphosphorylated peptide representing the same region was used (24), the ELISA signal was strong with or without TRH (data not shown). Together, the data in Fig. 1 show that Ab6959 specifically recognizes the phosphorylated TRH receptor and that there is no detectable constitutive phosphorylation of the receptor at residues 355-365.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. Phosphorylation of TRH receptors in different cell lines. CHO cells were transiently transfected or not with TRH receptor. GH-Y cells, which lack TRH receptors, and GH3 cells, which endogenously express TRH receptors, were grown to the same density. All cell lines were treated without or with 1 µM TRH for 5 min, and phospho-TRH receptor was detected by ELISA using different phosphosite-specific antibodies that were affinity-purified (Ab6959, -5025, -5211, and -5213) or used as crude serum (Ab5209). The TRH-induced increase in the ELISA signal of transfected over nontransfected CHO cells was normalized to the value obtained with Ab6959; analogously, GH3 cells were compared with GH-Y cells. Phosphopeptide sequences and their corresponding receptor regions are shown. An acetylated N-terminal Cys residue was added to the immunizing peptides for coupling, and the carboxyl terminus was amidated. The rat TRH receptor C-tail is shown. The amino acid sequence is highly conserved; the nonconserved region, which is absent in some species, is shadowed. Ser and Thr residues in the conserved region are highlighted in color corresponding to the phosphosite-specific antibodies generated to detect those residues.

View larger version (43K): [in this window] [in a new window]   FIGURE 3. Identification of TRH receptor phosphorylation sites. A, CHO cells were transfected with HA-tagged TRH receptors with or without Ala substitutions for the Ser and Thr residues in the 355-365 region and/or truncation at residue 370, as shown. Left, cells stably expressing TRH receptors were incubated with vehicle or 100 nM TRH for 5 min. Right, transiently transfected cells were incubated with vehicle or 1 µM TRH for 5 min. Phospho-TRH receptor was quantified by ELISA using Ab6959 and normalized to wild type (Wt) receptor-expressing cells. **, p < 0.01. B, HEK293 cells were transiently transfected with HA-tagged TRH receptors. Cells were labeled with 32P and incubated with or without 1 µM TRH for 5 min, and receptors were isolated by immunoprecipitation with anti-HA antibody and SDS-PAGE. Gels were used for immunoblotting with anti-HA antibody (top) or phosphorimaging (bottom). Similar results were obtained with CHO cells, but HEK293 cells were used here due to higher levels of 32P labeling. C, 32P incorporation as a percentage of wild type receptor from three experiments performed as in B. Densitometry from immunoblots was used to correct for differences in receptor expression. **, p < 0.01 versus wild type; ##, p < 0.01 between sets. D, GH3 cells transiently transfected without or with HA-tagged TRH receptor were treated with 1 µM TRH for 2 min. Aliquots of each lysate were immunoprecipitated with Ab6959, Ab5025, Ab5211, or Ab5213 and immunoblotted with anti-HA antibody. Shown are blots from the same experiment, representative of three independent experiments.

The differences between transfected and endogenous TRH receptors could be explained by alternative splicing, but this does not appear to be the case. Alternative splicing of the type 1 rat TRH receptor can give rise to mRNAs encoding a 412-amino acid long form or a 387-amino acid short form that is identical to the long form for the first 375 amino acids but differs in the last 12. Using two different primer pairs, we found that the long form comprises the vast majority of total TRH receptor mRNA in GH3 cells (data not shown), in agreement with previous reports for GH3 cells and pituitary tissue (25). If the protein concentrations are proportional to the message levels, the small amount of short receptor will not affect the results described here.

View larger version (44K): [in this window] [in a new window]   FIGURE 4. Importance of 355-365 region for internalization and desensitization. A-C, stable CHO cell lines expressing HA-tagged wild type (Wt) or mutant receptors at similar concentrations were used. A, an antibody prefeeding method described under "Experimental Procedures" was used to measure receptor internalization in response to 100 nM TRH. B, cells incubated with or without 100 nM TRH for 30 min were fixed with paraformaldehyde, permeabilized, and stained with anti-HA antibody. C, cells were incubated, and internalization was measured as in A. Levels of receptor expression were similar. *, p < 0.05; **, p < 0.01 compared with wild type. D, -Arr1/2KO MEFs were transfected with HA-tagged wild type or mutant TRH receptors plus vector alone or -arrestin 2. Cells were labeled with [3H]inositol and incubated with 10 mM LiCl with or without 100 nM TRH for 30 min when total 3H-labeled inositol phosphates were measured. The figure shows results for TRH-stimulated inositol phosphate production normalized to correct for small differences in surface TRH receptor expression, which was measured by ELISA with anti-HA antibody. *, p < 0.05; **, p < 0.01.

