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Endocrinology Vol. 145, No. 2 930-940
Copyright © 2004 by The Endocrine Society

Desensitization and Endocytosis Mechanisms of Ghrelin-Activated Growth Hormone Secretagogue Receptor 1a

Jesus P. Camiña, Marcos C. Carreira, Said El Messari, Catherine Llorens-Cortes, Roy G. Smith and Felipe F. Casanueva

Department of Medicine (J.P.C., M.C.C., F.F.C.), Research Area, Molecular Endocrinology Laboratory, Complejo Hospitalario Universitario de Santiago (CHUS) and University of Santiago de Compostela, E-15705 Santiago de Compostela, Spain; Institut National de la Sante et de la Recherche Medicale (S.E.M., C.L.-C.), Unite 36, College de France, Chaire de Medecine Experimentale, 75231 Paris cedex 05, France; and Huffington Center on Aging and Department of Molecular and Cellular Biology (R.G.S.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Felipe F. Casanueva, Molecular and Cellular Endocrinology Laboratory, Department of Medicine, Complejo Hospitalario Universitario de Santiago-Universidad de Santiago de Compostela, P.O. Box 56, E-15780 Santiago de Compostela, Spain. E-mail: endocrine{at}usc.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, a sequential analysis of pathways involved in the regulation of GH secretagogue receptor subtype 1a (GHSR-1a) signaling has been undertaken to characterize the process of rapid desensitization that is observed after ghrelin binding. This process was evaluated by studying the binding of [125I]ghrelin, measurement of intracellular calcium mobilization, and confocal microscopy. The results indicate that GHSR-1a is mainly localized at the plasma membrane under unstimulated conditions and rapidly desensitizes after stimulation. The agonist-dependent desensitization is not mediated by protein kinase C because phorbol ester, phorbol-12-myristate-13-acetate, failed to block the ghrelin-induced calcium response. The ghrelin/GHSR-1a complex progressively disappears from the plasma membrane after 20 min exposure to ghrelin and accumulates in the perinuclear region after 60 min. Colocalization of the internalized GHSR-1a with the early endosome marker (EEA1) after 20 min exposure to ghrelin suggests that endocytosis occurs via clathrin-coated pits, which is consistent with the lack of internalization of this receptor observed after potassium depletion. Different from other G protein-coupled receptors, GHSR-1a showed slow recycling. Surface binding slowly recovered after agonist treatment and returned to control levels within 360 min. Furthermore, inhibition of vacuolar H+-ATPases prevented recycling of the receptor, suggesting that the nondissociation of the ligand/receptor complex is responsible for this effect. The GHSR-1a internalization may explain the characteristic physiological responses mediated by this receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PULSATILE RELEASE of GH from anterior pituitary gland is regulated by the interplay of at least two hypothalamic hormones, GHRH and somatostatin, via their engagement with specific cell surface receptors on the anterior pituitary somatotroph cells (1). Release of GH in vivo can also be elicited by a group of synthetic GH secretagogues (GHSs), a family of synthetic peptidyl or nonpeptidyl compounds (2, 3). The neuroendocrine activities of GHSs are mediated by a G protein-coupled receptor (GPCR), the GHS receptor type 1a (GHSR-1a), which was cloned and identified in the pituitary and hypothalamus using MK-0677, a nonpeptidyl GHS (4, 5). GHSR-1a triggers calcium release from intracellular stores through the generation of inositol trisphosphate by means of protein subunit G{alpha}11 (5, 6). The intracellular calcium mobilization and subsequent entry of calcium from extracellular medium through voltage-operated L-type channels initiate the cellular responses to the stimulus (1). An endogenous ligand has recently been identified for this receptor: a peptide isolated from the human stomach called ghrelin (7). The discovery of this hormone is a good example of reverse pharmacology because starting with artificial compounds such as GHSs, it was possible to isolate the natural ligand via the discovery of the GHSR-1a.

Ghrelin is the first secreted natural peptide that possesses the hydroxyl group of Ser3 residue acylated by n-octanoic acid that is essential for its bioactivity. This hormone is mostly expressed and secreted by the stomach and to a lesser degree in the pituitary (8), placenta (9), testis (10), hypothalamus (11), heart, and kidney (12), although the amount secreted and the physiological relevance of its presence in these tissues has still to be addressed. This hormone releases GH both in vitro and in vivo, and this stimulatory effect on GH secretion is more potent than that of GHRH (13). This unambiguously demonstrates that ghrelin is involved in a novel GH-regulating system along with GHRH and somatostatin, although when administered in high doses, it is also able to modify the secretion of prolactin, ACTH, and splanchnic hormones. However, ghrelin, like the synthetic GHSR agonists, is more than a GHS because potent and relevant actions have been described on the control of appetite and energy homeostasis with ancillary actions on sleep regulation, reproduction, and cardiovascular functions (14). The discovery of this hormone is, thus, an important breakthrough in the understanding of the complex interplay between GH secretion and the integrated control of energy homeostasis (15).

