Agonist-Induced Endocytosis and Recycling of the Gonadotropin-Releasing Hormone Receptor: Effect of ß-Arrestin on Internalization Kinetics

Milka Vrecl, Lorraine Anderson, Aylin Hanyaloglu, Alison M. McGregor, Alex D. Groarke, Graeme Milligan, Philip L. Taylor and Karin A. Eidne

MRC Reproductive Biology Unit (M.V., L.A., A.H., A.M.M., P.L.T., K.A.E.) Centre for Reproductive Biology Edinburgh, EH3 9EW, United Kingdom
Molecular Pharmacology Group (A.D.G., G.M.) Division of Biochemistry and Molecular Biology Institute of Biomedical Life Sciences University of Glasgow Glasgow G12 QQ, United Kingdom


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study examined the dynamics of endocytotic and recycling events associated with the GnRH receptor, a unique G protein-coupled receptor (GPCR) without the intracellular carboxyl-terminal tail, after agonist stimulation, and investigated the role of ß-arrestin in this process. Subcellular location of fluorescently labeled epitope-tagged GnRH receptors stably expressed in HEK 293 cells was monitored by confocal microscopy, and the receptor/ligand internalization process was quantified using radioligand binding and ELISA. Agonist stimulation resulted in reversible receptor redistribution from the plasma membrane into the cytoplasmic compartment, and colocalization of internalized GnRH receptors with transferrin receptors was observed. Internalization experiments for the GnRH receptor and another GPCR possessing a carboxy-terminal tail, the TRH receptor, showed that the rate of internalization for the GnRH receptor was much slower than for the TRH receptor when expressed in both HEK 293 and COS-7 cells. TRH receptor internalization could be substantially increased by coexpression with ß-arrestin in COS-7 cells, while GnRH receptor internalization was not affected by coexpression with ß-arrestin in either cell type. Coexpression of the GnRH receptor with the dominant negative ß-arrestin (319–418) mutant did not affect its ability to internalize, and activated GnRH receptors did not induce time-dependent redistribution of ß-arrestin/green fluorescent protein to the plasma membrane. However, the ß-arrestin mutant impaired the internalization of the TRH receptor, and activated TRH receptors induced the ß-arrestin/green fluorescent protein translocation. This study demonstrates that, despite having no intracellular carboxy-terminal tail, the GnRH receptor undergoes agonist-stimulated internalization displaying distinctive characteristics described for other GPCRs that internalize via a clathrin-dependent mechanism and recycle through an acidified endosomal compartment. However, our data indicate that the GnRH receptor may utilize a ß-arrestin-independent endocytotic pathway.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH and its receptor are key mediators in the regulation of the reproductive process. After its pulsatile release from the hypothalamus, GnRH reaches the anterior pituitary gland where it interacts with specific high- affinity GnRH receptors causing the release of the gonadotropins, LH and FSH. Of the G protein-coupled receptors (GPCRs) cloned to date, the mammalian GnRH receptor is unique as it is the only receptor in which the functionally important intracellular cytoplasmic C-terminal tail is completely absent. It is truncated at the cell membrane immediately after the seventh transmembrane domain (1). The GnRH receptor is preferentially coupled to phosphoinositidase C via the Gq/G11 family of G proteins, and its stimulation by GnRH induces the production of inositol phosphates (IPs) and diacylglycerol, which results in the elevation of cytosolic calcium and the activation of protein kinase C (2). Agonist-induced receptor activation leads to a complex set of intracellular events including desensitization of GPCRs by receptor-G protein uncoupling followed by receptor internalization and recycling, which is now thought to be essential for the reestablishment of receptor responsiveness (3, 4, 5).

Experiments measuring endocytotic events at the level of the receptor have been performed for the ß2-adrenergic receptor (6, 7, 8), TRH (9), and muscarinic (10) and angiotensin II type 1A (AT1A) receptors (11). After agonist activation these receptors internalize into intracellular endosomes via clathrin-coated pits and subsequently recycle back to the plasma membrane as functional receptors. The binding of ß-arrestins to GPCRs has been shown to be a convergent step of GPCR signaling with ß-arrestins acting as adaptor-like proteins regulating the rate and specificity of receptor internalization (12, 13). GPCR phosphorylation appears to be a prerequisite for arrestin-receptor interaction (14), and it is proposed that ß-arrestin binding to ligand-activated GPCRs specifically directs the phosphorylated receptors into clathrin-coated pits due to its ability to interact with both the receptor and clathrin. Mutagenesis studies have revealed that the N-terminal half of nonvisual arrestins has the ability to recognize the agonist-activated GPCRs while the clathrin-binding domain is located in the COOH terminus of the molecule (15, 16). The role of ß-arrestin in promoting GPCRs internalization has been further confirmed using ß-arrestin dominant negative mutants to impair receptor internalization (17). Recently, the jellyfish (Aqueora victoria) green fluorescent protein/ß-arrestin fusion protein has been introduced as a sensitive tool for real time visualization of the receptor-mediated translocation of ß-arrestin in living cells (18). Such a process has been demonstrated for a number of ligand-activated GPCRs (19).

Sites located within the intracellular receptor loops, and in particular the intracellular C-terminal region, have been shown to be important in GPCR internalization, as receptor mutants with truncated C-terminal tails or lacking putative G protein-coupled receptor kinase phosphorylation sites displayed impaired internalization (13, 20, 21). We have demonstrated that the addition of a functional intracellular C-terminal tail to the GnRH receptor significantly increased internalization rates (22).