To estimate the fraction of total phosphorylation represented by the 355-365 region of the receptor, we transiently transfected cells with wild type and 4Ala-substituted full-length receptors and with wild type and 4Ala receptors truncated at residue 370 (371Stop and 4Ala371Stop, respectively). We then labeled cells with ³²P and purified receptors by immunoprecipitation with antibody to the HA epitope. The amount of ³²P incorporated into receptor was reduced by almost half in the 4Ala mutant and over 75% in the truncated 4Ala mutant (Fig. 3, B and C). This result implies that phosphorylation in the 355-365 region represents a substantial fraction of total TRH-induced receptor phosphorylation and that additional phosphorylation sites are located in or dependent upon the distal tail. As shown in Fig. 3B (top), only the wild type and 4Ala full-length receptors underwent a mobility shift in response to TRH.

Antibodies against four distinct receptor regions (Abs 6959, 5025, 5211, and 5213) were tested for their ability to immunoprecipitate TRH receptor from GH3 cells transfected with HA-tagged receptors (Fig. 3D). None of the antisera immunoprecipitated receptor from naive cells, confirming that there is no constitutive phosphorylation in the cytoplasmic tail and that the antibodies do not bind to nonphosphorylated receptor. Ab5025 and Ab5213 immunoprecipitated less HA-tagged TRH receptor than Ab6959 from TRH-treated GH3 cells (29 ± 11 and 24 ± 11%, respectively, compared with Ab6959; p < 0.01). There was no appreciable immunoprecipitation with Ab5211, similar to what was seen by ELISA with this antibody (Fig. 2). The results indicate that in pituitary GH3 cells, amino acids 355-365 form the major phosphorylation site, whereas more distal sites undergo a lesser degree of phosphorylation.

Functional Significance of Receptor Phosphorylation—Receptor phosphorylation is usually required for desensitization and internalization via -arrestin. To understand the functional significance of phosphorylation at residues 355-365, we characterized desensitization and internalization in CHO cell lines stably expressing either wild type or mutant receptors. CHO cells were used instead of GH3 cells to allow analysis of mutant receptors in the absence of endogenous receptors.

Internalization was studied using an antibody prefeeding method to measure receptor endocytosis. The wild type receptor internalized extensively, but the 4Ala mutant did not (Fig. 4A). The internalization defect in the 4Ala receptor was confirmed using two additional methods to follow the loss of receptors from the cell surface (data not shown). In one, cells were incubated with or without hormone, fixed, and then incubated with antibody to the N-terminal receptor epitope without permeabilization. In the other, cells were incubated with or without hormone, washed with a mild acid buffer to remove surface-bound TRH, and then incubated with [³H]MeTRH on ice such that only surface receptors bound.

Immunofluorescence microscopy was used to localize wild type and 4Ala receptors in naive and hormone-treated CHO cells. After 30 min of TRH exposure, wild type receptors had undergone endocytosis and were coalesced in a deep endocytic compartment, whereas 4Ala receptors were either on the membrane or in small vesicles close to the surface (Fig. 4B).

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Phosphorylation by downstream kinases. Phosphorylated TRH receptor was detected by ELISA using Ab6959 (A-C) or Ab5025 (D). A, GH3 cells were treated with 1 µM PMA, 1 µM ionomycin, 1 µM PMA plus 1 µM ionomycin with or without 100 nM TRH, or 100 nM TRH for 1 min. **, p < 0.01 versus naive cells. B, GH3 cells were treated with 10 nM BIM, 30 µM BAPTA/AM, or BIM plus BAPTA/AM for 15 min or 1 µM PMA overnight before 100 nM TRH was added for 20 s. After overnight incubation with PMA, PKC was undetectable by immunoblotting (not shown). *, p < 0.05; **, p < 0.01 compared with cells treated with TRH alone. C, CHO cells transiently transfected with HA-tagged wild type (Wt) or mutant TRH receptors were incubated with 1 µM PMA and 1 µM ionomycin for 1 min. **, p < 0.01 versus wild type. D, GH3 cells were treated without or with 100 nM TRH plus or minus 1 µM PMA and 1 µM ionomycin for 1 min. The substantial background ELISA signal with Ab5025 in naive GH3 cells was not due to receptor phosphorylation, because it was not reduced by alkaline phosphatase under conditions that reduced the TRH-stimulated signal (data not shown) and was not found by immunoprecipitation (Fig. 13A). **, p < 0.01; n.s., not significant.