The wide spectrum of ghrelin action is mainly mediated by GHSR-1a (4, 16, 17); therefore, elucidation of the mechanisms involved in the regulation of GHSR-1a should contribute to a better understanding of the physiological role of ghrelin as well as a characterization of the potential therapeutic use of its analogs, i.e. the known GHSs. The regulation of ghrelin receptor responsiveness potentially involves molecular events governing receptor signaling, desensitization, and down-regulation. Desensitization is a consequence of a combination of the uncoupling of the receptor from heterotrimeric G proteins and the internalization of cell surface receptors to intracellular compartments, which provides a mechanism for protecting the cell against receptor overstimulation. The down-regulation of receptors involves inhibition of mRNA expression and protein synthesis resulting from continuous exposure of cells to agonists (18, 19). These mechanisms as they pertain to ghrelin and GHSR-1a have not been characterized; therefore, in the present study, we investigated the dynamics of ghrelin-mediated receptor desensitization by monitoring ligand receptor trafficking.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human ghrelin was provided by Neosystem (Strasbourg, France), and GHRP-6 was provided by Clinalfa (Laeufelfingen, Switzerland). Human GHRH (1–29NH2) was purchased from Serono Laboratory (Madrid, Spain). L-{alpha}-Lysophosphatidic acid (oleoyl, LPA), adenosine, phorbol 12-myristate 13-acetate, cycloheximide, leupeptin, and concanamycin A were obtained from Sigma (St. Louis, MO). Polyallylamine hydrochloride was provided by Aldrich (Milwaukee, WI). Glass slides were provided by Nalge Nunc (Glostrup, Denmark). Fura-2 pentaacetoxymethylester (fura-2/AM) was obtained from Molecular Probes (Eugene, OR). [125I]ghrelin was from Amersham Pharmacia Biotech (Piscataway, NJ).

Cell cultures and transfection of HEK 293 cells
The human embryonic kidney (HEK) 293 cell line that stably expresses the human ghrelin receptor 1a (HEK-GHSR-1a) was cultured in 100-mm dishes in high-glucose DMEM plus 10% (vol/vol) fetal calf serum, glutamine, and penicillin-streptomycin solution plus 500 µg/ml geneticin G-418 to 70–80% confluence for 3 d as described previously (5). To measure the background binding, nontransfected HEK 293 was seeded in 100-mm dishes and cultured to 80% confluence for 2 d in DMEM supplemented with 10% (vol/vol) fetal calf serum. Media were supplemented with penicillin G (100 U/ml) and streptomycin sulfate (100 mg/ml). Cells were grown under a humidified atmosphere of 95% air and 5% CO2 at 37 C.

Cell cultures and transfection of Chinese hamster ovary (CHO) cells
Plasmid construction of the C-terminal tagging GHSR with enhanced green fluorescent protein (EGFP).
The human GHSR-1a cDNA [a generous gift from Dr. A. D. Howard (Department of Obesity Research, Merck Research Laboratories, Rahway, NJ) (5)] was amplified by PCR using the following sense and antisense oligonucleotide primers: 5'-ATTAAGCTTATGTGGAACGCGACGCCCAGC-3' (containing the nucleotides ATT, the HindIII restriction site sequence and nucleotides 1–21 of the GHS receptor cDNA type 1a) and 5'-TGTGGATCCGTATTAATACTAGTTTCCCATGT-3' (corresponding to nucleotides 1075–1098 of the GHS receptor cDNA type 1a, followed by the sequence of the BamHI restriction site, and three additional nucleotides, ACA). The PCR product (1112 bp) was gel purified and digested with HindIII and BamHI and was inserted between the HindIII and BamHI sites of plasmid pEFGP-N1 (Clontech, Palo Alto, CA) upstream of the 5' end of the EGFP cDNA. The construct was checked by sequencing.

CHO-K1 cells (American Type Culture Collection, Manassas, VA) were maintained in Ham’s F12 medium supplemented with 7.5% (vol/vol) fetal calf serum, 1 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were grown under a humidified atmosphere of 95% air and 5% CO2 at 37 C. To establish pure cell lines expressing the GHSR-1a-EGFP, 2.6 x 106 CHO-K1 cells were transfected with 1 µg of the plasmid, using a liposomal transfection reagent (Dosper liposomal transfection reagent; Roche Molecular Biochemicals, Indianapolis, IN). Transfected cells were selected on the basis of resistance to 750 µg/ml geneticin G418. Resistant cells were dissociated by trypsin and analyzed by fluorescence-activated cell sorting on an Epics EST flow cytometer equipped with an Autoclone cell sorter (Coulter France, Marjency, France) as described previously (20). After 7 d in culture, final selection of clones was achieved by assessing the intensity of membrane fluorescence in each clone using an BX 60 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a standard fluorescein isothiocyanate filter set. A subsequent fluorescence-activated cell sorting analysis was performed to check the purity and fluorescence intensity of the selected stable cell line (clone 1, GHS-1aR-EGFP CHO), which was used for all experiments.

Calcium measurements
Intracellular calcium measurements were performed in HEK-GHSR-1a cell suspensions using the fluorescent calcium probe fura-2 as described previously (21). Briefly, cells were resuspended in Krebs-Ringer-HEPES [KRH; containing (in mM): 125 NaCl, 5 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2 CaCl2, 6 glucose, and 25 HEPES/NaOH (pH 7.4)] and loaded with 3 µM fura-2/AM for 45 min at room temperature under gentle continuous mixing. Cell suspensions were diluted 1:4 with KRH and maintained at room temperature until use. For each measurement, around 5–6 x 105 cells were resuspended in 2 ml KRH and then placed in a cuvette positioned in a holder and thermostatically controlled at 37 C. The fluorescence signal was measured under continuous stirring in an LS-50B fluorometer (PerkinElmer, Boston, MA) in ratio mode, using 345 nm as {lambda}1ex, 380 nm as {lambda}2ex, and 490 nm as {lambda}em. Each measurement was calibrated by the cell lysis method (22).