The aim of this study was to examine endocytotic and recycling events of the GnRH receptor. Previous studies on internalization events connected with the GnRH receptor have employed radiolabeled ligand binding (23, 24, 25, 26, 27, 28), which is based on the assumption that ligand and receptor sort similarly through the endocytotic pathway. To test this assumption, we employed the receptor tagging methodology to determine the GnRH receptor endocytotic trafficking cycle at the level of the receptor. The role of ß-arrestin in promoting internalization in the GnRH receptor and in a GPCR possessing a C-terminal tail, the TRH receptor, was also determined.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characteristics of Hemagglutinin (HA)-Tagged GnRH Receptors
Insertion of the HA tag into the amino terminus of the rat GnRH receptor did not perturb either receptor-ligand binding or second messenger activation. Membranes prepared from HEK 293-B5 cells, which stably express HA-tagged GnRH receptors, bound the GnRH receptor agonist, [D-Trp6]-GnRH, with a dissociation constant (Kd) of 0.27 nM and a Bmax of 3.0 pmol/mg protein. These values were comparable to those obtained in a HEK 293 cell line stably expressing wild-type (WT) GnRH receptors (HEK 293-A2) with a Kd of 0.20 nM and a Bmax of 3.1 pmol/mg protein (29). EC50 values of 2.8 and 3.6 nM were obtained for GnRH-stimulated total IP production in HEK 293-B5 and HEK 293-A2 cell lines, respectively.

Visualization and Cellular Localization of GnRH Receptors
GnRH receptor distribution was examined in HEK 293 cells stably expressing HA-tagged GnRH receptors using indirect immunofluorescent staining. Fluorescently labeled HA-tagged GnRH receptors were predominantly distributed around the cell circumference in unstimulated HEK 293-B5 cells. Permeabilized, nontreated cells (Fig. 1aGo) showed similar fluorescent distribution consistent with localization of the receptor to the cell surface. GnRH agonist treatment (1 or 2 h at 37 C) elicited a redistribution of cellular immunostaining indicative of GnRH receptor internalization (Fig. 1bGo). The redistributed cytoplasmic signal showed a vesicular pattern, and in some cells positive staining was observed in the perinuclear region. TRH, another hypothalamic releasing hormone, had no effect on GnRH receptor distribution (Fig. 1cGo). The same was also observed for the GnRH receptor antagonist [Ac-3,4-dehydro-Pro1, D-p-F-Phe2, D-Trp3,6]-GnRH(GnRH-antag) (Fig. 1dGo), and pretreatment of cells with GnRH-antag prevented agonist-induced internalization (Fig. 1eGo). No specific staining was observed in untransfected HEK 293 cells (Fig. 1fGo).



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Figure 1. Visualization of GnRH Receptor Internalization by Confocal Microscopy

a, Untreated HEK 293-B5 cells stably expressing HA-tagged GnRH receptors; b, cells incubated with GnRH agonist, [D-Trp6]-GnRH; c, TRH; d, GnRH-antag ([Ac-3, 4-dehydro-Pro1, D-p-F-Phe2, D-Trp3, 6]-GnRH); e, GnRH-antag and GnRH; f, untransfected HEK 293 cells. All peptides were at a concentration of 1 µM and were incubated for 2 h at 37 C. Scale bar, 5 µm.

 
Colocalization studies using redistributed HA-tagged GnRH receptors with transferrin receptors were also carried out. Cells were first incubated with GnRH (1 µM for 1 h at 37 C), then fixed and incubated with both anti-HA and antitransferrin receptor antibodies. The receptors were detected using fluorescein-isothiocyanate (FITC) and Texas Red-conjugated secondary antibodies, respectively. Agonist-treated redistributed GnRH receptors (Fig. 2aGo) showed a similar distribution pattern with transferrin receptor (Fig. 2bGo), and colocalization is shown on Fig. 2cGo.



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Figure 2. Colocalization of Internalized GnRH Receptors with Transferrin Receptor

HEK 293-B5 cells were first treated with GnRH (1 µM for 1 h at 37 C), fixed, and then incubated with rabbit polyclonal anti-HA antibody (a) and mouse monoclonal antitransferrin receptor antibody (b). Agonist-induced redistributed GnRH receptors showed distribution patterns similar to those of transferrin receptors (c). Scale bar, 5 µm.

 
Time-Dependent GnRH Receptor/Ligand Internalization as Quantified by Radioligand Binding and ELISA
The quantitative radioligand assay is based on the assumption that receptor and ligand sort similarly and that the intracellular receptor/ligand complex can be determined by measuring intracellular ligand. ELISA measurements allow us to monitor the changes in surface-expressed HA-tagged receptor, and comparisons can be made with results obtained from the radioligand internalization kinetic studies. Figure 3Go shows a comparison between GnRH-induced internalization as quantified by radioligand binding and ELISA. The two methods gave comparable results regarding receptor/ligand internalization kinetics at 37 C in the presence or absence of hypertonic sucrose medium. The agonist-induced internalization process was almost completely suppressed at 4 C, and no internalization was observed for GnRH-antag as determined by radioligand assays (data not shown). ELISA measurements also demonstrated that the level of surface-expressed receptor after GnRH-antag treatment was comparable with the levels observed in untreated cells (Fig. 4Go).



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Figure 3. Comparison between GnRH Internalization Time Course as Quantified by Radioligand Binding and ELISA Techniques

HEK 293-B5 cells were incubated at 37 C either with 125I-labeled GnRH agonist or GnRH (1 µM) for the indicated time intervals. The amount of internalized receptor was then expressed either as a percentage of the total binding at that time interval or, in the case of ELISAs, calculated from decrease in the level of surface-expressed receptor after agonist treatment compared with untreated, control cells. The two assays were also performed in the presence of 0.4 M sucrose. Circles and squares represent ELISA and radioligand binding results at 37 C in the absence (closed symbols) and presence of 0.4 M sucrose (open symbols) respectively. All results are the mean ± SE from three independent experiments performed in triplicate.