To determine the importance of phosphorylation at residues 355-365 for desensitization, we measured signaling of wild type and mutant receptors in -Arr1/2KO MEFs. In these cells, wild type receptor signals strongly but does not internalize well; expression of -arrestin suppresses signaling and promotes receptor internalization. -Arrestin inhibited TRH-stimulated second messenger production by the wild type receptor and by the receptor truncated at residue 370, but it had little effect on signaling by either full-length 4Ala or truncated 4Ala receptors (Fig. 4D). The results show that phosphorylation of Ser and Thr residues in the 355-365 region is important for -arrestin-mediated desensitization. Both -arrestin 1 and -arrestin 2 inhibited TRH-stimulated inositol phosphate production effectively.³

Kinases Responsible for TRH Receptor Phosphorylation—GPCRs are typically phosphorylated by GRKs, which recognize the activated conformation of the receptor and/or by downstream kinases. The 355-365 region of the TRH receptor contains consensus PKC sites at residues 360 and 364, and activation of the receptor increases intracellular calcium and PKC activity. To determine whether the second messenger-activated PKC or calcium-sensitive pathways are responsible for the rapid phosphorylation of TRH receptors, GH3 cells were treated with the phorbol ester PMA and/or the calcium iono-phore ionomycin to activate downstream pathways or with the PKC inhibitor bisindolylmaleimide (BIM) and/or the intracellular calcium chelator BAPTA/AM, to inhibit signaling. Alternatively, cells were exposed to PMA overnight, which effectively depletes conventional and novel PKCs. PMA and ionomycin did not alter basal phosphorylation when added individually but caused a small increase when added together; this effect on basal phosphorylation was not additive with phosphorylation due to TRH (Fig. 5A). The effect of PMA and ionomycin was completely abolished in CHO cells when Ser³⁵⁵ was mutated to Ala, suggesting that PKC phosphorylates Ser³⁵⁵, although it is not a classical PKC site (Fig. 5C and supplemental Fig. 2). Overnight exposure to PMA did not affect TRH-induced phosphorylation (Fig. 5B), indicating that PKC is not required for phosphorylation. BIM and BAPTA/AM caused a slight decrease in TRH-induced phosphorylation that was probably due to drug toxicity, because it was irreversible and also seen with Ab5025 (data not shown), which recognizes a distal region of the receptor tail that is not phosphorylated in response to PMA and ionomycin (Fig. 5D). These data show that downstream kinases play at most a minor role in TRH-induced phosphorylation and suggest that a GRK is the relevant kinase.

GRK2, -3, -5, and -6 are widely expressed, and mRNA for each was detected by reverse transcription-PCR in GH3 cells (Fig. 6A). When GH3 cell lysates were blotted for GRK proteins, however, GRK2 was the major form, and GRK6 was barely detectable (supplemental Fig. 3). Pituitary cells have previously been reported to express GRK2; some studies have also identified GRK3 and GRK6 protein, but others have found only the mRNAs (26, 27).

Because GRK2 is the dominant GRK in GH3 cells, its importance for TRH receptor phosphorylation at residues 355-365 was analyzed by two methods. First, GH3 cells were transiently transfected with an HA-tagged TRH receptor and wild type or kinase-deficient, dominant negative GRK2. Cell lysates were immunoprecipitated with Ab6959 against phosphorylated TRH receptor and then immunoblotted with anti-HA antibody. As mentioned under "Experimental Procedures," Ab6959 was not effective on Western blots, but this co-transfection approach allowed analysis of tagged receptor in the small fraction of successfully transfected cells. Phosphorylated TRH receptors were immunoprecipitated from GH3 cells exposed to TRH, but not from unstimulated cells (Fig. 6B). When GRK2 was co-expressed with receptor, substantially more receptor was phosphorylated, but very little phosphorylated receptor was retrieved when dominant-negative GRK2 was co-expressed. Comparable results were seen when phospho-TRH receptor was detected by ELISA in CHO cells co-transfected with TRH receptor and wild type or dominant negative GRK2 (supplemental Fig. 2).