Whole-cell binding assays
Competition analysis.
Confluent HEK-GHSR-1a monolayer cells (about 70–80%) were resuspended in binding buffer containing DMEM, 1% (wt/vol) BSA (pH 7.4), centrifuged at 500 x g for 3 min at room temperature, washed twice, and then resuspended (1 x 100-mm plate/1.5 ml; 5–6 x 105 cells per aliquot) in binding buffer supplemented with 100,000 cpm/aliquot of [125I]ghrelin in the presence and absence of increasing concentrations of cold ghrelin (from 10-11 to 10-5 M) and unlabeled competitors (from 10-7 to 10-5 M) for 2 h at 4 C. Nonspecific binding was determined in parallel incubations of nontransfected HEK 293 cells with [125I]ghrelin, which was subtracted from total binding to yield specific binding values. After incubation, the medium was removed, and the cells were washed twice with binding buffer. Cell-surface-bound radioligand was removed by incubating the cells in 0.5 ml ice-cold acid buffer [0.5 M NaCl and 0.2 M acetic acid (pH 2.0)] for 10 min at 4 C. Under these conditions more than 90% of the surface [125I]ghrelin was removed. Finally, the cells were pelleted by centrifugation, and the supernatants were counted in a gamma counter.

125I-Ghrelin internalization assays.
Confluent HEK-GHSR-1a monolayer cells (70–80% confluence) were resuspended in binding buffer as described above and incubated in binding buffer supplemented with 100,000 cpm/aliquot of [125I]ghrelin for 2 h at 4 C. The media containing labeled ghrelin were removed, and cells were washed twice with ice-cold binding buffer. Fresh binding buffer (0.5 ml) was added and cells were incubated at 37 C for periods from 0 to 180 min. At each time point, cells were pelleted and the incubation medium was treated with 10% (wt/vol) trichloroacetic acid (TCA) at 4 C for 1 h. The TCA-insoluble materials were pelleted by centrifugation at 10,000 x g for 10 min at 4 C. Supernatant and insoluble fractions were counted to determine postactivation releasable label and TCA-insoluble material, respectively. Cell-surface-bound ghrelin was removed by resuspending the cells in ice-cold acid buffer for 15–20 min at 4 C. The cells were pelleted by centrifugation and the supernatants, corresponding to cell-surface-bound ghrelin, were counted. Finally, the pellet obtained was solubilized in lysis buffer (1% Nonidet P-40, 0.5% Triton X-100, and 1 M NaOH) and radioactivity, corresponding to internalized ghrelin, was measured (23, 24).

[125I]Ghrelin internalization in K+-depleted medium
Confluent HEK-GHSR-1a monolayer cells (70–80% confluence) were resuspended in HEPES-buffered saline [containing (in mM): 120 NaCl, 1.2 MgSO4, 1 EDTA, 15 CH3COONa, 1 CaCl2, 10 glucose, and 100 HEPES (pH 7.4) complemented with 1% (wt/vol) BSA]. Intracellular potassium depletion was carried out by incubating the cells for 5 min at 37 C in hypotonic medium [HEPES-buffered saline: water, 1:1 (vol/vol)], followed by 60 min of incubation in HEPES-buffered saline supplemented with (control) or without 10 mM K+ at 37 C. The cells were then washed and resuspended in HEPES-buffered saline supplemented with 100,000 cpm/aliquot of [125I]ghrelin for 2 h at 4 C. After incubation, the cells were washed and then incubated at 37 C for various periods of time. At each time point, the media were removed and incubated with 10% (wt/vol) TCA to determine postactivation releasable label and TCA-insoluble material. Cell surface and internalized ghrelin was measured as above (23, 25, 26).

Receptor recycling
Confluent HEK-GHSR-1a cells were resuspended in binding buffer and incubated with binding buffer containing 500 nM unlabeled ghrelin for 2 h at 37 C. After incubation, the cells were washed to remove the unlabeled ghrelin and incubated again at 37 C. At each time point, the cells were washed and incubated with 100,000 cpm/aliquot of [125I]ghrelin for 2 h at 4 C. The media containing labeled ghrelin were removed, and the cells were washed twice with ice-cold binding buffer and the cell surface-bound ghrelin was measured after treatment with ice-cold acid buffer. To evaluate the possibility of new receptor synthesis, in some experiments protein synthesis was inhibited by the addition of cycloheximide (200 µM), which was added to the cell culture medium 1 h before and maintained throughout the assay.

Inhibition of lysosomal pathways
Leupeptin (1 mM), NH4Cl (10 mM), and Concanamycin A (100 nM) were used as inhibitors of lysosomal pathway (27). These compounds were added to the HEK-GHSR-1a cells 1 h before and maintained in the medium throughout the experiment. The effect of these compounds was evaluated on receptor recycling. Cells were incubated with binding buffer containing unlabeled ghrelin (500 nM) for 2 h at 37 C. After incubation, the cells were washed and incubated with binding buffer for 3 h at 37 C. At this time, the incubation medium was supplemented with 100,000 cpm/aliquot of [125I]ghrelin and incubated for 2 h at 4 C. The media containing labeled ghrelin were removed, and the cells were washed twice with ice-cold binding buffer and the cell surface-bound ghrelin was measured after treatment with acid buffer.

Nonspecific binding, determined as radioactivity bound to nontransfected HEK 293 cells, represented about 8 ± 1% of the cell-associated radioactivity in all conditions studied.