 


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Figure 4. ELISA Measurement of Surface-Expressed GnRH Receptors after GnRH and GnRH Antagonist Treatment

The levels of surface-expressed receptors in HEK 293-B5 cells were monitored after treatment with either GnRH or GnRH-antag (1 µM for 1 or 2 h at 37 C). The results (mean ± SE) are expressed as a percentage of the value obtained in untreated cells from three independent experiments performed in triplicate.

 
Recycling of GnRH Receptors
To study the recycling of the internalized GnRH receptor, cells were initially treated with GnRH (1 µM for 2 h) and then allowed to recover after ligand removal and replacement with fresh medium. Receptor recycling was observed (by ELISA) within 15–30 min after ligand removal, and by 1 h almost all of the internalized receptor reappeared at the cell surface (Table 1Go). Coincubation of cells with the protein synthesis inhibitor, cycloheximide (10 µg/ml), inhibited neither agonist-induced internalization nor receptor recycling (Table 1Go), showing that during the course of the experiment no substantial de novo synthesis of the receptor occurred.


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Table 1. Recycling of GnRH Receptors

 
To test whether intracellular receptors represented a static pool or a fraction of dynamically endocytosed and recycling receptors, we performed recycling experiments in the continuous presence of agonist. In addition, we tested whether constitutive recycling proceeds through the acidified endosomal compartment by assessing the effect of NH4Cl, which interferes with the acidic pH found in endosomal vesicles (4). First, receptor internalization was induced by incubating the cells with 125I-labeled GnRH agonist (1 h at 37 C) to obtain a substantial intracellular receptor pool followed by an acid wash to remove surface-bound ligand. The absence of extracellular label during recycling ensured that no additional labeled intracellular receptors were generated, and the presence of a saturating concentration of unlabeled GnRH agonist prevented rebinding of any label that dissociates after recycling. Upon removal of labeled GnRH agonist and incubation of cells in normal medium, GnRH receptor recycled back to the plasma membrane. Under these conditions, the intracellular pool of receptors decreased by around 22% in 5 min (from 27.28 ± 0.66 to 21.24 ± 0.51%), and by 1 h the reduction was about 68% (from 27.28 ± 0.66 to 8.6 ± 1.33%). However, the addition of the 25 mM NH4Cl to the medium substantially inhibited the recycling of the GnRH receptor as evidenced by the reduction of the intracellular receptor pool by only 42% (from 27.28 ± 0.66 to 15.71 ± 0.64%) by 1 h.

Effect of ß-Arrestin on Internalization Kinetics of the GnRH and TRH Receptors
To address the role of ß-arrestin on the internalization kinetics of GnRH and TRH receptors we 1) performed internalization experiments in HEK 293 and COS-7 cells that express different levels of endogenous ß-arrestins, 2) studied the effect of both WT ß-arrestin and ß-arrestin dominant negative mutant [ß-arrestin (319–418)] coexpression on internalization of GnRH and TRH receptors in HEK 293 and COS-7 cells, respectively, and 3) followed the agonist-induced receptor-mediated redistribution of ß-arrestin/green fluorescent protein (GFP) conjugate to the plasma membrane. Studies performed using HEK 293 cells stably expressing either the GnRH or TRH receptors showed a striking difference between internalization kinetics of the two receptor types. A rapid decrease of surface-bound ligand was observed for TRH receptor with an estimated half-time (t1/2) of 2.2 min after TRH treatment. Internalization reached a steady state within the first 5–10 min after agonist exposure, and after this period the proportion of the internalized receptors was about 70% (data not shown). In contrast, the disappearance of GnRH receptors from the cell surface was delayed, with an estimated t1/2 of 20 min. Equilibrium between surface and intracellular receptors was reached only after 1 h of continuous agonist exposure with the estimated intracellular pool of receptor being around 30% (Fig. 3Go). The effect of different endogenous levels of ß-arrestin on receptor internalization rate was evaluated by performing experiments in COS-7 cells, which express about 70% less total ß-arrestins/mg protein than HEK 293 cells (30). Under conditions of low endogenous ß-arrestin expression, TRH receptor internalization was significantly slower in COS-7 (t1/2 of 5.7 min) than in HEK 293 cells. However, the rate was significantly increased by coexpression of ß-arrestin in COS-7 cells (t1/2 of 3.1 min) (Fig. 5Go). In contrast, coexpression of GnRH receptor with ß-arrestin in COS-7 cells had no effect on internalization kinetics (Fig. 5Go). Coexpression with ß-arrestin in HEK 293 cells did not promote internalization in either receptor (data not shown). We next assessed the ability of a dominant negative ß-arrestin mutant (ß-arrestin (319–418)), which has been previously characterized to retain clathrin binding but lack the receptor binding activity (17), to impair receptor internalization. COS-7 cells were transfected either with GnRH or TRH receptor alone or together with different ß-arrestin constructs and the intracellular receptor pool estimated after agonist treatment (15 min at 37 C). The coexpression of ß-arrestin (319–418) substantially reduced the effect of ß-arrestin on TRH receptor internalization in COS-7 cells (Fig. 6Go). The coexpression of ß-arrestin (319–418) only slightly decreased the basal agonist-induced internalization in COS-7 cells, whereas a significant decrease was observed in HEK 293 cells stably expressing the TRH receptor (Fig. 7Go). No significant effect of ß-arrestin construct coexpression on GnRH receptor internalization was observed either in COS-7 or in HEK 293 cells (Figs. 6Go and 7Go). In addition, the ability of coexpressed ß-arrestin/GFP conjugate in COS-7 cells to enhance the internalization of TRH, but not the GnRH receptor, was also confirmed (Fig. 6Go).



