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Originally published In Press as doi:10.1074/jbc.M407676200 on September 21, 2004

J. Biol. Chem., Vol. 279, Issue 51, 53806-53817, December 17, 2004
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Common Structural Basis for Constitutive Activity of the Ghrelin Receptor Family*

Birgitte Holst{ddagger}§, Nicholas D. Holliday¶, Anders Bach{ddagger}, Christian E. Elling||, Helen M. Cox¶, and Thue W. Schwartz{ddagger}||

From the {ddagger}Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200, Copenhagen, Denmark, Centre for Neuroscience Research, King's College London, Guy's Campus, London, SE1 1UL, United Kingdom, and ||7TM Pharma A/S, Fremtidsvej 3, DK-2970, Hørsholm, Denmark

Received for publication, July 8, 2004 , and in revised form, September 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the Gq, phospholipase C pathway was ~50% of their maximal capacity as determined through inositol phosphate accumulation. These two receptors also showed very high constitutive activity in activation of cAMP response element-driven transcription. GPR39 displayed a clear but lower degree of constitutive activity through the inositol phosphate and cAMP response element pathways. In contrast, GPR39 signaled with the highest constitutive activity in respect of activation of serum response element-dependent transcription, in part, possibly, through G12/13 and Rho kinase. Antibody feeding experiments demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically be tuned up and down depending on the size and hydrophobicity of the side chain in position VI:16 in the context of an aromatic residue at VII:09 and a large hydrophobic residue at VII:06. It is concluded that the three ghrelin-like receptors display an unusually high degree of constitutive activity, the structural basis for which is determined by an aromatic cluster on the inner face of the extracellular ends of TMs VI and VII.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In principle, all 7TM1 G protein-coupled receptors are to some degree constitutively active, in that they are able to change into an active conformation without the presence of the agonist ligand (1). For the majority of receptors, the constitutive activity is so low that it is not picked up by the various signaling assays employed. Some receptors, however, such as the {beta}2-adrenergic receptors show a low but significant degree of constitutive activity of perhaps 3-10% of the maximal signaling capacity depending on the system in which it is studied (2). A few endogenous receptors have been described to display very high constitutive activity (for example, the CB1 cannabinoid receptor (3) and certain sphingolipid receptors (4)). However, it is still debated whether these receptors are in fact truly constitutively active, because it is difficult to rule out the possible presence of lipid ligands in the cellular system in which they are studied. Nevertheless, high constitutive activity is indisputably a hallmark of many virally encoded 7TM receptors such as ORF74 from human herpes virus 8 and US28 from human cytomegalovirus (5). The virus has optimized, for example, ORF74 to signal constitutively with ~50% efficacy and to be modulated positively by endogenous angiogenic chemokines and negatively by endogenous angiostatic or modulatory chemokines (6, 7).

Recently, we discovered that the receptor for the hormone ghrelin also signals with ~50% of maximal activity in the absence of its peptide ligand (8). This receptor property remained unnoticed for many years because mobilization of intracellular calcium had been used almost exclusively to monitor the signal transduction activity of this receptor (9). When inositol (1,4,5)-triphosphate (IP) turnover was measured instead, it became clear that the ghrelin receptor in fact was highly constitutively active (8). Ghrelin is an acylated 28-amino acid peptide hormone that is released from endocrine cells of the stomach in the premeal situation and functions as an important orexigenic from the periphery that stimulates especially acute food intake. Neuropeptide Y/agouti-related peptide neurons of the arcuate nucleus are a major target of ghrelin in the hypothalamus. Ghrelin increases the firing rate and induces an increased expression and release of neuropeptide Y and agouti-related peptide (10, 11). Furthermore, ghrelin also seems to exert important actions on energy expenditure, both through receptors on adipocytes and through modulation of thermogenesis (12, 13). Thus, it has been suggested that the high constitutive signaling activity of the ghrelin receptor could serve as a signaling set-point, for example, in the control of appetite and energy expenditure, where it would counterbalance a large number of inhibitory hormones and neurotransmitters, such as leptin, insulin, and PYY3-36 (14).