Confocal microscopy
Approximately 2 x 105 CHO cells expressing the GHSR-1a-EGFP diluted in 0.25 ml Ham’s F12 medium were seeded on polyallylamine hydrochloride-coated (0.1 mg/ml for 30 min) 16-well glass slides. The cells were grown overnight in a humidified atmosphere of 95% air and 5% CO2 at 37 C. Cells were preincubated 120 min at 37 C with 90 µM cycloheximide in all experiments to prevent de novo protein synthesis. Cells were preincubated for 30 min at 4 C in ice-cold Earle’s buffer [containing (in mM): 140 NaCl, 5 KCl, 1.8 CaCl2, and 3.6 MgCl2 (pH 7.4) complemented with 0.2% BSA, 0.01% glucose, 90 µM cycloheximide, and 0.8 mM of 1–10 phenanthrolene] in the presence/absence of various peptides. Internalization was promoted by placing the cells at 37 C for 15 min. Thereafter, the cells were rinsed three times with ice-cold Earle’s buffer and subsequently fixed for 10 min with 4% paraformaldehyde dissolved in 0.1 M PBS (pH 7.4). The cells were rinsed again in cold Earle’s buffer, mounted using Vectashield (Vector Laboratories, Compiègne, France), and coverslipped for confocal microscopic examination. The cells were examined with a Leica TCS SP 2 (Leica Microsystems, Heidelberg, Germany) confocal laser-scanning microscope mounted on a Leica DM IRBE inverted microscope equipped with an argon/krypton laser. EGFP fluorescence was detected with 100% excitation at 488 nm, using a RSP 500 (dichroic) mirror and the spectrophotometer set to acquire emission between 530 and 560 nm. Optical sections (1024 x 1024) of individual cells were taken at the equatorial level (level of the nucleus), using a 63 x 1.32 NA oil-immersion objective.

Colocalization of the GHSR-1a-EGFP with intracellular markers
For immunofluorescence experiments, CHO cells stably expressing the GHSR-1a-EGFP were grown overnight on 14-mm round glass coverslips. To analyze trafficking of the internalized GHSR-1a, cells were incubated with 100 nM ghrelin for various times, washed three times in cold PBS (pH 7.4), and fixed 20 min at room temperature with 2% paraformaldehyde in PBS. Cells were rinsed with PBS, blocked for 30 min with PBS containing 5% BSA, permeabilized with 0.05% Triton X-100, and incubated with primary antibody diluted in the blocking buffer overnight at 4 C. The following primary antibodies were used: early endosome autoantigen 1 (EEA1) mouse monoclonal (1:250 final dilution; Transduction Laboratories, Lexington, KY) or cathepsin D goat monoclonal (1:200 final dilution; Santa Cruz Biotechnology, Santa Cruz, CA). After three rinsings with PBS containing 0.1% BSA, cells were incubated for 60 min with TRITC-conjugated donkey antigoat IgG (1:400, Molecular Probes) or Cy3-conjugates goat antimouse IgG secondary antibodies diluted in PBS containing 0.1% BSA. The cells were then rinsed twice with PBS and mounted with Mowiol.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To investigate the mechanisms modulating the desensitization and internalization of GHSR-1a, we monitored radioligand binding, confocal microscopy, and the intracellular calcium rise on receptor activation. For radioligand binding and intracellular calcium experiments, we used HEK-GHSR-1a cells, and for confocal microscopy assays, we used CHO cells that stably express the human GHSR-1a fused to EGFP (CHO-GHSR-1a cells).

Specific binding of ghrelin to GHSR-1a
The specific binding of [125I]ghrelin to HEK-GHSR-1a cells showed a dissociation constant of 1.5 nM derived from Scatchard plot analysis (Fig. 1AGo, inset). The specificity of ghrelin binding to cell surface was checked by competitive binding experiments using GHRP-6, a peptidyl GHS, GHRH, or adenosine. In agreement with previous studies in other cell types (7, 28, 29, 30, 31), bound [125I]ghrelin was completely displaced in a dose-dependent manner by both unlabeled ghrelin and GHRP-6 but not by GHRH or adenosine (Fig. 1AGo).



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  		FIG. 1. A, Competition analysis of [125I]ghrelin by different unlabeled competitors. Binding was measured at the cell surface as described in Materials and Methods, and the ordinate represents binding as a percentage of control (specific binding in the absence of unlabeled competitor). The inset (A) shows the Scatchard plot. B, Ghrelin-induced calcium mobilization in HEK 293 cells stably transfected with GHSR-1a. Cells were stimulated with 100 nM ghrelin, and changes in the intracellular Ca2+ were measured with the fluorescent probe fura-2. C, Dose response of ghrelin-induced Ca2+ transients. The results (mean ± SE of three independent experiments) are expressed as percentage of the maximum response.

 
Desensitization of GHSR-1a
Ghrelin doses administrated to HEK-GHSR-1a cells induced a biphasic increase of [Ca2+]i characterized by a prominent and transient rise, with no detectable lag period, which was followed by a sustained plateau (Fig. 1BGo). Ghrelin-induced calcium rise was dose dependent, as shown in Fig. 1CGo, with a half-maximal response at approximately 0.5 nM and maximal stimulation beyond 10 nM. This calcium mobilization is triggered by the activation of phosphatidylinositol (PI)-specific phospholipase C (PLC) that acts on phosphatidylinositol 4,5-biphosphate to generate inositol 1,4,5-triphosphate (IP3), which triggers the release of calcium from intracellular stores (5, 6). In addition to IP3, the action of PI-PLC generates diacylglycerol, a second messenger that activates protein kinase C (PKC). To evaluate the role of PKC on the ghrelin-activated signaling pathway, PKC was activated by pretreatment with phorbol ester, phorbol-12-myristate-13-acetate (PMA). As shown in Fig. 2Go, A and B, the ghrelin-induced calcium mobilization was unchanged after a brief pretreatment with PMA (1 µM, 5 min before ghrelin). Dosage and timing were effective because this pretreatment completely blocked the LPA-induced calcium transient (1 µg/ml; Fig. 2Go, C and D). LPA, a growth factor-like lipid used as internal control, produces IP3-dependent calcium mobilization through specific GPCRs regulated by PKC (32, 33). The effect of PMA-activated PKC was also evaluated by [125I]ghrelin binding. Treatment for 1 h with 1 µM PMA caused a nonsignificant reduction in ghrelin binding to the GHSR-1a (Fig. 2EGo). To gain additional insight into the mechanism of action of ghrelin, homologous and heterologous desensitization between ghrelin and LPA were assessed. As Fig. 2FGo shows, repeated administration of LPA induced homologous desensitization of its transmembrane signaling system, whereas it did not induce heterologous desensitization to ghrelin. When the order of stimuli was reversed (Fig. 2GGo), ghrelin induced a homologous desensitization of its receptor and was also able to cross-desensitize the LPA receptor. This point was concordant with the results described above, reflecting that GHSR-1a does not appear to be regulated by PKC, although this kinase may indiscriminately mediate agonist-independent phosphorylation of other PKC-regulated receptors, such as the LPA receptor(s), and initiates heterologous desensitization.



