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Endocrinology Vol. 145, No. 7 3095-3100
Copyright © 2004 by The Endocrine Society

BRIEF COMMUNICATION

Fate of Internalized Thyrotropin-Releasing Hormone Receptors Monitored with a Timer Fusion Protein

Laurie B. Cook and Patricia M. Hinkle

Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

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

Abstract

Trafficking of TRH receptors was studied in a stable HEK293 cell line expressing receptor fused to a Timer protein (TRHR-Timer) that spontaneously changes from green to red over 10 h. Cells expressing TRHR-Timer responded to TRH with an 11-fold increase in inositol phosphate formation, increased intracellular free calcium, and internalization of 75% of bound [3H][N3-methyl-His2]TRH within 10 min. After a 20-min exposure to TRH at 37 C, 75–80% of surface binding sites disappeared as receptors internalized. When TRH was removed and cells incubated in hormone-free medium, approximately 75% of [3H][N3-methyl-His2]TRH binding sites reappeared at the surface over the next 2 h with or without cycloheximide. Trafficking of TRHR-Timer was monitored microscopically after addition and withdrawal of TRH. In untreated cells, both new (green) and old (red) receptors were seen at the plasma membrane, and TRH caused rapid movement of young and old receptors into cytoplasmic vesicles. When TRH was withdrawn, some TRHR-Timer reappeared at the plasma membrane after several hours, but much of the internalized receptor remained intracellular in vesicles that condensed to larger structures in perinuclear regions deeper within the cell. Strikingly, receptors that moved to the plasma membrane were generally younger (more green) than those that underwent endocytosis. There was no change in the red to green ratio over the course of the experiment in cells exposed to vehicle. The results indicate that, after agonist-driven receptor internalization, the plasma membrane is replenished with younger receptors, arising either from an intracellular pool or preferential recycling of younger receptors.

TRH ACTS VIA a G protein-coupled receptor (GPCR) to stimulate release of TSH and prolactin from the anterior pituitary gland. The TRH receptor (TRHR) is coupled to Gq and TRH stimulates phospholipase C, leading to an increase in cytoplasmic calcium and protein kinase C activity (1). Signal generation via the TRH receptor has been investigated in detail, but the processes of receptor inactivation and reactivation have not been analyzed in as much depth. TSH is released in a pulsatile pattern in vivo (2), suggesting that the pituitary gland is normally exposed to TRH intermittently. For this reason, desensitization and resensitization of the TRHR are expected to be important in regulating the physiological response of pituitary cells to TRH.

Activated GPCRs are usually desensitized as a consequence of receptor phosphorylation and ß-arrestin binding (3, 4, 5). The agonist-occupied receptor is selectively phosphorylated by specific kinases, and ß-arrestin binds the phosphorylated receptor. This uncouples the receptor from G proteins and targets it to clathrin-coated pits. The net result is both desensitization, because signal transduction is terminated, and endocytosis of the agonist-receptor complex through a clathrin- and dynamin-dependent pathway. Depending on the receptor, ß-arrestin may dissociate before endocytosis or internalize with the receptor. Following endocytosis, GPCRs either recycle to the plasma membrane or enter a degradative pathway. A number of the signals that control this sorting decision have been identified, but receptor trafficking is incompletely understood and is the subject of intense investigation (3).

TRHR trafficking is believed to adhere to this canonical pathway. TRH binding is followed by receptor phosphorylation (6, 7), ß-arrestin binding (8, 9, 10), and rapid and extensive endocytosis (8, 10, 11, 12, 13, 14, 15). The receptor binds to both ß-arrestin1 and ß-arrestin2 and internalizes together with the arrestin (9). When TRH is removed from the medium, a majority of receptor binding activity is recovered at the cell surface and TRH signaling is restored (12, 13, 16). These findings have been interpreted as evidence that internalized receptor recycles, but recycling of the receptor protein itself has not been shown directly.

In this study, we have asked whether internalized TRHRs return to the cell surface following endocytosis of the hormone-receptor complex and subsequent withdrawal of the hormone. To do so, we have taken advantage of a Timer protein (17, 18) that provides an estimate of the age of the protein to which it is attached. We report that much of the internalized receptor is not recycled over the course of several hours. Instead, the plasma membrane pool appears to be largely replenished from an intracellular pool of newer receptors.