The ghrelin receptor belongs to a small family of receptors for peptide hormones and neuropeptides; besides ghrelin, the family includes motilin, neurotensin, and neuromedin U (NMU) (Fig. 1A). Like ghrelin, motilin is expressed in neuroendocrine cells of the gastrointestinal tract but acts mainly locally on neurons in the gut, where it stimulates meal-related secretions and motility of the intestine (15). The motilin receptor is completely silent with respect to constitutive signaling and consequently serves as a good control receptor in this study, as in previous studies (8). Neurotensin is both a gut hormone and a neuropeptide located in various parts of the central nervous system (16). The receptor that mediates most of the functions of neurotensin is neurotensin receptor 1. However, a structurally related receptor NT-R2 was recently cloned and was shown to bind neurotensin with lower affinity; it is interesting that neurotensin apparently has only minimal effects on the signaling of this receptor (17). It is noteworthy that in the context of the present study, NT-R2, like the ghrelin receptor, signals constitutively with ~50% of its maximal efficacy as determined by measurements of IP accumulation (17). NMU is a neuropeptide that acts through two structurally related receptors, NMU receptors 1 and 2 (18) (Fig. 1A). There are no indications in the literature or in our preliminary studies that these receptors are constitutively active.2



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  		FIG. 1.
The ghrelin receptor family. A, schematic phylogenic tree of the ghrelin receptor family indicating the relative relationship of the receptor. The black dots indicate the three receptors that either previously (the ghrelin receptor and NT-R2 (8, 17)) or in the present study (GPR39) have been demonstrated to display a high degree of constitutive signaling activity. B, serpentine model of GPR39. Residues that are identical among GPR39, the ghrelin receptor, and NT-R2 are highlighted in white on black. The generic numbering system for 7TM receptor residues described by Schwartz (59) is used throughout the article, and the proposed first residues of each transmembrane helix are indicated by 1.

 

Among the members of the ghrelin receptor family is an orphan receptor, GPR39, that, together with GPR38 (later deorphanized as the motilin receptor), were initially cloned as structural homologues to the ghrelin receptor (19) (Fig. 1, A and B). Very little information is available concerning GPR39 except that Northern blot analysis indicates that its peripheral expression is restricted to the stomach and the small intestine, whereas it is much more widely expressed in the central nervous system (19). Inspired by the knowledge of the high constitutive signaling of the ghrelin receptor and the NT-R2 receptor, we cloned the human GPR39 and discovered that it is also was highly constitutively active. In the present study, the signaling properties of the three constitutively active members of the ghrelin receptor family are characterized in parallel, and the internalization property of GPR39 is compared with that of the ghrelin receptor. Furthermore, through a mutational analysis of the opposing faces of TMs III, VI, and VII of the ghrelin receptor in particular, an aromatic cluster is identified that seems to be structurally important for the constitutive activity of this family of receptors. This was verified through a series of corresponding substitutions in both the ghrelin receptor and GPR39 at position VI:16 with residues of variable aromaticity and size through which the constitutive activity could systematically be tuned up and down.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Ghrelin, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P, motilin, and neurotensin were purchased from Bachem (Bubendorf, Switzerland). LY294002 and Y27632 were from Calbiochem. Pertussis toxin was purchased from Sigma Chemical Co.

Molecular Biology—The cDNA for the motilin receptor was provided by Bruce Conklin (The Gladstone Institute, San Francisco, CA) and the cDNA for the human herpes virus 8 encoded ORF74 receptor by Mette Rosenkilde (University of Copenhagen, Copenhagen, Denmark). The human ghrelin/growth hormone secretagogue receptor cDNA and the GPR39 were cloned by PCR from a human brain cDNA library. The cDNA was cloned into the eukaryotic expression vector pcDNA3 (Invitrogen, Carlsbad, CA). Mutations were constructed by PCR using the overlap expression method (20). The PCR products were digested with appropriate restriction endonucleases, purified, and cloned into pcDNA3. All PCR experiments were performed using Pfu polymerase (Stratagene, La Jolla, CA) according to the instructions of the manufacturer. All mutations were verified by restriction endonuclease mapping and subsequent DNA sequence analysis using an ABI 310 automated sequencer (Applied Biosystems, Foster City, CA).