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  		FIG. 2. Role of PKC on ghrelin receptor. PKC activity was stimulated by means of phorbol ester PMA and evaluated by changes in the intracellular calcium and [125I]ghrelin binding. Cells were stimulated with ghrelin (100 nM) (A) and LPA (1 µg/ml) (C) under control conditions. Ghrelin-induced intracellular calcium mobilization was not affected after preincubation in the presence of phorbol ester PMA (1 µM, 5 min) (B). In contrast, LPA-calcium signal was completely abolished (D). HEK-GHSR-1a cells were treated with PMA (1 µM) 1 h before incubation with [125I]ghrelin and maintained in the medium throughout the experiment. Data (mean ± SE) are expressed as percentage of radioactivity surface-bound found in untreated control cells (E). The repeated administration of LPA (1 µg/ml) caused homologous desensitization, although it did not cause heterologous desensitization to ghrelin (F). In contrast, the repeated administration of ghrelin (100 nM) caused homologous desensitization as well as heterologous desensitization to LPA (G).

 
Assessment of internalization of the ghrelin/GHSR-1a complex by radioligand binding studies and confocal microscopy
After incubation of HEK-GHSR-1a cells with [125I]ghrelin for 2 h at 4 C, the excess of ghrelin was removed, and the bound label was allowed to internalize at 37 C. The internalization process was stopped at fixed intervals and [125I]ghrelin associated to the cell surface was counted after acid wash. Intracellular bound and free ghrelin in the medium were measured after cell lysis and treatment with TCA (TCA-soluble and -insoluble material), respectively. As Fig. 3AGo shows, there was a time-dependent reduction in the amount of [125I]ghrelin associated to the surface receptors, whereas the amount of internalized label increased rapidly so that within 15–20 min 60% of the initial bound [125I]ghrelin was internalized. TCA-soluble 125I began to be detected in medium after 30 min, reaching a maximum at 60–90 min (Fig. 3BGo). The TCA-insoluble counts, representative of ghrelin dissociated from the receptor without internalization, reached a peak at 10 min and remained constant thereafter (10% of control membrane-bound label; Fig. 3BGo). HEK-293 cells showed an efficient endocytosis of GHSR-1a receptors as illustrated by the fact that 74% of the ghrelin was internalized within 20 min. After 60 min most of the counts were detected in TCA-soluble fraction, which might suggest that after internalization of ghrelin/GHSR-1a, complex ghrelin is rapidly degraded.



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  		FIG. 3. Kinetics of [125I]ghrelin internalization. [125I]ghrelin was bound to the HEK 293 cells transfected with GHSR-1a for 2 h at 4 C and washed at 4 C to remove the unbound ligand and then incubated at 37 C for the various times. At each time point, the incubation medium was removed and treated with TCA. After the cells were washed, surface [125I]ghrelin was removed by acid stripping, and the remaining cells were then solubilized in NaOH. Nonspecific binding was evaluated in HEK 293 cells (nontransfected with GHSR-1a, control cells). All figures are representative of four independent experiments. Results were expressed as percentage of radioactivity associated with the starting [125I]ghrelin found. Data are the means ± SE of triplicates. A, Time course of [125I]ghrelin internalization. {bullet}, Surface-bound; {blacksquare}, intracellular [125I]ghrelin in HEK 293 cells transfected with GHSR-1a. B, Time course of postactivation releasable [125I]ghrelin. {blacktriangleup}, TCA soluble in the medium; {diamondsuit}, TCA precipitable in the medium in HEK-GHSR-1a.

 
Internalization induced by activation of the GHSR-1a by ghrelin or GHS was further evaluated by confocal microscopy in CHO cells stably expressing the human GHSR-1a-EGFP. In the resting cells, fluorescence associated with the receptor was predominantly confined to plasma membrane (Fig. 4AGo). A slight fluorescent was also associated with the Golgi apparatus, even under treatment with cycloheximide. After exposure to ghrelin (100 nM) for 15 min, the fluorescence almost completely disappeared from the plasma membrane to be redistributed to a population of intracellular vesicles distributed throughout the cytoplasm (Fig. 4BGo and see Fig. 7Go). Ligand specificity of the internalization of the GHSR-1a is suggested by the fact that no redistribution of the fluorescent labeling could be observed in cells incubated with 100 nM des-n-octanoyl ghrelin, a ghrelin derivative lacking n-octanoyl modification on Ser3 and inactive on GH release (Fig. 4CGo). Moreover, a 15-min incubation of the transfected CHO cells in presence of hexarelin (100 nM), a synthetic GHS that binds to GHSR-1a, induced a similar decrease in cell surface labeling associated to a redistribution of fluorescence to scattered cytoplasmic vesicles (Fig. 4DGo). To identify the nature of these intracellular structures, a double-labeling study using an early endosomal marker (EEA1) showed a complete overlay between the internalized EGFP-tagged receptor and the EEA1 protein (see Fig. 8AGo). These data appear to suggest that endocytosis of the GHSR-1a may occur predominantly via clathrin-coated pits.