View larger version (63K): [in this window] [in a new window]   FIGURE 6. Role of GRKs in TRH receptor phosphorylation. A, mRNA from GH3 cells was incubated without or with reverse transcriptase (RT) and amplified using primers for GRK2, -3, -5, or -6. GRK plasmid cDNAs were used as controls. Gels shown are representative of two independent experiments. Only GRK2 was readily detected by Western blot (supplemental Fig. 3). B, GH3 cells transfected or not (NT) with HA-tagged TRH receptor, and GRK2 or dominant-negative (DN) GRK2 were stimulated with 100 nM TRH for 1 min. Phospho-TRH receptor was immunoprecipitated using Ab6959 crude serum and detected by Western blot using an anti-HA antibody. The blot shown is representative of two independent experiments. Overexpression of wild type GRK2 increased the amount of phospho-TRH receptor immunoprecipated by at least 2-fold, whereas dominant-negative GRK2 reduced pull-down by over 70%. C, GH3 cells incubated with GRK2 or cyclophilin B (CyPB) siRNA were treated with 100 nM TRH for 1 min, and phospho-TRH receptor was measured by ELISA using Ab6959. Shown are means ± range of duplicate determinations representative of three independent experiments. *, p < 0.05. D, representative immunoblot of cell lysates from C with anti-GRK2 or anti-CyPB antibodies. Densitometric analysis of three independent experiments showed that GRK2 protein levels were reduced by 46 ± 6% (p < 0.01) with GRK2 siRNA, with no significant changes with CyPB siRNA.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Time course of TRH receptor phosphorylation. A, left, GH3 cells were treated with 1-1000 nM TRH for various times. Right, dose-response curve of 1 min time points. Shown are means ± range of duplicate determinations representative of at least three independent experiments. B, left, GH3 cells were treated with 1 µM TRH for various times. Right, medium with freshly prepared TRH (fresh) or TRH that had been incubated with cells for 1000 min (recycled) was added to naive cells for 1 min to stimulate phosphorylation; n.s., not significant. A and B, phospho-TRH receptor was measured by ELISA with Ab6959.

Kinetics of TRH Receptor Phosphorylation—GH3 cells were treated with various concentrations of TRH, and phosphorylated TRH receptor was measured at different times in order to determine the kinetics of phosphorylation. Phosphorylation was extremely rapid, reaching a maximum by 30 s in GH3 (Figs. 7A and 13A) and CHO cells (supplemental Fig. 4A). Phosphorylation dropped gradually over the next 30 min in cells exposed to high concentrations of TRH but remained elevated for at least 16 h in the continued presence of agonist (Fig. 7B). The decline in ELISA signal after prolonged TRH exposure was not due to degradation of the peptide, because spent medium from cells incubated with TRH overnight was able to elicit the same response as freshly prepared TRH (Fig. 7B, right).

Comparison of the maximal phosphorylation obtained at different concentrations of TRH showed that the EC₅₀ was 30 nM (Fig. 7A, right), yet the K_d of TRH for its receptor in intact cells is 10 nM (22). This discrepancy is probably due to the fact that arrestins, which bind after the receptor is phosphorylated, increase the affinity of the receptor for TRH (6). Equilibrium binding experiments in whole cells measure the affinity of the agonist-receptor-arrestin complex, whereas phosphorylation assays measure a step that precedes arrestin binding and depends on the affinity of TRH for the receptor without arrestin. The EC₅₀ for phosphorylation is within the range reported for half-maximal inositol 1,4,5-trisphosphate production (28, 29).