Transfections and Tissue Culture—COS-7 cells were grown in Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal calf serum, 2 mM glutamine, and 0.01 mg/ml gentamicin. Cells were transfected using calcium phosphate precipitation method with chloroquine addition as described previously (21). For gene dose experiments, variable amounts of DNA were used. The amount of cDNA (20 µg/75 cm2) resulting in maximal signaling was that used for dose response curves. HEK293 cells were grown in Dulbecco's modified Eagle's medium with high glucose supplemented with 10% fetal calf serum, 2 mM glutamine, and 0.01 mg/ml gentamicin. Cells were transfected with Lipofect-AMINE 2000 (Invitrogen).

Phosphatidylinositol Turnover—One day after transfection, COS-7 cells were incubated for 24 h with 5 µCi of [myo-3H]inositol (Amersham Biosciences) in 1 ml of medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 0.01 mg/ml gentamicin per well. Cells were washed twice in buffer (20 mM HEPES, pH 7.4, supplemented with 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 10 mM glucose, and 0.05% (w/v) bovine serum) and were incubated in 0.5 ml of buffer supplemented with 10 mM LiCl at 37 °C for 30 min. After stimulation with various concentrations of peptide for 45 min at 37 °C, cells were extracted with 10% ice-cold perchloric acid followed by incubation on ice for 30 min. The resulting supernatants were neutralized with KOH in HEPES buffer, and the generated [3H]inositol phosphate was purified on Bio-Rad AG 1-X8 anion-exchange resin as described. Determinations were made in duplicate.

CRE and SRE Reporter Assay—HEK293 cells (30,000 cells/well) seeded in 96-well plates were transiently transfected. In the case of the CRE reporter assay, the cells were transfected with a mixture of pFA2-CRE binding protein and pFR-Luc reporter plasmid or serum response element (SRE)-Luc (PathDetect; Stratagene) and the indicated amounts of receptor DNA. After transfection, cells were maintained in low serum (2.5%) throughout the experiments and were treated with the respective inhibitor of intracellular signaling pathways. One day after transfection, cells were treated with the respective ligands in an assay volume of 100 µl of medium for 5 h. The assay was terminated by washing the cells twice with PBS and addition of 100 µl of luciferase assay reagent (LucLite; PerkinElmer Life and Analytical Sciences). Luminescence was measured in a TopCounter (Top Count NXT; PerkinElmer Life and Analytical Sciences) for 5 s. Luminescence values are given as relative light units.

Microscopy—The trafficking of cell surface receptors was observed by a live antibody labeling method. Stable HEK293 transfectants (selected in 0.8 mg/ml G418 sulfate) were grown to 50-70% confluence on poly-L-lysine-coated glass coverslips, transferred to humidified 35-mm Petri dishes, and treated for 1 h in serum-free Dulbecco's modified Eagle's medium at 37 °C. Cells were labeled with M2 anti-FLAG antibody (15 µg/ml; Sigma Chemical Co.) and transferrin-Texas Red (1:200; Molecular Probes, Paisley, UK) for 30 min at 4° or 37 °C in serum-free Dulbecco's modified Eagle's medium/1% bovine serum albumin (final volume, 25 µl). Subsequent treatments (15 min, 37 °C) with vehicle (5 µl of Dulbecco's modified Eagle's medium), ghrelin (final concentration, 100 nM), and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P (100 nM) were terminated by two washes with ice-cold PBS. Processing for immunofluorescence microscopy was performed at room temperature. Cells were fixed (2% paraformaldehyde in PBS, 15 min; quenched by PBS and 25 mM glycine; 2 x 5 min) and permeabilized (0.075% Triton X-100 in PBS, 5 min), and the primary antibody was then detected with goat anti-mouse IgG-Alexa 488 (1:200 in PBS/1% bovine serum albumin for 30 min; Molecular Probes). After removal of non-specifically bound secondary antibody (six washes), cells were postfixed in 2% paraformaldehyde, nuclear DNA stained with 4',6-diamidino-2-phenylindole (Sigma), and coverslips mounted in Mowiol 40-88 (Calbiochem). Immunofluorescence analysis was performed as previously described in detail (22). In brief, a vertical stack of 25-30 fluorescent images was acquired digitally on a Zeiss Axiovert 100 microscope (63x oil objective; Omega Optical excitation and emission filter sets), using Openlab 2.0 (Improvision, Coventry, UK) to direct a piezo z-axis drive in 0.2-µm steps. The central 15 images for each fluorophore were then deconvolved to remove out-of-focus light (Openlab) and reconstructed in three dimensions (3.0 µm z-section) in Volocity (Improvision).

