Regulated Dimerization of the Thyrotropin-Releasing Hormone Receptor Affects Receptor Trafficking But Not Signaling

Gyun Jee Song and Patricia M. Hinkle

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

Address all correspondence and requests for reprints to: Patricia M. Hinkle, Department of Pharmacology and Physiology, University of Rochester Medical Center, Box 711, Rochester, New York 14642. E-mail: Patricia_Hinkle{at}urmc.rochester.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To investigate the function of dimerization of the TRH receptor, a controlled dimerization system was developed. A variant FK506 binding protein (FKBP) domain was fused to the receptor C terminus and dimerization induced by incubating cells with dimeric FKBP ligand, AP20187. The TRH receptor-fusion bound hormone and signaled normally. Addition of dimerizer to cells expressing the receptor-FKBP fusion dramatically increased the fraction of receptor running as dimer on SDS-PAGE. AP20187 caused dimerization in a time- and concentration-dependent manner, acting within 1 min. Dimerizer had no effect on TRH receptors lacking the FKBP domain, and its effects were blocked by excess monomeric FKBP ligand. AP20187-induced dimerization did not cause receptor phosphorylation, inositol phosphate production, or ERK1/2 activation, and dimerizer did not alter signaling by TRH. Induced dimerization did, however, alter TRH receptor trafficking. TRH promoted greater receptor internalization in cells treated with AP20187 but not monomeric ligand, based on loss of surface binding sites and immunostaining. Dimerization increased the rate of internalization of TRH receptors and decreased the apparent rate of receptor recycling. AP20187 enhanced the small amount of TRH-induced receptor internalization when the receptor-FKBP fusion protein was expressed in cells lacking ß-arrestins. The results show that controlled dimerization of the TRH receptor potentiates hormone-induced receptor trafficking.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE TRH RECEPTOR is a member of the superfamily of G protein-coupled receptors (GPCRs). TRH, a hypothalamic neuropeptide, has well-characterized roles in controlling synthesis and release of prolactin and TSH from the anterior pituitary gland through its action at the TRH receptor. The TRH receptor, once occupied by agonist, activates phospholipase C (PLC) through Gq/11, leading to the formation of inositol (1, 4, 5)trisphosphate (IP3), which elevates the intracellular free Ca2+ concentration by mobilizing an IP3-sensitive Ca2+ store in the endoplasmic reticulum (1) and also activates protein kinase C and MAPK (2, 3).

Bioluminescence resonance energy transfer analysis has been used to demonstrate that the TRH receptor dimerizes and that agonist stimulation causes a time- and dose-dependent increase in the amount of energy transfer (4, 5). We used coimmunoprecipitation of receptors tagged with different epitopes to demonstrate that the TRH receptor exists as homodimers or oligomers (6).

The existence of GPCR dimers or oligomers is now well known with a large number of reports suggesting that dimerization is a general phenomenon for the GPCR superfamily (7, 8, 9, 10). Earlier direct evidence for GPCR dimers came from studies involving functional reconstitution after coexpression of two nonfunctional mutant or chimeric receptors. Maggio et al. (11) used two {alpha}2-adrenergic/M3 muscarinic chimeric receptors in which the C-terminal receptor portions (containing transmembrane domains VI and VII) were exchanged. Neither of the two chimeric receptors alone showed any detectable binding activity, but binding of both muscarinic and adrenergic ligands was restored upon coexpression. When expressed alone, the metabotropic {gamma}-aminobutyric acid receptor GbR1 is retained intracellularly as an immature protein because it has a carboxyl-terminal endoplasmic reticulum retention motif. The GbR2 reaches the cell surface but is not functional if it is expressed alone. If the two receptors are coexpressed, the GbR1 endoplasmic reticulum retention signal is masked by heterodimerization and a functional GbR dimer is targeted to the plasma membrane (12).

Many reports describe the use of coimmunoprecipitation to demonstrate the existence of GPCR dimers and most of these studies involve coexpression of differentially epitope-tagged receptors including {alpha}1a-and ß2 adrenergic receptor, D2 dopamine receptor, {delta}-opioid receptor, and chemokine receptor CCR2 (13, 14, 15, 16, 17). Biophysical techniques represent a powerful tool because they enable the detection and monitoring of GPCR dimers in living cells. Fluorescence and bioluminescence resonance energy transfer have been used to demonstrate dimers of yeast pheromone receptor and GnRH, TSH, ß2-adrenergic, oxytocin, and vasopressin V2 receptors (4, 18, 19, 20, 21). This evidence has raised several questions concerning the importance of dimer complexes for receptor biosynthesis, signaling and trafficking (9). Little is known about the consequences of homodimerization of the TRH receptor. The objective of this study was to determine the function of TRH receptor dimerization by establishing a model in which receptor dimerization can be controlled. We show that induced dimerization does not, by itself, elicit a signal, but it does alter ligand-induced receptor redistribution.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulated Homodimerization System
We used a regulated homodimerization system based on human FK506 binding protein (FKBP) 12 and its small molecular ligands. A variant of FKBP (FKBPv) containing a hemagglutinin (HA) epitope tag was fused to the C-terminal end of the TRH receptor. AP21998, a monomeric ligand that binds to a single FKBPv, was used as negative control drug in this study. AP20187, a membrane-permeant dimeric drug that was created by chemically linking two of monomeric ligands by a short linker (22), was used to induce dimerization of the FKBPv-TRH receptor fusion protein (Fig. 1AGo). AP20187 binds to the FKBP variant but it binds very poorly, with 1000-fold lower affinity, to endogeneous FKBP.