Subcellular Localization of Phosphorylated TRH Receptor—The subcellular localization of phosphorylated TRH receptor in GH3 cells was identified by immunofluorescence with Ab6959 after various times of incubation with TRH (Fig. 8). There was almost no fluorescence when cells were not exposed to agonist or when Ab6959 was blocked with immunizing peptide (Fig. 8, A and C, respectively). Phosphorylated receptor was easily visualized in cells incubated for 30 s with TRH when staining was confined to the plasma membrane (Fig. 8B). Phosphorylated TRH receptor was detected in endosomes deeper inside the cell after 10 min or longer incubations with TRH (Fig. 8D and data not shown). When cells were incubated with TRH for 1 min, washed, and incubated without agonist, immunofluorescence was nearly gone by 10 min (Fig. 8, E and F).

View larger version (69K): [in this window] [in a new window]   FIGURE 8. Subcellular localization of phospho-TRH receptor. A-F, GH3 cells were treated without or with TRH, fixed, blocked, and probed with either Ab6959 or Ab6959 preincubated with 4pp6959 (1 µg/ml). Primary antibody was detected using anti-rabbit secondary antibody conjugated to Alexa 546 (red); nuclei were stained with DAPI (blue). A, vehicle; B, 1 µM TRH for 30 s; C, 1 µM TRH for 30 s and 4pp6959; D, 1 µM TRH for 30 min; E, 100 nM TRH for 1 min; F, 100 nM TRH for 1 min followed by a 9-min washout. Images were exposed and processed identically.

View larger version (20K): [in this window] [in a new window]   FIGURE 9. Time course of TRH receptor dephosphorylation. GH3 cells were treated with 10-1000 nM TRH (A) for 5 min or 100 nM TRH (B) for 1-30 min. Cells were washed to remove TRH and incubated in agonist-free medium for various times. Shown is phospho-TRH receptor remaining after agonist removal, measured by ELISA with Ab6959.

As discussed above, TRH receptors are still at the plasma membrane after only 1 min with TRH but mostly internalized after 30 min. The results shown in Fig. 9 suggest that TRH receptors are more readily dephosphorylated at the plasma membrane. To test this idea, we used hypertonic sucrose to block internalization. Hypertonic sucrose completely blocked TRH receptor internalization at early time points (Fig. 10, A and C) and reduced agonist internalization by 50% after 30 min. When cells were incubated with TRH for 30 min and then agonist was removed, hypertonic sucrose accelerated dephosphorylation, supporting the conclusion that dephosphorylation takes place faster when receptors are at the surface (Fig. 10B). The same experiment was repeated with a 5-min incubation with TRH, when many receptors are still on the plasma membrane. In this case, hypertonic sucrose did not affect the rate of dephosphorylation (data not shown). Furthermore, the rate of dephosphorylation was unaffected by the time of TRH exposure in MEFs lacking both of the nonvisual arrestins, -arrestins 1 and 2, where rapid internalization does not occur. Following transfection of -Arr1/2KO MEFs with -arrestin, the rate of dephosphorylation at residues 355-365 was slower after 60 min of TRH exposure, when the majority of receptors are internalized, than after 1 or 5 min, when receptors are primarily at the plasma membrane (data not shown). This is in agreement with results we previously reported using mobility shift to follow dephosphorylation of transfected receptors in heterologous cells (6).

Whereas no GPCR-specific phosphatase has been identified, several receptors are dephosphorylated by a PP2A- or PP1-like phosphatase based on pharmacological inhibition (30-34). Several phosphatase inhibitors were tested for their ability to inhibit dephosphorylation of the TRH receptor. Cantharidin (up to 500 nM), microcystin LF (up to 2 nM), okadaic acid (up to 100 nM), and calyculin A (up to 5 nM) had no effect on dephosphorylation at concentrations sufficient to inhibit PP1 by at least 50% (cantharidin or microcystin LF) or to inhibit both PP1 and PP2A completely (okadaic acid or calyculin A). Cyclosporin A (1 µM), an inhibitor of PP2B (calcineurin), was also ineffective (data not shown). Based on these results, it seems unlikely that the TRH receptor is dephosphorylated by members of the PP1, PP2A, or PP2B families.