MAP Kinase Assay—COS-7 cells (seeding density, 150,000 cells/well) were transfected in the assay plates. Two days after transfection, the indicated concentrations of ligand were added to assay medium without any serum and incubated for 10 min at 37 °C. The reactions were stopped by removal of the medium and two washing steps with ice-cold PBS. The cells were lysed in sample buffer and separated on SDS/10% PAGE according to the method of Laemmli (23). Proteins were transferred onto nitrocellulose, and Western blot analysis was carried out using 1:5000 dilution of mouse monoclonal antiphopho-ERK1/2 antibody (Santa Cruz Biotechnology). Total ERK protein was determined using a 1:10,000 dilution of anti-ERK antibody (Santa Cruz Biotechnology). Blots were probed anti-mouse horseradish peroxidase-conjugated secondary antibodies, visualized using enhanced chemiluminescence reagent (Amersham Biosciences) and quantified by densitometric analysis. ERK1/2 phosphorylation was normalized according to the loading of protein by expressing the data as a ratio of phospho-ERK1/2 over total ERK1/2. Results were expressed as percentage of the value obtained in nonstimulated, mock-transfected cells.

Cell Surface Expression Measurement (ELISA)—Cells were transfected and seeded out in parallel with those used for IP accumulation assay. The cells were washed twice, fixed, and incubated in blocking solution (PBS/0.2% dry milk) for 60 min at room temperature. Cells were kept at room temperature for all subsequent steps. Cells were incubated 2 h with anti-FLAG (M2) antibody (Sigma Chemical Co) in 1:300 dilution. After three washes, cells were incubated with anti-mouse horseradish peroxidase-conjugated antibody (Amersham Biosciences) at a 1:4000 dilution. After extensive washing, the immunoreactivity was revealed by the addition of horseradish peroxidase substrate according to manufacture's instruction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Signaling through the Phospholipase C Pathway—Determination of IP accumulation was used as a measure of signaling through the G{alpha}q, phospholipase C pathway in transiently transfected COS-7 cells. As described previously (8), the ghrelin receptor displays a high degree of constitutive signaling activity through the phospholipase C pathway, demonstrated by the gene dose-dependent but ligand-independent increase in IP production (Fig. 2A). Gene-dosing experiments with GPR39 also demonstrated a dose-dependent increase in IP accumulation in cells expressing the GPR 39 receptor as opposed to cells transfected with the empty vector (Fig. 2B). Surface ELISA of the FLAG-tagged GPR39 receptor confirmed that the gene-dosing experiments in fact did result in a dose-dependent surface expression of the orphan receptor (Fig. 2B, inset). The lack of an endogenous ligand for GPR39 prevented us from evaluating the degree of constitutive activity compared with the maximal stimulation achieved by the agonist. However, as a result of our general probing of receptors with meal-ions (24, 25) we discovered that Zn(II) administered as ZnCl2 acted as a low potency agonist with an EC50 value of 34 µM in GPR39-transfected cells as opposed to non-transfected cells (Fig. 2C). The zinc ion increased the receptor-mediated signaling from 22 fmol/105 cells to 46 fmol/105 cells at the highest expression level (Fig. 2C). In parallel transfections of COS-7 cells, it was found that the ghrelin receptor displayed a higher level of constitutive activity compared with GPR39, although a slightly lower degree of surface expression was observed by ELISA. The highest achievable level of constitutive signaling activity observed for the ghrelin receptor was 45 fmol/105 cells (Fig. 2, A and C). Higher receptor gene doses resulted in lower responses in IP accumulation, and a similar phenomenon was observed in the in the downstream signaling pathways described below (data not shown). Addition of ghrelin further increased the IP signaling by ~2-fold, to 80 fmol/105 cells.