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Fig. 1. Function of HA-Tagged TRHR-FKBPv Fusion Protein

A, Schematic representation of controlled receptor dimerization. B, Cells transiently transfected with TRHR-FKBPv-HA construct were treated with 100 nM TRH. IP3 was measured at 1 min by radioreceptor assay. C, Cells were incubated with fura2 and Ca2+ responses of individual cells were determined by measuring the 340/380 fluorescence ratio. TRH (100 nM) was added as noted. Points represent the mean ± SEM of responses from 25 cells. D, Cells were fixed and stained with antibody against the HA epitope.

 
Characteristics of HA-Tagged TRHR-FKBPv Fusion Protein
We first confirmed that the HA-tagged TRHR-FKBPv fusion protein functions normally in transiently transfected Chinese hamster ovary (CHO) cells. TRH activates PLC through Gq/11 to produce IP3 and diacylglycerol. The ability of TRH to stimulate an increase in [3H]inositol phosphate production and free intracellular Ca2+ ion concentration (Fig. 1Go, B and C) was comparable to that observed with the native receptor. The TRHR-FKBPv-HA fusion protein was clearly localized at the plasma membrane (Fig. 1DGo). For subsequent experiments, we used stable cell lines of CHO cells expressing either an HA-tagged TRH receptor, which has no FKBP domain, or a TRH receptor fused to the FKBPv-HA construct. The stable lines expressing HA-TRHR or TRHR-FKBPv-HA bound 0.65 or 0.55 pmol of [3H]MeTRH/mg of protein, respectively (Table 1Go). These levels are below those of pituitary GH3 cells, which express endogenous receptors (0.5–1.2 pmol/mg protein). We measured the affinities of the receptor fusion protein for [3H]MeTRH by Scatchard analysis (Table 1Go). Both the HA-TRH receptor and FKBPv-HA-TRH receptor showed the same affinity in CHO cells and their affinities are comparable to those previously reported for TRH receptors lacking the FKBP domain (6). These results show that insertion of the fragment FKBPv-HA into the carboxyl terminus of the rat TRH receptor does not disturb either receptor ligand binding or second messenger activation.


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Table 1. Expression Levels and Affinity for Agonist of TRH Receptors

 
AP20187-Induced TRH Receptor Dimerization
To establish that the dimeric FKBPv ligand AP20187 induces dimerization of TRH receptors, cells stably expressing TRHR-FKBPv-HA were treated with TRH with or without the dimerizer. Cell lysates were prepared and receptors were immunoprecipitated with anti-HA antibody and then run on SDS-PAGE and immunoblotted with mouse anti-HA antibody. Immunoreactive bands of TRH receptor monomer were detected at 85–90 kDa and dimers were detected at 180–200 kDa, appropriate for the glycosylated fusion proteins. AP20187 caused receptor dimerization with or without TRH (Fig. 2AGo, lanes 3 and 4). When an excess (10 µM) of the monomeric ligand AP21998 was added together with the dimerizer, the dimer band disappeared, indicating that the monomeric drug competed effectively with the bivalent ligand (Fig. 2AGo, lane 5). The monomeric ligand AP21998 did not cause dimerization by itself (Fig. 2AGo, lanes 6 and 7). As shown in Fig. 2BGo, the ratio of the intensity of the dimer to monomer band increased from an average of 0.72 in untreated cells to 3.96 in cells exposed to dimerizer (n = 5). There was considerable variability among experiments in the fraction of receptor running as dimer, however (compare Fig. 2BGo with Fig. 3Go, B and C, for example). After deglycosylation, receptor ran at 50–60 kDa and 120 kDa, consistent with the predicted molecular masses of 60 kDa and 120 kDa for receptor monomer and dimer, respectively (Fig. 2CGo). Receptor phosphorylation occurs when cells are exposed to TRH and this phosphorylation causes a small up-shift in receptor mobility (6). This was previously documented by 32P-labeling and phosphatase reversal of the up-shift. After deglycosylation of TRHR-FKBPv-HA, the receptor monomer band overlapped with IgG heavy chain, particularly when phosphorylated (Fig. 2CGo). When cells were exposed to the dimerizer AP20187 by itself, the intensity of the dimer band increased but there was no up-shift characteristic of phosphorylation (Fig. 2CGo, lane 3). TRH caused a small up-shift in mobility of TRHR-FKBPv-HA (Fig. 2CGo, lanes 2, 4, and 6), indicating that the fusion receptor was normally phosphorylated in response to activation.



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Fig. 2. AP20187-Induced Dimerization of the TRH Receptor

A, CHO cells stably expressing TRHR-FKBPv-HA were treated with 100 nM TRH, 100 nM AP20187, and/or 100 nM (lanes 6 and 7) or 10 µM (lane 5) AP21998 at 37 C for 30 min. The cell lysates were immunoprecipitated and immunoblotted with anti-HA antibody. Mobility of molecular mass (MW) markers (kDa) is indicated. B, Bar graph shows the mean ± SEM of the ratio of the relative intensities of the dimer to monomer band averaged from five independent experiments (lanes 1–4) or duplicate experiments (lanes 5–7). When the fraction of receptor running as dimer in AP20187-treated dishes was compared with that in control dishes in the same experiment (i.e. lane 3 vs. lane 1 above), AP20187 increased the proportion of dimers an average of 11.6 ± 2.7-fold (n = 5). C, Immunopurified receptors treated with PNGase for 1 h and immunoblotted with anti-HA antibody. D, Dimer; M, monomer.