Receptor Rephosphorylation—We tested when receptors that had been dephosphorylated become capable of being reactivated and rephosphorylated. TRH was added to GH3 cells for either 30 s, after which receptors were at the cell surface, or for 30 min, after which most receptors should have undergone internalization. TRH was then removed, and the cells were allowed to recover for various times before TRH was reapplied. Receptors that had been extensively internalized were 90% dephosphorylated in the first 15 min after TRH withdrawal, but reapplication of TRH led to rephosphorylation of only about one-third of receptors (Fig. 11A), suggesting that they had become dephosphorylated in vesicles and had not yet recycled to the surface, where they could interact with membrane-impermeant TRH. Receptors that were primarily on the surface were capable of undergoing extensive rephosphorylation as soon as dephosphorylation had taken place (Fig. 11B). Predictably, the extent of rephosphorylation was intermediate following a 5-min initial exposure to hormone (data not shown).

View larger version (54K): [in this window] [in a new window]   FIGURE 10. Effect of inhibiting endocytosis on TRH receptor dephosphorylation. A-C, GH3 cells were incubated in medium without or with 0.44 M sucrose for 20 min when 100 nM TRH was added for 0-30 min. A, cells were washed on ice to remove TRH and incubated on ice with 10 nM [3H]MeTRH for 60 min to label receptor remaining at the surface after stimulation with TRH, shown as percentage of time 0. B, TRH was removed after 30 min, and cells were allowed to recover in medium without or with sucrose for various times. Phospho-TRH receptor was measured by ELISA with Ab6959. C, phospho-TRH receptor was visualized by immunofluorescence microscopy as described in the legend to Fig. 8.

View larger version (18K): [in this window] [in a new window]   FIGURE 11. TRH receptor rephosphorylation. GH3 cells were treated with 100 nM TRH for 30 min (A) or 30 s (B). Some dishes were washed to remove agonist, and cells were allowed to recover for various times. 100 nM TRH was reapplied for 30 s or 1 min to some dishes. Phospho-TRH receptor was detected by ELISA using Ab6959. Filled squares, TRH present throughout; open squares, TRH removed and cells incubated in hormone-free medium; filled triangles, TRH reapplied. *, p < 0.05; **, p < 0.01. A, mean ± S.E. of triplicate determinations representative of at least three independent experiments; B, mean ± range of duplicate determinations representative of two independent experiments.

TRH receptors are largely confined to cells that secrete prolactin and TSH, and prolactin-secreting cells are abundant in the pituitary glands of lactating animals. To confirm that receptor phosphorylation can be used to track TRH receptor activation, we stained adjacent sections of pituitary glands with antibodies against phosphorylated TRH receptor, prolactin, or TSH (Fig. 12B). As predicted, phosphorylated receptor staining occurred in the same regions as staining for the two pituitary hormones. Additionally, prolactin and TSH staining appeared lighter in TRH-treated animals, suggesting that the lactotrophs and thyrotrophs underwent degranulation upon exposure to TRH.

Phosphorylation at Other Sites—Hierarchical phosphorylation, where phosphorylation at one site requires prior phosphorylation at another, has been reported for several GPCRs (35-39). We tested whether one site is phosphorylated earlier than the other by measuring the rates of phosphorylation at residues 355-365 and 371-378 simultaneously using Abs 6959 and 5025, respectively (Fig. 13, A and B). We also tested whether phosphorylation in these two regions occurs at different concentrations of agonist (Fig. 13E), which might imply the action of a different kinase. Both the rate and EC₅₀ value for phosphorylation were the same for the two regions. The rate of dephosphorylation and the subcellular localization of receptors phosphorylated in the two regions were likewise identical (Fig. 12, C and F).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The data reported here provide compelling evidence that in pituitary GH3 cells and in normal pituitary tissue, the TRH receptor undergoes rapid and extensive phosphorylation at amino acids 355-365 in response to agonist. The use of mutants lacking Ser and Thr residues in this region also shows that phosphorylation in this region is critical for internalization and -arrestin-dependent desensitization. More distal sites of the receptor undergo a lesser degree of phosphorylation in GH3 cells, whereas in some heterologous systems, these sites are heavily phosphorylated (Fig. 2; data not shown). The basis for the differences in receptor phosphorylation is not obvious from characterization of the GRKs expressed in different cell lines. The discrepancies between phosphorylation at more distal sites suggest a need for caution when trying to identify phosphorylation sites using only heterologous expression systems. Nevertheless, the kinetics of phosphorylation and dephosphorylation in the 355-365 region were similar in GH3 and CHO cells, and GRK2 was the dominant kinase in both cell types (supplemental Figs. 2 and 4).