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  		FIG. 2.
Constitutive signaling of the ghrelin receptor as determined by analysis of inositol phosphate turnover. A, gene-dosing experiments with the ghrelin receptor in transiently transfected COS-7 cells: basal constitutive activity (black bars) compared with ghrelin (100 nM) agonism (white bars). A and B, left, basal and agonist-induced activity in mock-transfected cells (empty vector pcDNA3-transfected cells) are shown for the highest amount of cDNA (20 µg). Gene dosing experiments with mock transfection have also been performed; the results showed the same low signaling level for all the DNA concentrations as observed for 20 µg of pcDNA3. B, gene-dosing experiments with GPR39 in transiently transfected COS-7 cells: basal constitutive activity (black bars) compared with Zn(II) (100 µM; white bars), which was identified as an agonist for GPR39. The inset shows the expression of GPR39 at the cell surface as measured by ELISA performed in parallel with the inositol phosphate turnover. C, dose-response curve for ghrelin on the ghrelin receptor (circle), neurotensin on NT-R2 (triangle), and Zn(II) as ZnCl2 on GPR39 (square). Data are mean ± S.E. of three to five independent experiments performed in triplicate.

 
The NT-R2 receptor has previously been described to signal through the phospholipase C pathway with ~50% constitutive activity (17), which was in accordance with the observation that ligand-independent IP accumulation in COS-7 cells transfected with this receptor was similar to the levels observed in cells transfected with the ghrelin receptor (Fig. 2C). It is interesting that neurotensin did not act as an agonist, in that it did not further increase the signaling of the receptor above the basal level (Fig. 2C). In the study in which this phenomenon was initially described, the non-peptide ligand SR48.692 was used as a high potency agonist, which further increased the IP accumulation by ~2-fold (17). The SR48.692 ligand was unfortunately not available for the present study Thus, the ghrelin receptor and NT-R2 signaled with a similar high constitutive activity corresponding to ~50% of their maximal efficacy through the phospholipase C pathway. GPR39 also displayed constitutive activity through this pathway but only approximately half of that observed with the ghrelin and NT-R2 receptors.

Signaling through the ERK MAP Kinase Pathway—7TM receptors can activate ERK signaling through several different pathways (26). In the present study, we use the virally encoded receptor ORF74, which couples constitutively to ERK1/2 in COS-7 cells as a positive control (27). ORF74 has been shown to signal constitutively through MAP kinase in COS-7 cells but not in HEK293 cells, which we could confirm. We also found that the ghrelin receptor did not signal constitutively through MAP kinase in HEK293 cells (27).

Transfection with ORF74 increased the basal MAP kinase signaling by ~2-fold, and this could be further increased by the agonist GRO{alpha} (Fig. 3). It was surprising, however, that none of the ghrelin-like receptors, which all display clear constitutive signaling through the phospholipase C pathway and through several other downstream pathways (see below), showed any clear sign of constitutive signaling through ERK1/2 (Fig. 3). Transfection with the ghrelin receptor did increase the basal ERK activity marginally by around 15% (n = 6). However, stimulation with the agonist ghrelin increased ERK phosphorylation by almost 3-fold, which is in accordance with previously published results (28). Stimulation of GRP39-transfected COS-7 cells with ZnCl2 increased the ERK1/2 phosphorylation by ~70%. Transfection with NT-R2 seemed to decrease the basal ERK phosphorylation slightly (i.e. ~20% (n = 6)) compared with mock-transfected cells, and this effect was reversed by addition of neurotensin. Thus, none of the otherwise constitutively active members of the ghrelin receptor family showed clear ligand-independent signaling through the ERK1/2 signaling pathway.



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  		FIG. 3.
Mitogen-activated protein (MAP) kinase signaling. A, representative example of basal signaling and maximal agonist-induced response of ERK1/2 phosphorylation in COS-7 cells transiently transfected with ORF74, the ghrelin receptor, NT-R2, pcDNA3 (mock transfection), and GPR39. B, ERK1/2 phosphorylation corrected for the total ERK1/2 protein expression. Results are expressed as fractions of the value obtained with pcDNA3-transfected cells and represents the mean ± S.E. from six independent experiments.