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Figure 5. Effect of ß-Arrestin on Internalization of GnRH and TRH Receptors Transiently Expressed in COS-7 Cells

Receptors were coexpressed with a pcDNA3 expression vector together with 5 µg of empty pcDNA3 vector or pcDNA3 ß-arrestin. The time-dependent loss of the surface GnRH and TRH receptors was measured by changes in radioligand binding. Each point is the average ± SE of three independent experiments performed in triplicate. The amount of the receptors on the cell surface as a function of time was fitted in the four-compartment model (31 ).

 


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Figure 6. Effect of ß-Arrestin (319–418) Mutant on ß-Arrestin-Induced Internalization of GnRH and TRH Receptors Transiently Expressed in COS-7 Cells

Receptors (5 µg cDNA in pcDNA3/100 mm dish) were coexpressed with a pcDNA3 expression vector together with 5 µg of empty pcDNA3 vector or 1 µg pcDNA3 ß-arrestin, 1 µg pcDNA3 ß-arrestin/GFP, 5 µg pcDNA3 ß-arrestin (319–418), 1 µg pcDNA3 ß-arrestin, and 4 pcDNA3 ß-arrestin (319–418). The percentage of internalized receptors after 15 min agonist exposure at 37 C was determined by radioligand binding. Solid and hatched bars represent the data for the GnRH and TRH receptor, respectively. Each point is the average ± SE of three independent experiments performed in triplicate.

 


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Figure 7. Effect of ß-Arrestin (319–418) Mutant on the Internalization of GnRH and TRH Receptors Stably Expressed in HEK 293 Cells

HEK 293 cells expressing either GnRH or TRH receptors were coexpressed with 10 µg of empty pcDNA3 vector or 10 µg ß-arrestin (319–418) pcDNA3, and the percentage of internalized receptors was determined after 15 min agonist exposure at 37 C by radioligand binding. Solid and hatched bars represent the data for the GnRH and TRH receptor, respectively. Each point is the average ± SE of three independent experiments performed in triplicate.

 
Effect of ß-Arrestin on GnRH- and TRH Receptor Endocytosis and Recycling Rate Constants
To acquire further information on the effect of ß-arrestin on internalization kinetics for GnRH and TRH receptors, the four-compartment model described by Koenig and Edwardson (31) was applied in an attempt to model receptor intracellular trafficking. This model predicts four major pathways of receptor movement within the cells: 1) receptor endocytosis from the cell’s surface into endosomes, 2) recycling from endosomes back to plasma membrane, 3) receptor movement from endosomes to lysosomes for degradation, and 4) the delivery of newly synthesized receptor to the plasma membrane. Utilizing a numerical parameter-fitting routine, we calculated a complete set of rate constants for the receptor endocytosis (ke), recycling (kr), synthesis (ks), and degradation (kx), which are summarized in Table 2Go. Comparable rate constants derived from data using either quantitative radioligand binding or ELISA measurements were obtained for HA-tagged GnRH receptor in HEK 293-B5 cells [ke = 0.013 ± 0.001; kr = 0.032 ± 0.001 min-1; ks = 0; kx= 0.010 ± 0.001 min-1 (radioligand binding); and ke = 0.013 ± 0.001; kr = 0.028 ± 0.002; ks = 0; kx = 0.018 ± 0.001 min-1 (ELISA measurements)], confirming the use of radioligand-binding assays for assessing receptor intracellular trafficking. Similar rate constants (ke = 0.012 ± 0.001; kr = 0.033 ± 0.003; ks = 0; kx = 0.008 ± 0.001 min-1) were also obtained for WT GnRH receptor stably expressed in HEK 293-A2 cells. The GnRH receptor internalization rates were similar in both cell types (HEK 293 and COS-7) and also unaffected by different ß-arrestin expression levels (Table 2GoA). The ke for TRH receptor in COS-7 cells was nearly 4-fold lower than the corresponding value in HEK 293 cells. However, ß-arrestin coexpression in COS-7 cells resulted in an almost 2-fold increase in ke for TRH receptor (Table 2GoB). The obtained kx values were also substantially higher for TRH- than GnRH receptor in both cell types (Table 2Go, a and b). In addition, internalization rates obtained with {alpha}T3 cells (ke = 0.014 ± 0.002; kr = 0.032 ± 0.005) and GH3 (ke = 0.181 ± 0.025; kr = 0.078 ± 0.006) cells, which endogenously express GnRH and TRH receptors, respectively, were comparable to those observed for the respective receptor types stably expressed in HEK 293 cells. In all calculations, the fitted value of ks was not significantly different from zero, confirming the absence of synthesis of either receptor during the course of the experiment (data not shown).