View larger version (51K): [in this window] [in a new window]   FIGURE 12. TRH receptor phosphorylation in rat pituitary tissue. A and B, anesthetized animals were injected intraperitoneally with saline (n = 2) or 0.1 mg/kg TRH (n = 3), and after 5 min animals were sacrificed; perfusion, fixation, and slide preparation are described under "Experimental Procedures." A, adenohypophyseal region (x100 objective) showing nuclei stained with DAPI (blue) and phospho-TRH receptor stained with Ab6959 followed by Alexa 546-conjugated secondary antibody (red). B, adjacent sections (x16 objective) showing phospho-TRH receptor, prolactin, or TSH stained with their respective antibodies followed by Alexa 546-conjugated secondary antibody (red).

Because GRKs, like G proteins, recognize the activated state of the receptor, we thought that the receptor might display some basal phosphorylation due to its constitutive activity in the absence of TRH. Some GPCRs, such as the bradykinin B₂ receptor, are phosphorylated in the absence of agonist (12). With the TRH receptor, however, the low signal obtained with phosphosite-specific antibodies in naive cells was due to nonspecific antibody interaction with the cells, not receptor phosphorylation, because the signal did not depend on TRH receptor concentration, was not reduced by phosphatase treatment or excess immunizing peptide, and was not diminished by two inverse agonists tested at high concentrations. It is possible that the failure to detect constitutive phosphorylation was due to limited sensitivity of the phosphorylation assays compared with the reporter assays used to detect constitutive signaling. Alternatively, the time spent in an activated state or the conformation achieved by the unliganded receptor may be sufficient to activate G_q/11 but not to recruit GRK2.

The importance of phosphorylation in the 355-365 region for uncoupling from G proteins is highlighted by the finding that -arrestin suppresses TRH-induced inositol phosphate formation for wild type but not 4Ala receptors. Phosphorylation at amino acids 355-365 is also critical for receptor trafficking, because TRH-driven receptor endocytosis is strongly impaired for the 4Ala and T365A mutants. Importantly, a receptor truncated after residue 370 desensitized and internalized normally, indicating that distal phosphosites are not needed for these actions.

Several lines of evidence show that the TRH receptor undergoes additional phosphorylation in the distal tail. First, TRH-dependent ³²P incorporation in the 4Ala receptor is reduced by approximately one-half when the 4Ala receptor is truncated after residue 370. Second, the full-length receptor undergoes a mobility shift in response to TRH. This mobility shift is seen with the 4Ala mutant but not when 4Ala receptors are truncated after residue 370. Third, antibodies directed against phosphopeptides covering all of the potential phosphorylation sites in the cytoplasmic carboxyl terminus identify two additional phosphorylation regions in the distal tail.

View larger version (28K): [in this window] [in a new window]   FIGURE 13. TRH receptor phosphorylation in different regions of the C-tail. A-E, phospho-TRH receptor was detected by ELISA using Ab6959 (squares) or Ab5025 (circles) in GH3 cells treated with 1 µM TRH for various times (A and B); with 100 nM TRH for 5 min followed by a washout as described in Fig. 6 (C); without or with 2 µM TRH for 1 min (**, p < 0.01) (D); or 1-1000 nM TRH for 5 min (E). Shown are mean ± range of duplicate determinations representative of at least three independent experiments. No significant differences were observed between Ab6959 or Ab5025. F, GH3 cells were treated with 1 µM TRH for 0-10 min, and subcellular localization of phospho-TRH receptor (red) was detected by immunofluorescence with Ab6959 or Ab5025; nuclei were stained with DAPI (blue).

The TRH receptor is maximally phosphorylated within 30 s when TRH is added at high concentrations. Phosphorylation of other GPCRs is usually much slower; phosphorylation of the M2 muscarinic acetylcholine (44), ₂ adrenergic (45), A₃ adenosine (46), and CC chemokine 5 (15) receptors requires many minutes, and only the cone photoreceptor has been shown to reach maximal phosphorylation faster than the TRH receptor (47). The C5a receptor is phosphorylated very rapidly (t 20 s) by PKC on a Ser residue (48), but maximal phosphorylation at putative GRK sites requires several minutes (49). The speed of TRH receptor phosphorylation raises several questions, such as whether the receptor has time to signal and whether it spends most of its life in a desensitized state. The peak calcium response to high concentrations of TRH occurs within a second or two preceding phosphorylation. In the continued presence of TRH, inositol 1,4,5-trisphosphate remains slightly elevated, and calcium pools become profoundly depleted (50). This means that TRH continues to activate phospholipase C to some degree long after receptor phosphorylation has peaked. Sustained signaling could be due to a small fraction of receptors that are not phosphorylated or to continued signaling by phosphorylated receptors that are not bound to -arrestin.