 
Signaling through the CRE Pathway—Although the activity of the important transcription factor CRE binding protein is generally believed to be controlled by cAMP-dependent kinases, it can also be activated by downstream kinases of the Gq pathway, such as Ca2+/calmodulin kinase IV or protein kinase C (29, 30). As demonstrated previously (31), gene-dosing experiments with the ghrelin receptor performed in transiently transfected HEK293 cells result in a dose-dependent but ligand-independent stimulation of the CRE pathway as monitored by a reporter assay using CRE-driven luciferase activity (Fig. 4A). The CRE-dependent transcriptional activity could be further increased by ghrelin and could be decreased by the inverse agonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P. However, the effects of the agonist and inverse agonist were smaller in the CRE reporter assay than in the IP assay, probably because of the high degree of amplification in the reporter assay. The NT-R2 also showed gene dose-dependent signaling through the CRE pathway as a ~5-fold increase in the CRE-mediated luciferase activity was observed in cells transiently transfected with this receptor (Fig. 4B). Similar to what was observed when measuring inositol phosphate accumulation, neurotensin was also a neutral ligand in the CRE assay (17). Transfecting with increasing amount of GPR39 cDNA induced only an ~2-fold increase in CRE-mediated luciferase activity (Fig. 4C), and the addition of Zn(II) induced only a very limited stimulation of the CRE-mediated luciferase activity. The motilin receptor served as a useful negative control, without constitutive activity in the gene-dosing experiments; importantly, however, upon stimulation with the agonist motilin, CRE activity was observed that reached levels similar to those observed with the unstimulated ghrelin receptor and NT-R2, indicating that a similar level or receptor expression was in fact reached (Fig. 4D). Thus, whereas both the ghrelin receptor and NT-R2 displayed a high level of constitutive activity through the CRE pathway, GPR39 was only signaling with marginal constitutive activity through this pathway.



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  		FIG. 4.
Induction of CRE-dependent gene transcriptional activity by the ghrelin receptor (A), the neurotensin receptor (B), GPR39 (C), and by the motilin receptor (D). Gene-dosing experiments were performed in transiently transfected HEK293 cells and the basal, ligand-independent signaling activities (black bars) of the respective receptors and the signaling in the presence of a maximal dose of the relevant full agonist: ghrelin (10-7 M), neurotensin (10-6 M), ZnCl2 (10-4 M), and motilin (10-7 M) (white bars) was measured by a CRE binding protein-luciferase reporter assay (for details, see "Experimental Procedures"). The ghrelin receptor was also treated with the previously identified inverse agonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P (10-6 M) (hatched columns) (8). Representative of at least four independent gene dosage experiments, where all four constructs assayed in parallel and each point performed in quadruplicate in 96-well plates are shown. RLU, relative light units, as measured in a PerkinElmer TopCounter.

 
Signaling through the SRE Pathway—Transcriptional regulation through the SRE pathway has been proposed to be stimulated by various G protein systems, including G{alpha}13, G{alpha}I, and G{beta}{gamma} (32-35).

Both the ghrelin receptor and NT-R2 demonstrated an increase of more than 10-fold in ligand-independent SRE signaling in gene-dose experiments performed in transiently transfected HEK293 cells (Fig. 5, A and B). However, a very large additional response in SRE activity was observed upon stimulation with ghrelin and a clear but relatively small effect of the inverse agonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P was also observed in cells transfected with the ghrelin receptor (Fig. 4A). In the case of SRE as well, no effect of neurotensin was observed (Fig. 5B). In contrast to what was observed in the IP and CRE assays, GPR39 showed a much higher constitutive activity than the ghrelin receptor in the SRE assay as a more than 30-fold stimulation in ligand-independent SRE activity was observed in the gene dose experiment with GPR39 compared with mock-transfected cells (Fig. 5C). Thus, the constitutive SRE signaling of GPR39 almost corresponded to the maximal agonist induced signaling observed with the ghrelin receptor. The motilin receptor was silent with respect to ligand-independent SRE activity, but motilin increased the transcriptional activity 12-fold above basal level, corresponding to the levels of ligand-independent SRE activity observed with the ghrelin and neurotensin receptors.



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  		FIG. 5.
Induction of SRE-dependent gene transcriptional activity by the ghrelin receptor (A), the neurotensin receptor (B), GPR39 (C), and by the motilin receptor (D). Gene-dosing experiments were performed in transiently transfected HEK293 cells and the basal, ligand-independent signaling activities (black bars) of the respective receptors and the signaling in the presence of a maximal dose of the relevant full agonist: ghrelin (10-7 M), neurotensin (10-6 M), ZnCl2 (10-4 M), and motilin (10-7 M) (white bars) was measured by a SRE-luciferase reporter assay (for details, see "Experimental Procedures"). The ghrelin receptor was also treated with the inverse agonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P (10-6 M) (hatched columns). Representative experiments of at least four independent gene dosage experiments, where all four constructs assayed in parallel and each point performed in quadruplicate in 96-well plates are shown. The insets demonstrate the effect of treatment with inhibitors (Y-27632, LY-294200, and PTX) expressed as a fraction of the basal activity. RLU, relative light units, as measured in a PerkinElmer TopCounter.