Materials and Methods

Development of lines of HEK293 cells stably expressing TRHR-Timer and hemagglutinin (HA)-tagged TRHRs has been described (6, 19). The TRHR-Timer cell line expresses the entire rat TRHR sequence lacking a stop codon with the Timer protein at the carboxyl terminus. Cells were maintained in DMEM or DMEM/F12 medium supplemented with 5% FBS at 37 C in a humidified 95% air and 5% CO2 environment.

To monitor inositol phosphate production, cells were labeled overnight with 2.5 µCi/ml [3H]inositol in F10 medium containing 5% FBS. Dishes were incubated with LiCl and TRH as described, washed, and total [3H]inositol phosphates and [3H]phospholipids were measured by minor modifications of published procedures (20).

To measure intracellular calcium, cells were incubated in 15 mM HEPES-buffered Hanks’ balanced salt solution (HBSS) (pH 7.4) and loaded with 4 µM fura-2AM (Molecular Probes, Eugene, OR). Coverslips were rinsed with HBSS and placed in Sykes-Moore chambers (Bellco Glass, Vineland, NJ). Ratios of 510-nm emission at dual excitation wavelengths (340 and 380 nm) were acquired every 3 sec as described (21). Images were analyzed using MetaFluor software from Universal Imaging (Downingtown, PA). Data are expressed as 340- to 380-nm ratios normalized to the baseline ratio.

In receptor cycling experiments, cells were maintained in Krebs buffered ringers solution (KBRS) [117 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 20 mM HEPES, 0.1% BSA, and 3 mM glucose (pH 7.4)]. To measure radioligand binding to intact cells, dishes were incubated in serum-free media or HBSS at pH 7.4 containing [3H][N3-methyl-His2]TRH ([3H]MeTRH) (60–100 Ci/mmol; DuPont/NEN Life Science Products, Boston, MA) with or without a 1000-fold molar excess of nonradioactive TRH at either 0 or 37 C for the times indicated. Nonspecific binding was between 1 and 7% of total binding and has been subtracted. Dishes were then placed on ice and washed three times with ice-cold 0.15 M NaCl. To determine internalized [3H]MeTRH, cells were then washed with ice-cold acid/salt buffer (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) to extract surface ligand, and the cells were solubilized and counted to measure internalized hormone (22).

Microscopy was carried out on a Nikon Diaphot inverted microscope with a x100 objective, 150-W xenon lamp, and Princeton Instruments (Princeton, NJ) Micromax camera using fluorescein [fluorescein isothiocyanate (FITC)] and rhodamine [tetramethylrhodamine isothiocyanate (TRITC)] filters from Chroma Technology (Rockingham, VT). Images were analyzed using Metamorph software from Universal Imaging. Nuclei were stained with Hoechst 33258 (Molecular Probes) and observed at 350-nm excitation and 430-nm emission. All microscopy was performed at 37 C. For immunocytochemistry, cells on coverslips were fixed and stained for the HA epitope as described (23).

Results

Experiments were performed using HEK293 cells stably transfected with plasmid encoding TRHR-Timer. The cell line expressed 0.5–1 pmol/mg protein [3H]MeTRH binding sites, slightly below the concentration of endogenous receptors reported in pituitary cell lines (24). The functionality of TRHR-Timer was assessed by measuring the ability of TRH to stimulate inositol phosphate production and a calcium transient. In cells that had been metabolically labeled with [3H]inositol, TRH caused an 11-fold increase in the formation of [3H]inositol phosphates, typical of the response seen in cells expressing the wild-type receptor using this assay (Fig. 1AGo). In cells loaded with the calcium indicator fura2, TRH elicited a sharp increase in the intracellular free calcium concentration equivalent to that generated by wild-type receptor (Fig. 1BGo). The ability of the TRHR-Timer to undergo agonist-mediated endocytosis was measured using resistance to a low-pH, high-salt wash as a measure of internalization. After [3H]MeTRH binding, 80% of receptor internalized within 10 min (Fig. 1CGo). Again, these properties are indistinguishable from those of the wild-type receptor and are consistent with reports by Milligan’s group (10, 11) that addition of a green fluorescent protein to the carboxyl terminus of the TRH receptor does not interfere with receptor signaling and trafficking.