 


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Fig. 3. Concentration Dependence and Time Course of AP20187-Induced TRH Receptor Dimerization

A, Cells stably expressing TRHR-FKBPv-HA were incubated with 0.1–100 nM AP20187 for 30 min. B, Cells were treated with 10 or 100 nM AP20187 for 0–60 min. Cell lysates were immunoprecipitated with anti-HA antibody. Samples (20 µl) were immunoblotted with anti-HA antibody. C, AP20187 (100 nM) was added to live cells (Cells) or cell lysates (Lys) before immunoprecipitation and immunoblotting. Arrows show receptor monomers and dimers. MW, Molecular mass.

 
AP20187-induced dimerization of the TRH receptor increased in a time- and concentration-dependent manner (Fig. 3Go). Exposure of cells to AP20187 resulted in a rapid increase in receptor dimerization visible within 1 min. To exclude the possibility that the observed dimerization of the TRH receptor was due to dimerization after solublization, we added AP20187 to cell lysates before immunoprecipitation. In this case, dimer formation was not observed (Fig. 3CGo, lane 1). AP20187 had no effect on receptor dimerization in cells expressing HA-TRHR without a FKBP domain (data not shown).

Effect of Dimerization on TRH Binding, TRH-Induced PLC Activation and Ca2+ Response
To determine whether dimerization alters receptor affinity for TRH, we incubated cells stably expressing TRHR-FKBPv-HA with 100 nM AP20187 for 2 h, conditions that cause maximal apparent dimerization. We then measured equilibrium binding of [3H]MeTRH, with or without dimerizer, in an additional 1 h incubation and obtained dissociation constant (Kd) values of 1.81 ± 0.57 and 1.85 ± 0.58 nM for untreated and dimerizer-treated cells, respectively (mean ± SE of three experiments). Bmax (maximal binding capacity) values in control and dimerizer-treated cells were compared in the same experiments; maximum binding in AP20187-treated cells averaged 88.1 ± 8.2% that observed in untreated cells. The half time for dissociation of [3H]TRH from membrane sites, measured as described in Materials and Methods, was the same, 21 min, in cells that had been preincubated with or without 100 nM AP20187. This is consistent with the lack of effect of the dimerizer on equilibrium binding. Although the extent of dimer formation in the intact cell is unknown, it seems likely that AP20187 did not change the number of molecules of peptide bound per receptor, because inducing dimerization did not alter the affinity of receptors for TRH and had little effect on total binding.

To learn whether dimerization itself activates PLC, we measured IP3 mass, total inositol phosphate production and translocation of a green fluorescent protein (GFP)-tagged PLC{delta}-pleckstrin homology (PH) domain. Cells stably expressing the TRHR-FKBPv-HA construct were treated with TRH, dimerizer, or dimerizer plus TRH for 1 min. TRH increased IP3 mass, as expected, but dimerizer had no effect on IP3 mass by itself and did not change the peak response to TRH (Fig. 4AGo). Furthermore, in metabolically labeled cells, exposure to dimerizer for a total of 2.5 h had no influence on basal or TRH-induced total inositol phosphate accumulation (Fig. 4BGo).



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Fig. 4. Effect of Dimerizer on PLC Activity

Cells stably expressing TRHR-FKBPv-HA were treated with 100 nM TRH, 100 nM AP20187, or TRH and AP20187 for A, 1 min or B, 30 min. A, IP3 was measured by radioreceptor assay. B, Total [3H]inositol phosphate accumulation was measured as described in Materials and Methods. Results shown are the mean ± SEM of triplicate observations from one of two experiments. C, Translocation of GFP-tagged PLC{delta}-PH domain by dimerizer and TRH. CHO cells stably expressing TRHR-FKBPv-HA were transiently transfected with GFP-tagged PLC{delta}-PH domain. After 24 h, cells were placed in HBSS and observed microscopically. Shown is a series of images of the same cells before (left), 1 min after the addition of 100 nM AP20187 (middle) and 1 min after further addition of 1 µM TRH (right) at 37 C. D, Line intensity histograms showing pixel intensity across line scans of the original image, in arbitrary units.

 
Next, we examined whether stimulation of cells with TRH or dimerizer changed the localization of a GFP-tagged PLC{delta}-PH domain reporter. The fusion protein of the PLC{delta} PH domain and GFP is prominently localized on the membrane of resting cells due to its interaction with membrane phosphatidylinositol(4,5)bisphosphate (Fig. 4CGo, left panel) (23). As shown in Fig. 4CGo, TRH caused a translocation of the reporter from the plasma membrane to the cytosol but dimerizer did not change the localization of the GFP-tagged PLC{delta}-PH domain. The redistribution of fluorescence is more clearly seen in line intensity histograms (Fig. 4DGo). Together, these results indicate that dimerization itself does not activate PLC.

We also asked whether dimerization causes Ca2+ release or changes the TRH-induced rise in intracellular free Ca2, because this is the most sensitive TRH response. Fura 2-loaded cells were exposed to vehicle, TRH, AP20187, or AP21998. TRH stimulated a rise in intracellular free Ca2+ that averaged 3.3 times basal and was observed in 82% of cells (Table 2Go). The dimeric FKBPv ligand AP20187 by itself caused a slight increase in intracellular Ca2+ in 24% of cells and the monomeric ligand AP21998 in 15% of cells (Table 2Go). The Ca2+ response to FKBPv ligands averaged 30% above baseline and was so small that we were unable to establish whether it resulted from release of intracellular Ca2+ or influx of external Ca2+. The Ca2+ response with TRH and dimerizer was not significantly different from that induced by TRH only.