 
To determine whether the SRE activation was caused by a functional coupling of the receptors to G{alpha}i/o proteins, we treated the cells with pertussis toxin (PTX; 100 ng/ml for 24 h). This treatment did not affect the constitutive activity of SRE-mediated transcription for the ghrelin receptor or for GPR39, but PTX reduced the constitutive SRE activity mediated by NT-R2 by ~50% (Fig. 5, insets). Because G{alpha}12/13 are often functionally coupled to the Rho signaling pathway, we used a specific Rho kinase inhibitor (Y-27632) to probe this pathway. Y-27632 decreased the basal GPR39-mediated SRE activity by ~60%, whereas the ghrelin receptor and NT-R2 mediated SRE activity was inhibited by only ~20%. A similar inhibition of the GPR39-induced SRE activity was observed by treatment of the cells with the PI3K inhibitor LY-294002. It is noteworthy that in cells transfected with either the ghrelin receptor or with NT-R2, a slight increase in the basal SRE signaling was observed by addition of the PI3K inhibitor (Fig. 5, A and B, insets). Thus, GPR39 signals with an extraordinarily high constitutive activity through the SRE pathway compared with NT-R2 and the ghrelin receptor; the latter did display a strong ligand-mediated signal in this pathway.

Constitutive Internalization of Receptors—The ghrelin receptor and GPR39 were FLAG-tagged at their extracellular N termini, and the ability of the receptors to internalize was studied in stably transfected HEK293 clones. To specifically examine the fate of receptors expressed at the cell surface, antibody feeding experiments were performed with the M2 antibody (recognizing the FLAG tag), which was added to the cell medium before ligand stimulation, fixation, and detection. When cells were incubated with the M2 antibody at 4 °C (to prevent internalization) the immunoreactivity was confined to the plasma membrane (data not shown). However, when cells expressing the FLAG-tagged ghrelin receptor were incubated at 37 °C for 45 min with the M2 antibody, but no ligand added, the majority of the immunoreactivity was at the end of the incubation period found intracellularly in a vesicular pattern (Fig. 6, top row). Thus, in HEK293 cells, the ghrelin receptor is constitutively internalized. Numerous intracellular puncta contained both FLAG immunoreactivity and transferrin-Texas Red, a marker for clathrin-coated internalization and recycling endosomes. Treatment with the ghrelin agonist (100 nM for the last 15 min before fixation) did not alter this pattern of presumably endosomal receptor localization (Fig. 6, second row). Addition of the inverse agonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P, however, induced a substantial redistribution of the ghrelin receptors to the plasma membrane (Fig. 6, third row). Ghrelin receptors were also trapped at the cell surface by treatment with concanavalin A (0.3 mg/ml), a nonspecific inhibitor of internalization, which also prevented transferrin endocytosis (data not shown). In parallel experiments, internalization of FLAG-tagged {beta}2-adrenergic receptors to transferrin-positive endosomes was observed only in the presence of the agonist isoproterenol (data not shown).



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  		FIG. 6.
Internalization properties of the ghrelin receptor and of GPR39. Internalization was studied by antibody feeding experiments performed for 45 min with the M2 antibody in HEK293 cells stably transfected with either the FLAG-tagged version of the ghrelin receptor (top three rows), GPR39, or [AsnVI:16Phe]GPR39 (bottom two rows). After fixation and permeabilization, the localization of the M2 antibody was done with an Alexa 488-labeled second antibody (green). Cells were co-exposed to Texas Red-labeled transferrin, which is constitutively internalized by the ubiquitous transferrin receptor. The immunofluorescent analysis was performed on a Zeiss Axiovert 100 microscope, in which out of focus light was deconvoluted from stacks of vertical images and a central 3.0-µm z-section was reconstructed using Improvision's Volocity software (see "Experimental Procedures"). These sections, viewed from above, are illustrated for representative single cells from three independent experiments. The ghrelin receptor was exposed to either the agonist, 10-7 M ghrelin (second row), or the inverse agonist, 10-6 M [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P (third row) for the last 15 min of the incubation period.