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Table 2. Internalization Rate Constants for GnRH and TRH Receptors

 
Cellular Distribution of ß-Arrestin/GFP Fusion Protein in HEK 293 Cells
To further study the role of ß-arrestin in GnRH and TRH receptor internalization, GFP has been coupled to ß-arrestin (ß-arrestin/GFP fusion protein), thus allowing us to visualize the agonist-induced translocation of ß-arrestin in cells expressing GPCRs. ß-Arrestin/GFP retained its biological activity with respect to its ability to enhance the TRH receptor internalization in COS-7 cells (Fig. 6Go). Confocal microscopy showed cytosolic distribution of ß-arrestin/GFP in untreated HEK 293 cells expressing either TRH (Fig. 8aGo) or GnRH receptor (Fig. 8cGo). After agonist stimulation, a time-dependent TRH-mediated redistribution of ß-arrestin/GFP from the cytosol to the cell membrane was observed (Fig. 8bGo), while no increase in membrane-associated fluorescence indicative of ß-arrestin/GFP translocation was observed in GnRH receptor-expressing cells even after prolonged exposure to GnRH (Fig. 8dGo). Similar results were obtained in COS-7 cells transiently expressing either TRH or GnRH receptor (not shown).



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Figure 8. Confocal Microscopy of ß-Arrestin/GFP Cellular Distribution in HEK 293 Cells Stably Expressing Either GnRH or TRH Receptors

HEK-293 cells stably expressing either TRH (a and b) or GnRH receptors (c or d) were transfected with cDNA for ß-arrestin/GFP. ß-Arrestin/GFP distribution is initially cytosolic in untreated cells (a and c). After agonist stimulation (TRH, 1.5 min; GnRH, 10 min), TRH-mediated redistribution of ß-arrestin/GFP from the cytosol to the membrane is shown (b), while no redistribution of ß-arrestin/GFP is observed for GnRH receptor expressing HEK 293 cells (d). Scale bar, 5 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previous studies on the GnRH receptor have not measured actual receptor internalization, but rather the internalization of labeled ligand. Using immunocytochemical methodology and a cell line stably expressing epitope-tagged GnRH receptors, this study reports direct visualization of GnRH receptor internalization. In unstimulated cells expressing HA-tagged GnRH receptor, the signal was confined mainly to the cell membrane. After treatment with agonist, GnRH receptors were internalized in a time-dependent manner with internalization being demonstrable after 20 min but pronounced after 1–2 h. The signal corresponding to the internalized receptor also showed a high degree of spatial correlation with transferrin receptor.

The agonist-induced internalization time course for GnRH receptors was further quantified by two different methods; radioligand binding and ELISA, of which the latter is not influenced by the state of receptor/G protein coupling (32). Relatively low internalization rates were confirmed using both methods. Radioligand binding studies in both primary (23) and immortalized {alpha}T3–1 gonadotrope cells (24), as well as in GH3 lactotropes stably transfected with the GnRH receptor (25), also demonstrate a surface GnRH receptor loss over a similar time frame.

Endocytosis by a clathrin-mediated pathway is temperature dependent and can also be inhibited by hypertonic sucrose (33), which induces abnormal clathrin polymerization and subsequently reduces the number of clathrin-coated pits. Another well established marker for endocytosis via clathrin-coated pits is the transferrin receptor (34). We observed that GnRH-induced internalization was inhibited both at 4 C and in the presence of sucrose. The location of redistributed GnRH receptors also overlapped substantially with that of the transferrin receptors. Therefore, our results suggest that agonist-triggered endocytosis of the GnRH receptor probably occurs via a clathrin-dependent mechanism.

After internalization, receptors can either be sorted into endosomes for recycling back to the cell surface or, alternatively, may undergo degradation within lysozomes (31). After agonist removal, GnRH receptors reappeared at the cell surface, a process unaltered by cycloheximide, suggesting that GnRH receptor recycling from endosomes, rather than de novo receptor synthesis, was involved. However, if recycling had occurred only after agonist removal, we would have anticipated a linear increase in the intracellular pool of receptor. Our observations show that this is not the case, and the recycling data confirmed that in the continuous presence of agonist, a dynamic equilibrium between endocytosed and recycling receptors was reached. GPCR internalization is now thought to be associated primarily with receptor resensitization, which is achieved through endosomal sorting and recycling of functional receptor back to the plasma membrane (4, 35). Recycling of the GnRH receptor was inhibited by NH4Cl, indicating the involvement of acidified endosomal compartments in receptor trafficking, as has been previously shown for ß2-adrenergic (4) and angiotensin receptors (11).

The efficiency of GPCRs endocytosis is not only receptor- but also cell type-dependent and should reflect differences in endogenous levels of certain intracellular proteins. HEK 293 and COS-7 cells express high and low levels of ß-arrestins, respectively (30), and thus provide us with an opportunity for examining the effect of different endogenous ß-arrestin expression levels on GPCR internalization dynamics in a natural cellular environment. Therefore, we determined internalization kinetics for two functionally distinct members of the GPCR family; the GnRH receptor and also the TRH receptor, which possesses a functional carboxy-terminal tail. Additional reasons to choose TRH receptor were that its endocytosis displays characteristics of the clathrin-mediated pathway (9), and the importance of the carboxy-terminal tail in agonist-induced internalization was previously confirmed using this receptor (20).

The ke and kr values were similar for the WT and epitope-tagged GnRH receptor stably expressed in HEK 293 cells and for WT GnRH receptor transiently expressed in COS-7 cells, and this ke is also comparable with a reported value for WT GnRH receptor in COS-7 cells (28). Considering the reported differences in endogenous ß-arrestin levels between the two cell types, a higher GnRH internalization rate in HEK 293 than in COS-7 cells was expected, which was not the case. The rate constants for the GnRH receptor in both cell types were much lower than those obtained for TRH receptor and also those reported for some other GPCRs (8, 36); however, they are still within the range of internalization rates reported for clathrin-mediated endocytosis in nonneuronal cells (37). TRH receptor internalization rates were cell type dependent: lower in COS-7 than in HEK 293 cells; however, overexpression of ß-arrestin in COS-7 cells enhanced TRH receptor kinetics to levels approaching those in HEK 293 cells. The receptor degradation can increase in the presence of agonist (36); therefore, it is necessary to take degradation into account in any attempt to model intracellular trafficking. The rate of TRH receptor degradation (kx) was higher than kx for GnRH receptor in both cell types, whereas the synthesis rate constant (ks) was negligible for either receptor type. A loss of approximately 10% of surface GnRH receptor was observed during the recycling experiments in the absence or presence of the protein synthesis inhibitor cycloheximide. This result is in close agreement with the kx and ks values for GnRH receptor obtained from the four-compartment model, which predicts that 30–40% of receptor in the endosomal compartment is destined for degradation, and no new synthesis of receptor occurs during the course of the experiment.