Dephosphorylation takes place within a few minutes when receptors experience a brief pulse of agonist but much more slowly when the receptor has been occupied for longer times. The time frame of internalization is consistent with the switch from rapid to slow dephosphorylation, and inhibiting endocytosis prevents the shift to slow dephosphorylation. In principle, this difference could be due to some additional modification or protein interaction that follows the initial phosphorylation, but it seems more likely that it is the result of a change in the receptor's subcellular localization.

It has been suggested that dephosphorylation of GPCRs occurs only after receptors have internalized, based on the report that at least one site on the ₂-adrenergic receptor is dephosphorylated more readily following endocytosis (9, 51). This is not the case for all GPCRs, however, because the rate of dephosphorylation of the D1 dopamine receptor is unaffected by receptor internalization (52). In the case of the TRH receptor, internalization actually slows dephosphorylation. To our knowledge, this is the first report of a receptor that is more readily dephosphorylated at the plasma membrane.

It seems likely that internalized TRH receptors are dephosphorylated on cytoplasmic endosomes after acidification and dissociation of ligand and -arrestin and that they must then cycle to the plasma membrane before they can be activated again by TRH. It is not known whether dephosphorylation influences receptor trafficking. Receptors that had undergone dephosphorylation while on the surface of GH3 cells were ready for a second pulse of agonist, as indicated by rephosphorylation of receptors. Physiological fluctuations in TRH levels in hypophyseal portal blood are not readily measured, and it is not known whether the pituitary must respond to rapid changes in TRH concentration. The ability to recover quickly may be particularly important for the TRH receptor in the central nervous system, where its role in neurotransmission may require rapid responsiveness.

The use of novel phosphosite-specific polyclonal antibodies described here makes it possible to study TRH receptor phosphorylation in native cells and in tissue. The rapid responsiveness of the receptor to the presence or absence of agonist and the lack of constitutive phosphorylation render receptor phosphorylation a useful marker of receptor activity in vivo. This approach allowed us to ask whether receptors are normally quiescent, barely activated, or strongly stimulated by TRH reaching the pituitary from the hypothalamus. Two important findings were obtained. First, receptors are measurably active in a resting animal. Second, injection of a dose of TRH expected to activate most receptors results in a dramatic increase in pituitary phospho-TRH receptor staining. We can conclude that TRH receptors in an untreated animal are partially "on" but very far from maximally activated. Analysis of phosphorylated TRH receptors in animals in different physiological and pathological states holds great promise for dissecting the contribution of TRH signaling pathways in vivo. More generally, the use of phosphosite-specific antibodies to monitor receptor activation in vivo should have broad applicability.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health (NIH) Grant DK19974 (to P. M. H.), an NIH cardiovascular research training grant and a Pharmaceutical Manufacturers' Association predoctoral fellowship (to B. W. J.), and an Endocrine Society Summer Research Fellowship (to E. K. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4.

¹ To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, University of Rochester Medical Center, Box 711, Rochester, NY 14642. Tel.: 585-275-4933; Fax: 585-273-2652; E-mail: Patricia_Hinkle{at}urmc.rochester.edu.

² The abbreviations used are: BIM, bisindolylmaleimide; GPCR, G protein-coupled receptor; GRK, GPCR kinase; HA, hemagglutinin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TRH, thyrotropin-releasing hormone; MeTRH, methylhistidine-TRH; TSH, thyroid-stimulating hormone; MEF, mouse embryo fibroblast; BAPTA/AM, 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; DAPI, 4',6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary; siRNA, small interfering RNA; RIPA, radioimmune precipitation.

³ B. W. Jones, G. J. Song, and P. M. Hinkle, unpublished data.

	ACKNOWLEDGMENTS

 
We thank David C. Lawrence for excellent technical assistance.

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