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Table 2. Summary of Ca2+ Responses of CHO Cells Stably Expressing TRHR-FKBPv-HA

 
Effect of Dimerization on TRH-Induced ERK1/2 Activation
ERK1/2 has previously been shown to be activated by TRH (2, 3). To ask whether receptor dimerization leads to activation of ERK1/2, we used antiphospho-(p44/p42) ERK1/2 antibody and antitotal ERK1/2 antibody for immunoblotting. TRH activated ERK1/2 with a maximal response at 5 min. Neither dimerizer nor monomeric ligand caused ERK1/2 phosphorylation alone or altered the TRH response (Fig. 5Go).



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Fig. 5. Effect of Dimerizer and TRH on ERK1/2 Activity

A, Cells stably expressing TRHR-FKBPv-HA were incubated with 100 nM AP20187, AP21998, or TRH for the indicated times. B, Cells were incubated for 5 min with 100 nM AP20187, AP21998, or TRH in the combinations shown. Total ERK1/2 and phosphorylated (p-)ERK1/2 were detected in cell lysates by Western blotting using anti-ERK1/2 and antiphosphorylated ERK1/2 antibodies. C, Bar graph shows the mean ± range of densitometry results from two independent experiments. The relative intensity of the phosphorylated ERK1/2 is expressed as the fold increase in the phosphorylated to total ERK1/2 ratio.

 
Effect of Dimerization on ß-Arrestin Binding
After activation, the TRH receptor undergoes phosphorylation, and ß-arrestin is translocated from the cytosol to the membrane, where it binds the phosphoreceptor and directs its endocytosis. Cells stably expressing TRHR-FKBPv-HA were transiently transfected with ß-arrestin2-GFP, which was visualized by fluorescence microscopy in live cells. Addition of TRH caused the expected rapid translocation of GFP-tagged ß-arrestin2 from the cytoplasm to the plasma membrane or endosomal vesicles within 1 min. However, dimerizer by itself did not change the distribution of ß-arrestin2-GFP even after prolonged treatment (Fig. 6Go). In cells exposed to AP20187 or AP21998, TRH again caused redistribution of GFP-tagged ß-arrestin2 within 1 min (data not shown).



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Fig. 6. Effect of Dimerizer and TRH on Translocation of ß-Arrestin2-GFP

CHO cells stably expressing TRHR-FKBPv-HA were transfected with plasmid encoding ß-arrestin2-GFP and visualized by fluorescence microscopy. Panels A and B show the same cells expressing ß-arrestin2-GFP either A, before or B, after addition of 100 nM TRH. Panels C and D show the same cells expressing ß-arrestin2-GFP before and after addition of 100 nM AP20187 at 37 C.

 
Effect of Dimerization on Receptor Internalization and Recycling
Multiple approaches were used to determine whether AP20187 brings about receptor redistribution or changes TRH-induced internalization. First, we measured the loss of surface binding sites in cells exposed to TRH. Cells were pretreated with dimerizer or monomeric ligand for 2 h and exposed to TRH for an additional 30 min to induce internalization. To halt further redistribution of receptors and remove TRH bound to surface receptors, cells were placed on ice, washed, and then incubated with a mild acid solution to remove any membrane-bound TRH. Control experiments showed that this procedure removed 85.4 ± 0.2% of specifically bound TRH from untreated cells and 86.4 ± 0.4% from cells that had been treated with AP20187. Cells were then incubated with [3H]MeTRH at 4 C to quantify plasma membrane receptors.

Dimerizer alone had no effect on the number of surface receptors (Fig. 7Go), whereas TRH promoted receptor internalization. Interestingly, TRH caused a significantly greater loss of surface receptors when cells had been exposed to dimerizer, indicating that dimerization potentiates TRH-dependent receptor internalization or inhibits receptor recycling. This experiment was repeated using a pool of colonies expressing the TRHR-FKBPv-HA construct with the same result; TRH decreased plasma membrane receptors by 43.6 ± 1.4%, whereas AP20187 plus TRH decreased receptors by 63.0 ± 0.6%.



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Fig. 7. Effect of Dimerizer on the Internalization of TRHR-FKBPv-HA

Cells stably expressing TRHR-FKBPv-HA were incubated with 100 nM unlabeled TRH, AP20187 or AP221998 in the combinations shown for 30 min. As described in Materials and Methods, dishes were washed with a mild acid solution to remove any surface-bound TRH and the concentration of membrane receptors was measured by labeling with [3H]MeTRH at 4 C. Shown are results of a representative experiment performed in triplicate. Similar findings have been obtained in three independent experiments. *, P < 0.01 vs. all other groups.

 
We measured the rate at which surface receptors disappeared by preincubating cells with or without dimerizer and then adding TRH. At intervals, we again titrated the number of plasma membrane receptors with [3H]MeTRH at 0 C after removing any bound TRH with mild acid. As shown in Fig. 8AGo, internalization of receptor was significantly accelerated by AP20187. To exclude the possibility that overall receptor density was altered by AP20187 treatment, we measured the total number of receptors using ELISA. Based on this assay, AP20187 did not affect the concentration of receptors over a 30-min period (Fig. 8BGo).