The GnRH receptor internalization kinetics in the two cell types were similar, and no obvious role of ß-arrestin in this process was observed. This observation was further supported by results obtained with ß-arrestin dominant negative mutant [ß-arrestin (319–418)], which efficiently impaired agonist-induced internalization of the TRH but not of the GnRH receptor. The applicability of this construct for studying agonist-induced GPCRs internalization in different cell lines has been previously determined (17). In addition, we constructed the ß-arrestin/GFP fusion protein, which enabled us to assess translocation of ß-arrestin/GFP complex to the plasma membrane in response to GPCR activation. The construct was fully functional with respect to its ability to promote TRH receptor internalization, and the time- and agonist-dependent redistribution of ß-arrestin/GFP to the plasma membrane was easily observed by confocal microscopy. However, no evident redistribution of ß-arrestin/GFP was observed in cells expressing the GnRH receptor. The results obtained with ß-arrestin constructs could suggest either that GnRH receptor internalization is not ß-arrestin-dependent or that ß-arrestin, which preferentially binds phosphorylated receptor (14), has a low affinity for GnRH receptor due to the lack of a tail and therefore of potential phosphorylation sites. Truncation of the carboxy-terminal tail of the ß2-adrenergic receptor has been shown to reduce ligand-induced internalization by 50%, an effect that is reversed when the receptor is coexpressed with ß-arrestin, and coexpression of the WT receptor with inactive mutants of ß-arrestin reduced internalization by 70% (13). Similarly, the removal of the phosphorylation sites in the carboxyl tail of the ß2-adrenergic receptor reduces receptor internalization to levels comparable to those observed for the GnRH receptor (30). This observation indicates that GPCR kinase-mediated phosphorylation is not absolutely required as a signal initiating internalization, but only as a factor determining its rate.

The obvious question is can clathrin-dependent endocytosis be ß-arrestin independent, since ß-arrestins specifically target activated receptor into clathrin-coated vesicles? In HEK 293 cells expressing AT1A receptor, agonist-promoted sequestration of this receptor was shown to be ß-arrestin independent, although it can be recruited to this pathway by overexpression of ß-arrestin (38). Furthermore, it has been suggested that AT1A receptor internalization is clathrin-mediated, since colocalization with transferrin receptor in the same cell line was observed (11). Similarly, we showed that the GnRH receptor undergoes agonist-induced receptor internalization (albeit at a slower rate than other GPCRs) probably via a clathrin-dependent mechanism and provided evidence for the dynamic nature of this process. However, it seems likely that the GnRH receptor endocytotic pathway is ß-arrestin and cell type independent as overexpression of ß-arrestin in different cell types does not affect internalization kinetics.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Chamber slides, inositol-free DMEM, penicillin, and streptomycin were obtained from GIBCO (Paisley, UK). Transfectam was obtained from Promega (Southampton, UK). All other tissue culture reagents and media were supplied by Sigma Cell Culture (Dorset, UK). Anti-HA mouse monoclonal antibody (clone 12CA5) and anti-HA rabbit polyclonal antibody were obtained from Boehringer Mannheim (East Sussex, UK) and Babco (Richmond, CA), respectively. Antitransferrin receptor mouse monoclonal antibody (clone Ber-T9) was obtained from Dako A/S (Glostrup, Denmark); mouse/goat serum from the Scottish Antibody Production Unit (Carluke, UK), and Citifluor from Chem Lab (Canterbury, UK). The Bio-Rad protein assay kit and Dowex resin were obtained from Bio-Rad (Herts, UK). 3,3',5,5'-Tetramethylbenzidine liquid substrate system was obtained from Sigma (Dorset, UK). TRH-(3-Met-His2)-[3H] (250 mCi) was from NEN Life Science Product (Hertfordshire, UK). All other compounds and reagents were obtained from either Sigma (Dorset, UK), Calbiochem (Nottingham, UK), or Amersham (Buckinghamshire, UK). HEK 293 and COS-7 cells were obtained from the European Collection of Animal Cell Cultures, Centre for Applied Microbiology and Research (Salisbury, UK).

Cell Culture
Cell lines were routinely grown in complete DMEM containing 10% (vol/vol) heat inactivated FCS, glutamine (0.3 mg/ml), penicillin (100 IU/ml), and streptomycin (100 µg/ml) and incubated at 37 C in a humidified atmosphere of 5% (vol/vol) CO2 in air.

Derivation of Epitope-Tagged GnRH Receptor-Expressing Cell Lines
A double-stranded oligonucleotide fragment corresponding to the sequence of the HA tag (YPYDVPDYA) was synthesized and ligated in frame, into the amino terminus of the rat GnRH receptor in the vector pcDNA3. Linearized HA-tagged GnRH receptor plasmid DNA was then stably transfected into human embryonal 293 cells (HEK 293) cells using Transfectam. Receptor-containing clones were then selected after the treatment of cell cultures with geneticin (1 mg/ml), expanded, and maintained in complete DMEM containing geneticin (500 µg/ml). HA-tagged GnRH receptor-containing clones were subsequently identified using previously described GnRH receptor ligand-binding assays (39) and total inositol phosphate assays (40). The clone HEK 293-B5, expressing the HA-tagged GnRH receptor, was chosen for further study.