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  		FIG. 1. TRH signaling in cells expressing TRHR-Timer. A, Cells expressing TRHR-Timer were metabolically labeled with [3H]inositol and then incubated for 30 min with 10 mM LiCl with or without 100 nM TRH. TRH had no effect on untransfected cells (not shown). Bars show the amount of [3H]inositol phosphates ([3H]InsPs) produced (mean ± SE of triplicate determinations). B, Cells were loaded with fura2 and incubated in HBSS. TRH (100 nM) was added at the time noted, followed by 2 mM Bapta and 1 µM ionomycin (Iono) to chelate extracellular calcium and release calcium from intracellular stores. Points show mean ± SE from 33 cells. Untransfected cells showed no calcium response to TRH (not shown). C, Cells were incubated with 5 nM [3H]MeTRH with or without excess unlabeled TRH for 1 h at 0 C to occupy surface receptors. Dishes were then washed twice and incubated at 37 C for various times before the fraction of specifically bound radioligand that had been internalized was determined. Values are mean ± range of duplicates.

 
To evaluate recycling of TRH binding sites, we incubated cells with 100 nM TRH for 20 min, conditions that bring about maximal internalization, then removed the hormone, washed, and continued the incubation. At intervals, we measured the density of TRHRs on the plasma membrane by incubating cells with [3H]MeTRH at 0 C. Because TRH does not cross the membrane, this assay only detects surface binding sites. After a 20-min exposure to TRH at 37 C, 75–80% of surface binding sites disappeared (Fig. 2Go), in agreement with the data described above. When TRH was then removed and the cells incubated in hormone-free medium, approximately 75% of the [3H]MeTRH binding sites reappeared at the surface over the next 2 h. This experiment suggests that TRHR-Timer recycles to the surface with a half-time of about 30 min, in agreement with published results obtained with the endogenous receptor in pituitary cells (16). To distinguish between recycling and de novo protein synthesis, we repeated the experiment in the presence of the protein synthesis inhibitor cycloheximide. Both the TRH-dependent disappearance of receptors from the plasma membrane and recovery of binding sites after TRH washout occurred with similar kinetics when protein synthesis was blocked.



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  		FIG. 2. Recycling of TRHR-Timer binding activity. Filled circles, Cells expressing TRHR-Timer were incubated at 37 C in KBRS containing either no hormone or 100 nM TRH for 20 min. Dishes were washed twice and either collected immediately or incubated further with KBRS before determination of surface TRH binding sites. Specific surface binding of [3H]MeTRH was measured by incubating cells on ice in buffer containing 5 nM [3H]MeTRH with or without excess unlabeled TRH for 1 h. Filled squares, The experiment was repeated using dishes that had been incubated with 10 µg/ml cycloheximide for 4 h before and throughout the experiment. This concentration of cycloheximide inhibited incorporation of [3H]leucine into acid-insoluble material by over 95%. Results show mean ± SE of three determinations and are expressed as the percentage of hormone binding in dishes not exposed to TRH; where error bars are not visible, errors were within symbol size. When TRH was present throughout the experiment, [3H]MeTRH binding remained at 20% of that present in cells not exposed to hormone throughout the experiment. When no TRH was present, the number of binding sites did not change significantly over 2.5 h.

 
Trafficking of TRHR-Timer was next monitored microscopically after addition and withdrawal of TRH. The Timer protein fluoresces green when first synthesized and subsequently undergoes 85% conversion to red with a reported half-time of 9–10 h in a spontaneous reaction (17, 18). As evident from the pictures at time zero in Fig. 3AGo, the TRHR-Timer was predominantly localized at the plasma membrane and both relatively new (green) and older (red) receptors were visible. In addition, some receptor was visible intracellularly in apparent vesicles. Fluorescence intensity of the TRHR-Timer was dim and exposure times needed to be at least 10 times longer than those used for cells expressing a similar concentration of the corresponding green fluorescent protein- TRHR construct.