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Fig. 8. Effect of Dimerizer on Receptor Internalization and Total Receptor Concentration

A, Cells stably expressing TRHR-FKBPv-HA were preincubated with or without 100 nM AP20187 for 2 h when TRH was added for the indicated times at 37 C. Surface receptors were then measured as described in Materials and Methods. Points show the mean ± SEM of triplicate dishes. Similar results have been obtained in three independent experiments. B, Cells were stimulated with TRH with or without AP20187 for 30 min at 37 C. Total receptor concentration was measured by ELISA after cell permeabilization with NP-40 as described in Materials and Methods.

 
Next, we monitored receptor internalization with immunocytochemistry. TRH receptors were clearly localized on the plasma membrane of unstimulated cells (Fig. 9AGo). After incubation with TRH for 30 min, bright punctate intracellular staining was observed (Fig. 9CGo), indicating that the TRH receptor had undergone internalization. Dimerizer had no effect on localization of receptor even with more than a 2-h incubation (Fig. 9BGo). With TRH and dimerizer treatment, TRH receptors were again observed in endosomal vesicles (Fig. 9DGo). To quantify receptor internalization, we counted cells with receptor clearly localized on the plasma membrane vs. cells with receptor localized partially or entirely intracellularly. With dimerizer and TRH treatment, 85% of cells showed completely or partially internalized receptor, compared with 50% of cells treated with TRH only, indicating that dimerizer increased the fraction of receptor in endocytic vesicles in CHO cells stably expressing TRHR-FKBPv-HA (Fig. 9EGo). Dimerizer had negligible effect on receptor internalization in cells expressing an HA-tagged TRH receptor without a FKBP domain (Fig. 9EGo). TRH-dependent receptor internalization was consistently about 20% higher in cells expressing the HA-tagged receptor than in those expressing the FKBPv-receptor fusion.



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Fig. 9. Effect of Dimerizer on the Localization of TRHR-FKBPv-HA

CHO cells stably expressing TRHR-FKBPv-HA were treated for 30 min with A, no drug; B, 100 nM AP20187; C, 100 nM TRH; or D, AP20187 and TRH; and then fixed and stained with monoclonal antibody against the HA epitope. E, The percentage of cells with predominantly surface TRH receptor. Slides were coded and then evaluated independently by two observers unaware of treatment group.

 
To examine the roles of dimerization in receptor recycling, CHO cells stably expressing TRHR-FKBPv-HA were incubated with TRH to internalize the ligand-receptor complex and washed with a mild acid solution to remove surface-bound TRH. Cells were then incubated with or without dimerizer for different periods, and the number of surface binding sites was measured by incubating with [3H]MeTRH at 0 C. AP20187 delayed the restoration of plasma membrane receptors (Fig. 10Go).



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Fig. 10. Effect of Dimerizer on Recycling of TRHR-FKBPv-HA Fusion Protein

Cells stably expressing TRHR-FKBPv-HA were incubated with 100 nM TRH for 30 min to allow internalization. Cells were washed and incubated in HBSS with or without 100 nM AP20187 for the indicated times at 37 C, when recycled receptors were quantified as described in Materials and Methods. Points show cell-associated radioactivity as a percent of the zero time value. Points represent the mean ± SEM of triplicates. Results were similar in two independent experiments.

 
Effect of Dimerizer in Cells Lacking ß-Arrestins
ß-Arrestin functions as an adapter protein in GPCR internalization (24, 25, 26). Overexpression of ß-arrestin increases the rate of internalization of the TRH receptor, and absence of ß-arrestin limits receptor endocytosis (3, 5, 26). To evaluate whether the effects of dimerization on TRH receptor cycling depend on ß-arrestin, we expressed the TRHR-FKBPv-HA construct in fibroblasts from knockout mice lacking ß-arrestins 1 and 2 (ß-arr1/2-KO cells) and analyzed internalization. After 30 min stimulation with TRH, only 19% of surface receptors were internalized in the absence of ß-arrestin, and dimerizer by itself did not increase receptor endocytosis (Fig. 11Go). Addition of dimerizer and TRH increased internalization in each of three independent experiments, suggesting that dimerizer-mediated receptor internalization can still occur in the absence of ß-arrestin.



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Fig. 11. Effect of Dimerizer on the Internalization of TRHR-FKBPv-HA in ß-arr1/2-KO Cells

ß-Arr1/2-KO cells were cotransfected with TRHR-FKBPv-HA and GFP. Transfection efficiency averaged 10–20% for these mouse embryo fibroblasts. Cells were incubated with 100 nM TRH plus AP21998, 100 nM AP20187, TRH plus AP20187 or AP21998 for 30 min at 37 C and washed with cold saline. The density of surface receptors was measured as described in Materials and Methods. The results summarize data from three independent experiments, each done in triplicate. *, P < 0.05 vs. either AP21998- or AP21087-treated cells.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Many GPCRs have been shown to undergo homodimerization, but it remains uncertain whether dimerization is constitutive or regulated and whether dimerization has a specific function. To clarify possible functions of GPCR dimerization, we developed a system to allow artificially induced dimerization of the TRH receptor. Exposure of cells to the AP20187 dimerizer dramatically increased the intensity of the dimer band. This dimerization was specific because it was not observed unless the variant FKBP domain was fused to the receptor, was blocked by an excess of a monomeric FKBP ligand, and was not observed if dimerizer was added to cell lysates. The results suggest that either the dimerizer causes receptor dimerization, or it stabilizes a preexisting dimeric form of the receptor.