Detection and Visualization of HA-Tagged GnRH Receptors
HA-tagged GnRH receptors were detected using indirect immunocytochemistry on cell monolayers. Trypsinized cells were plated into eight-well chamber slides at a density of 2.5 x 104 cells per well in complete DMEM. After 2 days, cells were washed twice with 0.01 M PBS, pH 7.4, and treated as required in HEPES-modified DMEM with 0.1% BSA, pH 7.4, before fixing with freshly prepared 4% paraformaldehyde for 30 min at room temperature. To reduce the nonspecific binding, cells were incubated in blocking solution (PBS containing 1% BSA and 10% normal serum, pH 7.4) for 30 min. If permeabilized cells were required, to enable the visualization of internalized HA-tagged GnRH receptors, nonionic detergent (Nonidet P-40) was added to the blocking solution to a final concentration of 0.2%. Subsequently, cells were washed with PBS (three times) before incubating with primary antibody in blocking solution overnight at 4 C. After washing (four times in PBS) cells were incubated with goat antimouse FITC-conjugated secondary antibody (20 µg/ml) for 60 min at room temperature. Slides were then washed (four times in PBS), mounted in Citifluor, and sealed with coverslips. Cells were examined under an oil immersion objective (x60) using a Zeiss LMS 510 confocal laser microscope and a filter selective for FITC fluorescence. Optical sections (0.45 µm) were taken, and representative sections corresponding to the middle of the cells were presented. After indirect immunofluorescent staining, no specific fluorescence was observed in untransfected HEK 293 cells, or in HEK 293-B5 cells treated with secondary FITC-linked goat antimouse IgG antibody only.

Colocalization of HA-Tagged GnRH Receptors with Transferrin Receptor
For colocalization of epitope-tagged receptor with transferrin receptor, the cells were first treated (1 µM GnRH for 1 h at 37 C), fixed, and incubated with both rabbit polyclonal anti-HA antibody and mouse antihuman transferrin receptor antibody overnight at 4 C. The epitope-tagged receptors and the transferrin receptors were detected using antirabbit FITC and antimouse Texas Red-conjugated secondary antibody (as described previously). Cells were then viewed as previously described using filters selective for either rhodamine or fluorescein fluorescence.

Total IP Assays
Assays were performed in 24-well plates containing 1.5 x 105 cells per well, and total IPs were extracted and separated as described previously (40).

Iodination of GnRH Agonist and Antagonist
Iodinated radiolabeled GnRH analogs were prepared using the glucose oxidase/lactoperoxidase method and purified by chromatography on a Sephadex G-25 column in 0.01 M acetic acid/0.1% BSA. The specific activities of the 125I-des-Gly10,[D-Trp6]-GnRH (GnRH agonist) and 125I-[Ac-3,4-dehydro-Pro1, D-p-F-Phe2, D-Trp3,6]-GnRH (GnRH-antag) tracers were 53 µCi/µg and 28.2 µCi/µg, respectively, and were calculated from self-displacement assays using either rat pituitary homogenates or COS-7 cells transiently transfected with the WT GnRH receptor cDNA. Assays were incubated for 2 h at 4 C before filtration through a cell harvester using Whatman GFB filter paper.

Receptor Internalization and Recycling Assays
Receptor internalization and recycling assays were based on protocols described by Lauffenburger and Linderman (41). Briefly, HEK B5–293 cells were plated at a density of 1.5 x 105 cells per well in 24-well plates. After 2 days, cells were washed once with assay medium (HEPES-modified DMEM with 0.1% BSA, pH 7.4) before being incubated with 125I-labeled GnRH agonist or antagonist (100,000 cpm/well) in 0.5 ml assay medium for time intervals ranging from 5 min to 2 h at either 4 C or 37 C. To test the effect of hypertonic medium, assays were also performed in the presence of sucrose; in this case, the cells were pretreated with assay medium containing 0.4 M sucrose for 20 min at 37 C, the sucrose concentration being maintained during the ligand treatment. At appropriate times, cells were transferred onto ice and washed twice with ice-cold PBS. Subsequently, the extracellular receptor-associated ligand was removed by washing once with 1 ml of acid solution (50 mM acetic acid and 150 mM NaCl, pH 2.8) for 12 min. The acid wash was collected to determine the surface-bound radioactivity, and the internalized radioactivity was determined after solubilizing the cells in 0.2 M NaOH and 1% SDS (NaOH/SDS) solution. Nonspecific binding for each time point was determined under the same conditions in the presence of 10 µM unlabeled agonist or antagonist. After subtraction of nonspecific binding, the internalized radioactivity was expressed as a percentage of the total binding at that time interval. All time points were performed in triplicate for at least three separate experiments. To compare the agonist-induced internalization kinetics of the GnRH receptor with another member of the GPCR superfamily, HEK E2–293 cells, which stably express WT TRH receptor (42), were also assayed under identical conditions using 3H-labeled TRH agonist. To evaluate the effect of ß-arrestin on internalization, COS-7 cells and HEK 293 cells stably expressing either GnRH or TRH receptor (2 x 106/100 mm dish) were transiently transfected with various constructs using Transfectam. Cells were then split into 24-well plates, and internalization assays were performed with the appropriate labeled agonist as described above.