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  		FIG. 3. Microscopic analysis of TRHR-Timer cycling. A, Cells expressing TRHR-Timer were incubated at 37 C in KBRS buffer containing 100 nM TRH for 20 min. The chamber was rinsed twice and incubation continued in buffer alone. Images were captured at intervals before and after TRH addition and washout using FITC (green) and TRITC (red) filter sets. Images show the same field of cells, which moved over the course of the experiment. The lower panels show the ratio of intensities at red/green (TRITC/FITC) wavelengths. Exposure times and image processing were identical for all frames in each series. The ratio of red to green fluorescence intensity, in arbitrary units after subtraction of background regions, declined from an average of 1.36 at the plasma membrane untreated cells to 1.07 in cells exposed to TRH and allowed 2 h to recover. The large intracellular vesicles seen at the end of the washout period had an average ratio of 1.30. B, The experiment was repeated except that cells were treated with vehicle alone for the first 20 min. Settings are identical with those described for panel A. C, Cells were incubated with 100 nM TRH for 20 min at 37 C, washed, and then incubated in buffer alone for 60 min. Images were obtained using FITC and TRITC filters and cells were then stained with Hoechst dye to identify nuclei, displayed in blue. a, Green (newer receptor); b, red (older receptor); c, blue (nuclei); d, green/blue merge; e, red/blue merge. The intensities of red and green in this panel were adjusted to show the merge with blue and cannot be compared directly with those in panels A and B.

 
When hormone was added, extensive movement of receptors into cytoplasmic vesicles took place quickly, such that most receptor appeared intracellularly by 20 min. The cells also underwent changes in shape, which we find when we add TRH to any HEK293 cell line expressing a TRHR. These microscopic findings corroborate the biochemical evidence for internalization of the hormone-receptor complex. TRH was then withdrawn and the cells were washed on the microscope stage and incubated in hormone-free medium. Images were captured at intervals after TRH washout. Receptors became visible plasma membrane after several hours, again consistent with the [3H]MeTRH binding data. Two findings were unexpected, however. First, much of the internalized receptor remained intracellular in vesicles that gradually condensed to larger structures deeper within the cell. The compartment containing receptors was distinct from the nucleus, as shown by Hoechst staining of nuclei of cells expressing TRHR-Timer (Fig. 3CGo). Second, the receptors that moved to the plasma membrane were younger (more green) than those initially present. This is best seen in the bottom panels of Fig. 3AGo, which show the ratio of fluorescence intensities at red/green wavelengths. The receptors that remained in the cytoplasm were older than those that repopulated the membrane. Results with a control dish exposed to vehicle only are shown in Fig. 3BGo. In control cells, TRHR-Timer remained at the cell surface and there was no change in the red/green ratio over the course of the experiment, although there was some cell movement.

To eliminate the possibility that the carboxy-terminal Timer protein altered receptor trafficking, we studied a HEK293 line stably expressing a TRHR tagged with the HA epitope at the extracellular amino terminus, also expressed at levels close to those in normal pituitary cells (6). In these experiments, HA-TRHRs were identified in fixed cells by immunostaining. As shown in Fig. 4Go, HA-TRHRs were initially on the plasma membrane and underwent extensive endocytosis within 20 min. Three hours after removal of hormone, HA-TRHRs were again visible at the surface, but intense receptor staining was also present in the perinuclear region, exactly as found with the TRHR-Timer protein. Cells that received TRH continuously for 3 h had a punctate appearance, suggesting that there was some receptor cycling and newly internalized receptor in smaller endocytic vesicles. All of the internalized receptor in cells that had been exposed to TRH for 20 min 2 h before staining was in late endosomes deeper within the cell.



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  		FIG. 4. Microscopic analysis of HA-TRHR cycling. Cells expressing HA-TRHR were incubated at 37 C in KBRS buffer with no additions, with 100 nM TRH for 20 min or 3 h, or cells were incubated with 100 nM TRH for 20 min, rinsed twice, and incubated in buffer alone for an additional 3 h. Cells were washed, fixed, and stained for the HA epitope.