Dimerization did not cause receptor phosphorylation, indicating that dimerization by itself does not force the receptor into an activated conformation recognized by receptor kinases. Consistent with this, receptor dimerization was not sufficient to initiate signal transduction by the TRH receptor, based on second messenger (IP3) formation, Ca2+ signaling, and ERK1/2 activation. Furthermore, dimerization neither potentiated nor inhibited TRH signaling. Although this suggests that dimerization of receptors does not activate them, this conclusion must be qualified because it is not known whether the structure of AP20187-stabilized receptor dimers accurately reflects the structure of receptor dimers found in cells.

Induced dimerization of the TRH receptor-FKBPv-HA fusion did, however, affect TRH-induced receptor redistribution. The intracellular localization of a GPCR is determined by multiple pathways of movement of receptors within cells including receptor synthesis, endocytosis, recycling to the plasma membrane, and degradation. AP20187 did not alter localization of the TRH receptor by itself. However, when cells were subjected to dimerizer and then exposed to TRH, the TRH receptor underwent more extensive redistribution to endocytic vesicles. The response was specific because AP20187’s effect was not observed in cells expressing a TRH receptor without the variant FKBP binding domain and because the monomeric FKBP ligand did not promote receptor endocytosis. Induced dimerization of receptor increased the net movement of receptors from the plasma membrane to an intracellular compartment and delayed the rate of receptor recycling after hormone withdrawal. These results suggest that induced dimerization alters both the rate of receptor internalization and the net rate of receptor recycling. GPCRs undergo both homodimerization and dimerization with other partners. The results described here raise the possibility that the availability of TRH receptors to bind and respond to TRH could be affected by formation of homo- or heterodimers that would affect receptor cycling. Regulated dimerization of another GPCR, the platelet activating factor (PAF) receptor, has been achieved by constructing a fusion protein of the receptor and bacterial DNA gyrase B and dimerizing through the addition of coumermycin (27). Regulated dimerization of the PAF receptor did not initiate signal transduction but did cause endocytosis of the receptor and potentiate PAF-induced internalization of in CHO cells.

Once activated, the TRH receptor undergoes phosphorylation, binds with high affinity to either ß-arrestin1 or ß-arrestin2 and undergoes clathrin- and dynamin-dependent endocytosis (24, 28, 29). Although ß-arrestin internalizes with it, the TRH receptor is extensively recycled after TRH removal, making it an exception to the general rule that GPCRs that internalize with ß-arrestin are degraded rather than recycled. To explain the effect of dimerizer on agonist-promoted receptor trafficking, we hypothesized that dimerization increased receptor affinity for ß-arrestin, accounting for the increased rate of internalization and decreased recycling. However, using GFP-labeled ß-arrestin, we found no evidence that dimerization per se causes ß-arrestin recruitment to the receptor or that dimerized receptors bind ß-arrestin more avidly after TRH activation. We also observed an effect of AP20187 on receptor internalization in cells from mice lacking both nonvisual arrestins.

Our results show that forced dimerization increases the fraction of TRH receptor in endocytic compartments, but it is not yet clear how dimerization is involved in regulating receptor cycling. One hypothesis is that dimerization changes TRH receptor conformation to cover some as-yet-unidentified sequence required for rapid recycling. Specific sequences present in the cytoplasmic tail have been reported to be required for efficient recycling of the ß2-adrenergic receptor and {delta}-opioid receptor (30, 31). An alternative explanation is that dimerized TRH receptor has higher affinity for critical regulators such as Rab11a or myosin Vb (25, 32). Rab 11a activity is required for muscarinic M4 receptor recycling because expression of a constitutively GDP-bound form of Rab11a severely impairs receptor recycling (32). Myosin Vb is an effector molecule that selectively interacts with the active Rab11a (33). Expression of the myosin Vb tail inhibits recycling of transferrin and M4 receptors (32, 33).

Many growth factor receptors act as dimers and high-affinity binding sites on these receptors occur only in their dimeric forms. Regulated dimerization systems have been developed for growth factor receptors with intrinsic tyrosine kinase activity, including the platelet-derived growth factor (34), insulin (34), erbB (35), and fibroblast growth factor receptors (36), for receptors that associate with intracellular tyrosine kinases, including the T cell (37) and erythropoietin (38) receptors, and for the FAS death receptor (39). In these model systems, forced dimerization of receptors stimulates signaling in the absence of ligand. In contrast, regulated dimerization of two GPCRs, PAF (27) and, as shown here, TRH receptors, is insufficient to activate downstream signaling pathways but does increase the net internalization of the ligand-receptor complex. Based on these results, we speculate that dimerization of GPCRs in general may potentiate agonist-induced receptor endocytosis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
CHO cells were obtained from the American Type Culture Collection (Manassas, VA). Sources of reagents were: pcDNA3, BamHI, KpnI, primers, pfu DNA polymerase, and LipofectAMINE (Invitrogen, Carlsbad, CA), PNGaseF (New England Biolabs, Beverly, MA), TRH, G418 and protease inhibitor mixture (Calbiochem, San Diego, CA), HA11 monoclonal anti-HA antibody (Covance, Berkeley, CA), cycloheximide, BSA, and paraformaldehyde (Sigma, St. Louis, MO), horseradish peroxidase-conjugated antimouse IgG and IP3 Biotrack Assay Kit (Amersham Biosciences, Piscataway, NJ), chemiluminescence reagent, [3H]MeTRH, [3H]TRH and [3H]inositol (PerkinElmer Life Science, Boston, MA), fura 2/AM and rhodamine red goat antimouse IgG (Molecular Probes, Eugene, OR), Protein A/G Plus agarose for immunoprecipitation (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ERK1/2 antibody and antiphopho-ERK1/2 antibody (Cell Signaling, Beverly, MA), and BM-blue-POD (3,3',5,5'-tetramethylbenzidine) solution (Roche Diagnostics, Indianapolis, IN). AP20187 and AP21998 were kindly provided by Ariad Pharmaceuticals Inc. (Cambridge, MA). The ß-arrestin2-GFP was a generous gift from Dr. Marc Caron (Duke University, Durham, NC). GFP-conjugated PLC{delta}-PH-domain was kindly provided by Dr. Tomas Balla (National Institutes of Health, Bethesda, MD). Mouse embryonic fibroblasts from knockout mice that lack both ß-arrestin1 and 2 (ß-arr 1/2-KO) were provided by Dr. Robert J. Lefkowitz (Duke University Medical Center) (40).