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  		FIG. 4. Localization and endocytosis of GHSR-1a-EGFP receptors in CHO cells. The localization of the GHSR-EGFP expressed in stably transfected CHO cells was visualized by confocal microscopy in cells incubated for 15 min at 37 C in the absence (A) or presence (B) of 100 nM ghrelin, 100 nM des-n-octanoyl ghrelin (C), and 100 nM hexarelin (D).

 


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  		FIG. 7. Time course of GHSR-1a-EGFP internalization in CHO cells after stimulation with ghrelin. The localization of the GHSR-1a-EGFP expressed in stably transfected CHO cells was visualized by confocal microscopy in cells incubated for different times at 37 C in the absence or presence of 100 nM ghrelin. In absence of ligand, the fluorescent labeling appeared at the cell surface (A). After a 20-min incubation with 100 nM ghrelin, the fluorescent labeling disappeared from the membrane, whereas cytoplasmic fluorescent vesicles were visualized inside cells (B). After a 40-min incubation, the fluorescent vesicles migrated in the direction of the nucleus (C), and a perinuclear localization was observed after 60 min (D). After 120 min incubation, the fluorescent vesicles were concentrated near the membrane, and a moderate labeling appeared at the cell surface (E).

 


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  		FIG. 8. Double visualization of the GHSR-1a-EGFP with endosomal and lysosomal markers. A, CHO cells stably expressing the GHSR-1a-EGFP were treated with 100 nM ghrelin for 20 min. The GHSR-1a-EGFP (green) is extensively colocalized with the early endosome marker EEA1 (red) in small vesicles dispersed peripherally throughout the cell. The colocalization was visualized as yellow. B, After 60 min stimulation of cells with 100 nM ghrelin, the GHSR-1a-EGFP (green) shows no colocalization with cathepsin D, a lysosomal marker (red).

 
Involvement of clathrin-mediated endocytosis
To determine whether the GHSR-1a internalization was mediated by clathrin-coated pits, similar studies were performed using potassium-depleted medium, a condition that significantly affects the formation of clathrin-coated pits and internalization through this pathway (23). As Fig. 5AGo shows, potassium depletion significantly inhibited the internalization process of [125I]ghrelin, with a 65% reduction at 20 min. This reduction was associated with a blockade in the disappearance of [125I]ghrelin from the cell surface (Fig. 5BGo). After 60 min, no difference in cell membrane-bound radioactivity was found, which might indicate that the number of clathrin-coated pits remaining after potassium depletion may be sufficient to maintain a slow internalization.



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  		FIG. 5. Effect of potassium depletion on the internalization of [125I]ghrelin. Cells were preincubated with [125I]ghrelin for 2 h at 4 C and then incubated at 37 C in medium with or without K+ for the indicated times. At each time point, cells were washed, and surface [125I]ghrelin was removed by acid stripping (B), and the remaining cells were then solubilized in NaOH (A). Results are expressed as mean ± SE of triplicates.

 
Recycling of GHSR-1a
Because the results described above suggest recycling through a process involving clathrin-coated pits, the capability for recycling after internalization was studied. Recycling of the internalized receptor to the surface was evaluated after a pulse of 500 nM of unlabeled ghrelin for 2 h at 37 C and the recovery of [125I]ghrelin binding was assessed at increasing intervals of time. The pulse with a saturating dose of unlabeled ghrelin was able to completely reduce the available binding sites to the ligand, which were 9 ± 1% of the control value at the beginning of the chase period, and remained severely depleted for about an hour (Fig. 6AGo). At 3 and 6 h, recovery of [125I]ghrelin binding to the membrane fraction was 62% and 89% of control, respectively, suggesting that the receptor had been either recycled or synthesized de novo. The latter possibility is unlikely because continuous incubation of the pulsed cells with 200 µM cycloheximide, a protein synthesis inhibitor, did not modify the recovery rate of binding sites. In fact, ghrelin-induced calcium rise showed a similar dynamics (Fig. 6BGo). After 3 h of incubation with ghrelin, the calcium response was recovered to an extent of 65% and was fully restored after 6 h (98% of untreated control cells).



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  		FIG. 6. Kinetic of recycling of GHSR-1a. Recycling of GHSR-1a to the plasma membrane was evaluated in cells treated with ghrelin (500 nM) for 2 h at 37 C to internalize the receptor. After washout the extent of recycling to the plasma membrane was assessed at 37 C for the indicated times by binding of [125I]ghrelin to cell surface. Cycloheximide (200 µM) was used to evaluate the possible contribution of new receptor synthesis (A). Functionality of GHSR-1a was evaluated by ghrelin-induced calcium rise under the same experimental conditions (B).

 
Confocal microscopy studies further validated the hypothesis that receptors were being recycled because large fluorescent vesicles reappeared just under and partially at the cell surface after 2 h of incubation with ghrelin, suggesting that the label had become associated again with the plasma membrane (Fig. 7Go). The possibility that internalized receptors could be targeted to lysosomes was assessed by evaluating colocalization of the internalized receptor with cathepsin D, a lysosomal aspartyl protease marker of lysosome-directed pathway. After prolonged ghrelin treatment (1 h), GHSR-1a-EGFP-labeled receptors exhibited a perinuclear localization but did not colocalize with cathepsin D (Fig. 8BGo).