 
Discussion

The experiments described here allow several conclusions: 1) TRHRs reach the plasma membrane within a few hours of synthesis, because green (new) receptors were visible at the plasma membrane, i.e. they reached the cell surface before they converted to red. 2) Both young and old receptors undergo rapid and extensive endocytosis when TRH binds. 3) If TRH is removed following receptor endocytosis, a significant fraction of internalized receptors do not recycle over the course of several hours but remain in vesicles that coalesce and move to a perinuclear region deep inside the cell. 4) If TRH is removed after receptor endocytosis, the plasma membrane is replenished with receptors younger than those that underwent endocytosis. Our data do not allow us to determine the long-term fate of internalized TRHR-Timer, and we cannot be certain whether older receptors would eventually have recycled or been degraded. GPCRs usually recycle to the plasma membrane from early endosomes, but recycling from late endosomal compartments is also well established (3, 25). Several technical issues limited the duration of our experiments, including the health of cells maintained in balanced salt solution and photobleaching from repetitive data acquisition.

Several caveats are important in interpreting these studies. Although the TRHR-Timer was not expressed at abnormally high levels and appeared to behave like the endogenous receptor in terms of its signaling, ligand binding, internalization, and apparent recycling, it is still possible that the normal receptor in pituitary cells behaves differently. It is unlikely that the addition of the Timer protein itself altered receptor trafficking, however, because an HA-tagged receptor behaved similarly. DsRed is an obligate oligomer in vitro, which could also influence receptor function (26). The TRHR is known to oligomerize and the fraction dimerized increases with agonist binding (6, 27), but the importance of receptor oligomerization for receptor signaling and trafficking is unknown (28). The Timer protein cannot provide information about events much faster than the 10–12 h required for conversion from green to red.

There are several potential explanations for the finding that younger receptors replenish the plasma membrane pool after agonist-mediated internalization. The plasma membrane may be replenished with newer receptors from an intracellular pool. Any intracellular pool must be diffusely localized, probably in the endoplasmic reticulum and/or Golgi apparatus, because TRHR-Timer staining in naive cells is most intense on the plasma membrane. This model demands that the cell be able to sense that the plasma membrane TRHR pool has been depleted in response to TRH, and the signals that might be involved are unknown. Another model is that, after TRH-driven receptor internalization, younger receptors recycle more efficiently than older receptors. This could happen if younger receptors are modified in a manner that selects them for recycling or, conversely, if older receptors undergo some modification that preferentially targets them to a degradative pathway following endocytosis. Posttranslational modifications of the TRHR are thought to include N-glycosylation (29), palmitoylation (although this has not been demonstrated), phosphorylation (6, 7), and ubiquitination (19). Ubiquitin modification regulates the trafficking of several receptors including the yeast GPCR ste2p (30), but no ubiquitin was detected on plasma membrane TRHRs and no changes in receptor ubiquitination were seen in response to TRH. Because young and old TRHR-Timer proteins internalized equally well, it is unlikely that they differed in either phosphorylation or ß-arrestin interactions. It may eventually be possible to isolate modified receptors tagged with the Timer protein and estimate their age based on spectral properties, but our initial attempts to do so have been unsuccessful.

In summary, we have demonstrated that trafficking of the TRH is more complex than previously appreciated. Receptors undergo extensive ligand-driven endocytosis, but recycling and replenishment of the plasma membrane occur by a process that enriches the surface in newer receptors. This may be a rational mechanism that allows a long-lived cell to replace receptors gradually as they are used, perhaps averting age-related damage. Additional work will be required to establish the mechanism of TRHR cycling and signals required for recruitment of new TRHRs to the cell surface.

Footnotes

This work was supported by a grant from the National Institutes of Health (DK19974, to P.M.H.) and a Pharmaceutical Manufacturers’ Association Predoctoral Fellowship (to L.B.C).

Abbreviations: FITC, Fluorescein isothiocyanate; GPCR, G protein-coupled receptor; HA, hemagglutinin; HBSS, Hanks’ balanced salt solution; KBRS, Krebs buffered ringers solution; [3H]MeTRH, [3H][N3-methyl-His2]TRH; TRHR, TRH receptor; TRITC, tetramethylrhodamine isothiocyanate.

Received March 9, 2004.

Accepted for publication April 23, 2004.

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