TRH Receptor Constructs
The plasmid pC4-Fv1E encoding the FKBP F36V variant protein followed by a carboxy terminal HA epitope tag was a generous gift from Ariad Pharmaceuticals Inc. To construct the FKBPv-HA plasmid, the fragment of FKBPv-HA was amplified by PCR with a 5' primer (5'-ATTGGATCCGGCTTCTAGAGGAGTGC-3') and a 3' primer (5'-CTGAAGTTCTCAGGATCCTCAC-3') that introduced a BamHI restriction enzyme site. The products were digested and ligated into a mammalian expression vector, pcDNA3 (FKBPv-HA in pcDNA3). To make the TRHR-FKBPv-HA construct, the TRH receptor gene without stop codon flanked by KpnI restriction sites was amplified and the fragment of the TRH receptor was ligated into the 5' end of the FKBPv-HA digested with KpnI. The sequence of TRHR-FKBPv-HA was confirmed by nucleotide sequencing. The 2HA-TRHR construct has been described previously (6).

Cell Culture and Transfection
Monolayer cultures of CHO cells were grown in DMEM/F12 (Invitrogen Life Technologies, Grand Island, NY) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) at 37 C in a humidified 95% room air, 5% CO2 incubator. Cells were transfected with plasmids encoding TRHR-FKBPv-HA (1 µg/ml) using Lipofectamine according to the manufacturer’s instructions. Cells were used for experiments 1–2 d after transfection. To create cell lines stably expressing TRH receptors, cells were transfected, and 800 µg/ml G418 were added to the medium to start selection 24 h later. G418-resistant colonies were amplified and then screened for expression of TRH receptors.

Radioreceptor Assay of IP3. Cells plated in 35-mm dishes were washed twice with Hanks’ balanced salt solution containing 15 mM HEPES (pH 7.4) (buffered HBSS) and incubated at 37 C with or without dimerizer or TRH for 1 min. The medium was aspirated, 0.8 ml of 20% ice-cold trichloroacetic acid was added, and the dish was put on ice immediately. Cells were scraped off the dish, transferred to an Eppendorf tube, and centrifuged at 12,000 x g for 1 min. The supernatant was extracted with 2 vol trichlorotrifluorethane/trioctylamine (3:1), and the aqueous phase was saved. The radioreceptor assay was performed using IP3 Biotrack Assay Kit.

Total inositol phosphate production
Stably transfected CHO cells expressing TRHR-FKBPv-HA construct were plated on 35-mm dishes and washed once with saline and labeled for 18–24 h with 2.5 µCi/ml myo-[3H] inositol in F10 medium. After labeling, cells were washed once with saline and pretreated with dimerizer or vehicle for 2 h in serum-free medium containing 20 mM LiCl. Cells were then exposed to TRH with or without AP20187 in medium containing LiCl for 30 min and then lysed with 1 ml of 0.05 N formic acid on ice for 30 min. The lysates were loaded on 1 ml Dowex Ag-1X8 columns that were washed with 12 ml of 0.1 N formic acid. Total inositol phosphates were then eluted with 3 ml of 0.05 N formic acid, 1.2 M ammonium formate.

Single Cell Cytoplasmic Ca2+ Imaging
Ca2+ imaging was carried out as described by Ashworth and Hinkle (41). Cells plated on coverslips were loaded with 4 µM Fura 2/AM. The coverslip was washed and put into a Sykes-Moore chamber (Bellco Glass, Vineland, NJ) on a Nikon inverted microscope on a heated stage at 37 C. The 340/380 nm fluorescence ratios were acquired every 3 sec and analyzed using MetaFluor software from Universal Imaging (Downingtown, PA).

Immunocytochemistry and Live Cell Microscopy
To visualize the sequestration of TRH receptors in CHO cells, cells grown on 25-mm coverslips were treated as described, washed once with PBS and fixed for 20 min at room temperature with 4% paraformaldehyde in PBS. Cells were incubated with blocking buffer containing 5% goat serum and 0.2% Nonidet P-40 (NP-40) in PBS. A mouse monoclonal anti-HA antibody, HA11, was added at a dilution of 1:1000 in the same buffer for 1–3 h and the coverslips were washed, incubated with rhodamine-conjugated antimouse secondary antibody (1:500) and then washed and mounted (42).

For live cell imaging, cells stably expressing TRHR-FKBPv-HA were plated on coverslips and transfected with GFP-tagged ß-arrestin2 or GFP tagged PLC{delta}-PH domain. After 18 h, coverslips were rinsed with buffered HBSS and placed in Sykes-Moore chambers. Cells were examined on a heated stage using a fluorescein filter. Digital images were captured and analyzed using MetaMorph software from Universal Imaging.