The effects of endosomal function inhibitors on the recycling of GHSR-1a to the plasma membrane were also studied. Both the weak base NH4Cl and a specific inhibitor of vacuolar H+-ATPases, concanamycin A, are known to interfere with the acidic pH of the late endosome and lysosomes. As shown in Fig. 9Go, treatment of HEK-GHSR-1a cells with these substances inhibited the recycling of internalized GHSR-1a as measured by the recovery of surface receptors within 3 h after a saturating pretreatment with ghrelin (500 nM, 2 h). These results suggested that the lack of acidification might prevent dissociation of the ligand/receptor complex, thus blocking the receptor recycling of the internalized receptor. Curiously, leupeptin, an inhibitor of lysosomal proteolysis, also inhibited the recycling of the internalized receptor (Fig. 9Go); this could be due either to an unspecific effect on the dynamic of receptor recycling or to a relationship between degradation of the peptide ligand and receptor recycling.



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  		FIG. 9. Effect of inactivation of lysosomal pathway on ghrelin receptor recycle. Ghrelin receptor recycling was evaluated in cells treated with various compounds interfering with lysosomal pathway: leupeptin (1 mM), NH4Cl (10 mM), and concanamycin A (100 nM). Cells were pretreated with the indicated inhibitors and stimulated with ghrelin (500 nM) for 2 h at 37 C, and after washout ghrelin bound to cell surface was measured. Values shown are mean ± SE from experiments performed in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although binding of agonists to GHSR-1a has been characterized (4, 16, 17, 24, 28, 29, 34, 35, 36, 37) and some aspects of the signal transduction pathway have been studied, an analysis of the cell biology processes and the dynamics of receptor trafficking had not been investigated (15). To study ghrelin-induced endocytosis of this receptor, we selected two cell lines (HEK 293 and CHO) that were engineered to stably express GHSR-1a. In the case of the CHO cells, the expressed GHSR-R1a was tagged with EGFP. We show that ghrelin/GHSR-1a complex appears to be internalized via clathrin into vesicles that become early endosomes by dropping their clathrin coat. Ligand and receptor dissociate in an acidified endosomal compartments. Afterward the ligand appears to be degraded in lysosomes, whereas the receptor recycles to the plasma membrane. In this way, recovery of cell surface receptors would minimally require the synthesis of new receptors.

GHSR-1a transduces information provided by its own endogenous ligand, ghrelin (5, 7), and a group of synthetic compounds, comprised as GHSs, developed as GH releasers over the past 25 yr. Although ghrelin is not related structurally to the synthetic GHSs, both types of molecules bind to the receptor with high affinity. Our results clearly indicate that [125I]ghrelin binding in HEK-GHSR-1a showed typical properties of a ligand-receptor interaction, namely high affinity and saturability. The observation that this binding was completely displaced by unlabeled GHRP-6, a peptidyl GHS, is in agreement with previously reported works in other cell systems (16, 29, 34). There is an ongoing controversy about whether this receptor is the sole receptor for GHSs or just one of a group of receptors for such ligands. The existence of different receptors types might be endorsed by the differences in the binding activities reported for ghrelin and different GHSs (34). However, site-directed mutagenesis and molecular modeling studies demonstrated the existence of distinct regions, suggesting an overlapping in the agonist-binding site (38). Added to this complexity, GHSR-1a appears to show other binding sites different from the characterized GHS binding pocket as could be demonstrated for adenosine, an agonist of this receptor (30, 31). In fact, no competition with [125I]ghrelin was observed in the presence of unlabeled adenosine in HEK-GHSR-1a, consistent with the activation of GHSR-1a through a different site. This point was not surprising, taking into account the lack of close homology between GHSR-1a and the most homologous adenosine receptors (~28% identity and ~38% similarity with the A2b and A3 receptors) (31).

Binding of ghrelin as well as GHSs to GHSR-1a leads to activation of the receptor-associated PI-PLC/PKC pathway (5, 6). In HEK-GHSR-1a cells, the addition of ghrelin induced a biphasic increase of [Ca2+]i in which the spike phase reflects IP3-mediated intracellular mobilization of calcium, and the plateau phase reflects the influx of extracellular calcium across the plasma membrane. Focusing on the mobilization of intracellular calcium, a rapid homologous desensitization of ghrelin-induced calcium mobilization was observed. One of the mechanisms for the desensitization of GPCRs is through a feedback regulation by the second messenger-stimulated kinases, such as PKC. This type of receptor regulation is a property that has generally been ascribed to heterologous or agonist-independent desensitization (18, 39, 40). Because the system did not appear to be under regulation of PKC and because phorbol ester PMA failed to block the ghrelin-induced calcium response, the agonist-dependent desensitization does not appear to be mediated by second messenger-dependent PKC. However, ghrelin-activated PKC might mediate the agonist-independent desensitization of other receptors and induce the heterologous desensitization, as was observed for the cross-desensitization of LPA receptor(s). In addition, GHSR-1a internalization was not promoted by PMA treatment, which suggests that the internalization of this receptor is PKC independent. These results suggest that the major cellular mechanism that might be mediating agonist-specific or homologous desensitization is phosphorylation by a GPCR kinase (18, 39, 41).