For the recycling experiment, HEK 293-B5 cells were preincubated with 125I-labeled GnRH agonist (1 h at 37 C) to get a substantial intracellular pool, followed by an acid wash to remove surface-bound ligand; cells were then incubated in medium containing saturating concentrations of unlabeled agonist for varying periods of time (5–60 min at 37 C) to monitor the decrease in the intracellular pool due to recycling of the ligand. To test whether the recycling proceeded through acidified endosomal compartments, 25 mM NH4Cl was added to the incubation medium during the recycling period, and the decrease in the intracellular receptor pool was monitored as described above. From the data obtained, the endocytosis (ke), recycling (kr), synthesis (ks), and degradation (kx) rate constants were calculated using the four-compartment model described by Koenig and Edwardson (31).

ELISA
ELISA assays for the measurement of surface-expressed HA-tagged GnRH receptors and quantification of receptor internalization were based on the method described previously (43). Cells were plated out at a density of 5 x 104 cells per well in 48-well plates. After 2 days, cells were treated as required in either HEPES-modified DMEM with 0.1% BSA, pH 7.4, or hypertonic sucrose medium for varying periods of time at 37 C before fixing with freshly prepared 4% paraformaldehyde for 10 min at room temperature. Cells were then washed three times in PBS, blocked (PBS containing 10% normal serum, pH 7.4), and incubated with a 1:200 dilution of primary anti-HA monoclonal antibody in blocking buffer overnight at 4 C. Subsequently, cells were washed with PBS (three times) and incubated for 1 h at 37 C in a 1:2000 dilution of a horseradish peroxidase-conjugated sheep antimouse IgG. After final washes in PBS (six times) and 0.9% NaCl (once), the reaction was developed using the 3,3',5,5'-tetramethylbenzidine liquid substrate system. The enzymatic reaction was stopped after 30 min at room temperature with 0.5 N H2SO4, and a 100 µl sample was taken for colorimetric measurement at 450 nm using a Labsystem multiscan MCC/340 reader. For the recycling experiment, a preliminary treatment (1 µM GnRH for 2 h at 37 C) was followed by an acid wash (50 mM acetic acid and 150 mM NaCl, pH 2.8) for 5 min to remove surface-bound ligand, and the cells were incubated in serum-free HEPES-modified DMEM/BSA medium at 37 C for varying periods of time (5 min to 1 h) to monitor the recovery of surface-expressed receptors. Cycloheximide (10 µg/ml), when used, was present throughout the whole experiment. Untransfected HEK 293 cells were assayed concurrently to determine background. All experiments were done in triplicate.

Construction of the ß-Arrestin-GFP Expression Construct
Production and subcloning of the ß-arrestin/GFP fusion protein were performed in two separate steps. In the first step, the coding sequence of a modified form of GFP (44) was altered by PCR amplification. Using the amino-terminal primer 5'-CCGCTCGAGAGTAAAGGAGAAGAACTTTTCAC-3' a XhoI restriction site (underlined) was introduced adjacent to the sequence of codon 2 of GFP. The ATG initiator codon was removed. Using the carboxyl-terminal primer 5'-TGCTCTAGATTATTTGTATAGTTCATCCATGCC-3' an XbaI restriction site (underlined) was introduced after the stop codon. The amplified fragment of GFP digested with XhoI and XbaI was ligated into the pcDNA3 expression vector (Invitrogen, San Diego, CA) digested with XhoI and XbaI. To obtain the ß-arrestin/GFP fusion protein, the coding sequence of ß-arrestin (45) was amplified by PCR. Using the amino-terminal primer 5'-AAAAAAGCTTTCTACCATGGGCGACAAAGGGACAC-3', a HindIII restriction site (underlined) and a partial Kozak site were introduced in front of the initiator Met of ß-arrestin. Using the carboxyl-terminal primer 5'-AACTCGAGTCTGTTGTTGAGGTGTGGAGAGC-3' a XhoI restriction site (underlined) was introduced just in front of the stop codon of ß-arrestin. Finally, the GFP construct in pcDNA3 was digested with XhoI and HindIII and was ligated together with the PCR product of the ß-arrestin amplification that was digested with HindIII and XhoI. The open reading frame so produced represents the coding sequence of ß-arrestin/GFP. This construct was fully sequenced before its expression and analysis. HEK 293 cells (1.5 x 106/60 mm dish) stably expressing either GnRH (B5 cells) or TRH (E2 cells) receptor were transfected with 2.5 µg of ß-arrestin/GFP cDNA using Transfectam. After 24 h, cells were plated into eight-well chamber slides, and treatments were carried out 48–72 h after transfection. The cells were then fixed with 4% paraformaldehyde, mounted, and sealed with coverslips. Confocal microscopy was performed as described previously.


    ACKNOWLEDGMENTS
 
The authors would like to thank Professor J. F. Benovic (Jefferson Medical College, Philadelphia) who kindly provided us with cDNA for ß-arrestin and ß-arrestin (319–418) dominant negative mutant, Professor S. V. Bavdek for support and encouragement, Dr. B. Byrne, Mr. R. Sellar, and Ms. S. Nettleship for expert technical assistance, and Dr. H. Rahe for critical evaluation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. K. A. Eidne, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Centre for Reproductive Biology, EH3 9EW Edinburgh, United Kingdom. E-mail: keidne{at}hgmp.mrc.ac.uk

M. Vrecl is financially supported by the Ministry of Science and Technology of the Republic of Slovenia.

Received for publication July 10, 1998. Accepted for publication August 18, 1998.


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