Immunoprecipitation and Immunoblotting
Cells were solubilized by incubation for 10 min in ice-cold lysis buffer [150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1 mM EDTA and 1:1000 protease inhibitor cocktail (pH 8.0)]. The cell lysates were centrifuged at 14,000 x g at 4 C and supernatants were incubated with HA11 antibody (1:5000) overnight at 4 C and 20 µl of protein A/G beads were then added and incubation continued for 1 h. For deglycosylation, immunopurified receptors in 50 µl were treated with 500 U PNGase according to the manufacturer’s instructions. Immunopurified receptors or deglycosylated receptors were boiled for 2 min and resolved on 10% SDS-PAGE as described previously (43). Proteins were transferred onto a nitrocellulose membrane, which was then subjected to two sequential 2-h incubations with mouse anti-HA antibody at 1:5000 and horseradish peroxidase-conjugated antimouse IgG antibody at 1:2000 and immunoactivity was detected by chemiluminescence. All blots are representative of experiments repeated two to five times. To quantify the intensity of receptor bands, films were scanned, background was subtracted, and the intensity of receptor bands was determined.

Detection of Phosphorylated ERK1/2
Cells were serum-starved for 16 h and treated with drugs or hormone as described. Dishes were placed on ice, and immediately lysed with sodium dodecyl sulfate (SDS) sample buffer (150 µl/60 mm dish). The activity of the p42/p44 (ERK1/2) was determined by immunoblotting with antiphospho-ERK1/2 antibody (1:5000). Samples of the cell extracts were run on separate gels and blotted with the anti-ERK1/2 antibody (1:1000) against total ERK to ensure that the amounts of ERK protein were equal.

Measurement of TRH Binding, Internalization, and Cycling
To measure binding of the high affinity agonist MeTRH, 35-mm dishes were incubated with [3H]MeTRH, with or without a 1000-fold excess of unlabeled peptide, in serum-free medium. Dishes were washed three times with ice-cold saline and cells taken up in 0.1% SDS and radioactivity counted. To measure the TRH dissociation rate, cells were preincubated with or without AP20187 for 30 min and then incubated with 5 nM [3H]TRH for 30 min on ice to label surface sites. Dishes were then washed three times with cold saline. HBSS containing 100 nM unlabeled TRH with or without 100 nM AP20187 was added and the cells were returned to 37 C for 0–60 min. At each time point, the HBSS buffer was collected and counted to quantify dissociated [3H]TRH, and strong acid/salt solution [0.2 N acetic acid, 0.5 M NaCl (pH 2.5)] was added to strip any surface-bound [3H]TRH.

To measure receptor internalization, cells were incubated with 100 nM TRH or 100 nM AP20187 plus 100 nM TRH for 2.5–60 min at 37 C. Cells were transferred to ice and washed twice with ice cold saline and then incubated for 5 min at 0 C with a mild acid solution that removes surface-bound hormone without damaging receptor [0.025 M acetic acid, 1 mM CaCl2, 0.5 M NaCl (pH 5.0)] (29). Dishes were then washed again with saline and incubated with 5 nM [3H]MeTRH at 0 C for 1 h to label only surface sites. Cells were then washed three times with cold saline, taken up in 0.1% SDS and radioactivity counted. To measure recycling, cells were pretreated with 100 nM TRH at 37 C for 30 min to produce a substantial intracellular pool, washed with mild acid solution as described above to remove surface-bound ligand, and then incubated at 37 C in medium containing dimerizer or not for times from 2.5–60 min. At intervals, cells were washed again with a mild acid solution and cold saline. To quantify the receptors that had recycled to the plasma membrane, cells were incubated with [3H]MeTRH at 0 C for 1 h, washed, solubilized and counted. Results were analyzed by ANOVA with Tukey’s test.

Measurement of Total Number of the TRH Receptors Using ELISA
Cells stably expressing TRHR-FKBPv-HA were grown in 35-mm dishes and treated with TRH and/or dimerizer for 30 min at 37 C. Cells were fixed for 20 min with 4% paraformaldehyde in PBS (pH 7.4), permeabilized with 5% goat serum and 0.2% NP-40 for 20 min at room temperature, and incubated with anti-HA antibody at 1:5000. Cells were washed three times in PBS, and incubated with horseradish peroxidase-conjugated antimouse secondary antibody (1:5000). Antibody binding was visualized by adding 0.3 ml substrate solution for horseradish peroxidase (BM-blue-POD solution). After incubation for 20 min at room temperature, the color reaction was terminated by adding 0.3 ml of 2 M sulfuric acid and 0.3 ml of samples was transferred into a 96-well plate, and the plate was read at 450 nm in a microplate reader.


    ACKNOWLEDGMENTS
 
Acknowledgments

We are grateful to Ariad Pharmaceuticals Inc. (Cambridge, MA), www.ariad.com/regulationkits, for generously providing plasmids and FKBP ligands. We also appreciate the excellent technical assistance of Marta Crowe.


    FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK19974 (to P.M.H.).

First Published Online July 14, 2005

Abbreviations: ß-arr 1/2-KO, ß-Arrestin1 and 2 knockout; CHO, Chinese hamster ovary; FKBP, FK506 binding protein; FKBPv, variant of FKBP; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HA, hemagglutinin; HBSS, Hanks’ buffered salt solution; IP3, inositol(1,4,5)trisphosphate; NP-40, Nonidet P-40; PAF, platelet activating factor; PH, pleckstrin homology; PLC, phospholipase C; SDS, sodium dodecyl sulfate.

Received for publication March 21, 2005. Accepted for publication July 5, 2005.


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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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