Receptor desensitization together with its internalization represents an important physiological mechanism that modulates receptor responsiveness and acts as an information filter for intracellular signaling. The kinetic studies of GHSR-1a internalization were carried out by two different methods: radioligand binding in HEK-293 cells stably expressing the human GHSR-1a and confocal microscopy in CHO cells stably expressing the human GHSR-1a tagged at its C terminus with EGFP. After treatment with ghrelin, GHSR-1a was internalized in a time-dependent manner clearly indicated by a rapid decrease in the number of radioligand binding sites at the surface associated with an increase inside the cell showing a maximum at about 20 min on ghrelin stimulation. This observation was supported by confocal microscopy analysis carried out in CHO cells, demonstrating a surface GHSR-1a loss over a similar time frame (20 min) after ghrelin stimulation. Two main pathways have been described for the internalization of GPCRs after their activation (18, 39): via clathrin-coated pits (42) and through caveolae (43, 44). The inhibition of internalization after potassium depletion, a condition known to decrease the number of surface clathrin-coated pits, strongly suggests that the ghrelin-GHSR-1a complex is internalized principally by a clathrin-mediated mechanism.

After ligand-receptor complex is internalized via clathrin pathway into vesicles, GHSR-1a can either be sorted into endosomes to be recycled back to plasma membrane or, alternatively, may be degraded within lysosomes (18, 39). The recovery experiments argue in favor of receptor recycling. First, in HEK-GHSR-1a cells, the level of receptors on the cell surface rose once again to close to 100% after 360 min of agonist removal, a process unaltered by cycloheximide, suggesting that GHSR-1a recycling from endosomes, rather than de novo receptor synthesis. This is endorsed by the observation that after the ghrelin pulse, fluorescence associated with GHSR-1a-EGFP in CHO cells reappears at the membrane with similar kinetics. Second, recycling of the GHSR-1a at cell surface was prevented by inhibitors of endosomal acidification, NH4Cl and concanamycin, which caused retention of ghrelin-receptor complex within the cell presumably by preventing degradation of ghrelin. Third, the fluorescence emitted by EGFP-labeled receptor in CHO cells was associated with punctuate cytoplasmic structures, which colocalize with EEA1. This colocalization with a marker of early endosomes suggests that endocytosis occurs via clathrin-coated pits and uses the endosomal trafficking pathway. Furthermore, on the CHO-GHSR-1a cellular model, double labeling with cathepsin D, showed that the EGFP-labeled receptor did not colocalize with this lysosomal marker, indicating that GHSR-1a is not targeted to lysosomal compartments. The experimental design used for confocal microscopy (labeling of the receptor itself, not the ligand) does not allow excluding a possible degradation of the ligand. Thus, it seems likely that most of GHSR-1a appearing at the cell surface came from endocytosed receptors, resulting in complete restoration of binding capacity and functionality. Interestingly, physiological experiments have shown that the GH response after two consecutive pulses of ghrelin is blunted when both pulses are separated by a 60-min interval but is restored to its full initial amplitude when the second pulse is administered after 180, 240, or 360 min (45). This observation fits in well with the kinetics of receptor recycling described in the present work. In this sense, GHSR-1a showed an extremely slow recycling (3–6 h), compared with other GPCRs (46). This pattern of receptor resensitization might be determined by the association of ß-arrestin with GHSR-1a during clathrin-mediated endocytosis because the formation of a stable receptor-ß-arrestin complex appears to retard the resensitization, whereas the formation of a transient complex favors rapid recycling to the plasma membrane (39, 46, 47).

The good agreement obtained independently by the various models used in this study for evaluating internalization kinetics suggest that complementary data obtained from both cellular models can appropriately be compared, even if the cell lines could not be assumed to behave exactly in the same way. The timing of binding disappearance in HEK-GHSR-1a cells coincides with that of membrane-associated GHSR-1a fluorescence in CHO cells as well as with the functional calcium response. Progressive recovery of the receptor expression at the plasma membrane was also observed within the same time range when evaluated by binding studies, internalization of the fluorescent receptor, and the intracellular calcium mobilization.

In conclusion, we have provided an initial characterization of the ability of the GHSR-1a to mediate ghrelin uptake and degradation through a rapid internalization pathway essentially involving clathrin-coated pits, dissociation of ligand-receptor complex in early endosomes, and reappearance of the receptor by a recycling pathway. This route may explain the characteristic physiological responses mediated by this receptor. Thus, recycling of GHSR-1a may provide a basis for the ghrelin-mediated GH secretion through this receptor in normal and pathological conditions.


   Acknowledgments
 
We are very grateful to Professor C. Kordon for critical reading of this manuscript, advice, and encouragement. The expert technical assistance of Mary Lage is greatly acknowledged.


   Footnotes
 
This work was supported by grants from the Fondo de Investigación Sanitaria and the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Red de Grupos de Tratamiento de la Obesidad (G03/028), Red de Centros de Metabolismo y Nutrición (C03/08), Secretaría Xeral de Investigacion e Desenvolvemento (PGIDIT02BTF91801PR), Xunta de Galicia, and the Ministerio Español de Ciencia y Tecnologia. J.P.C. is a recipient of a research contract from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, in the research area of the Complejo Hospitalario Universitario de Santiago.

Abbreviations: CHO, Chinese hamster ovary; EEA1, early endosome autoantigen 1; EGFP, enhanced green fluorescent protein; fura-2/AM, fura-2 pentaacetoxymethylester; GHS, GH secretagogues; GPCR, G protein-coupled receptor; GHSR-1a, GHS receptor type 1a; HEK, human embryonic kidney; IP3, inositol 1,4,5-triphosphate; LPA, L-{alpha}-lysophosphatidic acid; PI, phosphatidylinositol; PKC, protein kinase C; PLC, phospholipase C; PMA, phorbol-12-myristate-13-acetate; TCA, trichloroacetic acid.

Received July 30, 2003.

Accepted for publication October 14, 2003.


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