 
In contrast to the ghrelin receptor, GPR39 was almost exclusively located at the plasma membrane even when the incubation with the M2 antibody was performed at 37 °C for 45 min (Fig. 6). This was also the case in HEK293 cells stably transfected with a FLAG-tagged version of the [AsnVI:16Phe]GPR39 mutant receptor, which signals with the same high basal activity as the ghrelin receptor through the Gq, phospholipase C pathway (see below).

Thus the ghrelin receptor is constitutively internalized and this can be prevented by the inverse agonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P, whereas the homologous GPR39 receptor, which also is highly constitutively active, does not undergo constitutive internalization.

Mutational Analysis of the Structural Basis for the Constitutive Activity of the Ghrelin Family of Receptors—Previous studies using metal-ion site engineering have indicated that activation of 7TM receptors involves an inward movement of TMs VI and VII toward TM III in the main ligand-binding pocket at the extracellular ends of these helices (25).2 Based on the assumption that the high constitutive activity of the ghrelin, NT-R2, and GPR39 receptors could be a result of an increased tendency of TMs VI and VII to "dock" with each other and with the inner face of TM III, we performed a mutational analysis, mainly of residues located at the inner faces of these transmembrane segments initially in the ghrelin receptor (Fig. 7A). Residues were substituted either with Ala or with the corresponding residues of the silent motilin receptor (31) (Figs. 4 and 5). IP accumulation was chosen as the signaling read out because this is most closely associated with the receptor and the G protein. As shown in Fig. 7B, the constitutive activity of the ghrelin receptor was somewhat reduced in several of these mutants, and it was totally eliminated in four of the mutants: GlnIII:05 to Leu; PheVI:16 to Ala; ArgVI:20 to Ala; and Phe-VII:06 to Ala. However, in three of these mutants, the ability of the agonist to stimulate the receptor was also eliminated, which made interpretations of the results difficult. For example, the lack of signaling could be the result, in principle, of a lack of sufficient surface expression of the mutant receptor. But in the case of the PheVI:16-to-Ala substitution, a selective effect on the constitutive activity of the receptor was observed, in that the ghrelin stimulation of receptor signaling was intact (Fig. 7B). This is a particularly interesting position, because an activating metal-ion site has been built in the {beta}2-adrenergic receptor from this position to two positions in TMs III and VI, respectively.



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  		FIG. 7.
Mutational analysis of the ghrelin receptor, with focus on the inner faces of the extracellular ends of TMs III, VI, and VII. A, helical wheel diagram of the ghrelin receptor. Residues that could be mutated without eliminating the constitutive activity are indicated in black on gray. Two addition residues (Glu-196 and Arg-198) in extracellular loop 2 located on either side of the Cys residue that forms a disulfide bridge to CysIII:01 were also substituted because they are believed to be located just above the main ligand binding pocket. In white on black are the three Phe residues that were subjected to more elaborate mutagenesis plus the two other residues, GlnIII:05 and ArgVI:20, which were also hits for constitutive activity. B, inositol phosphate accumulation in COS-7 cells transiently transfected with the wild-type (WT) and a series of mutant forms of the ghrelin receptor. Black columns indicate the basal, constitutive signaling activity, and the open bars represent the signaling activity in response to the agonist ghrelin (10-7 M) expressed as percentage of wild-type ghrelin receptor.

 
As shown in Fig. 8A, when a large aromatic Tyr residue (as found in the neurotensin 2 receptor) was introduced at position VI:16 of the ghrelin receptor, the mutant receptor displayed constitutive activity similar to that of the wild-type ghrelin receptor, which has a structurally similar Phe at this position. However, when a small polar residue such as Asn (as found in the GPR39 receptor) was introduced at position VI:16, this resulted in an intermediate but clear constitutive activity, which corresponded well with the constitutive activity observed in the GPR39 receptor (Fig. 8A). The surface expression of the mutant receptors was similar to that of the wild-type receptor as reflected in the similar maximal signaling efficacy measured during stimulation with the ghrelin agonist (Table I). Only the FVI:16A mutation displayed a somewhat lower Emax compared with the wild-type receptor. In contrast, the other mutant receptor with a very low basal signaling (FVII:09A) was in fact highly expressed on the surface, with a higher Emax in response to ghrelin than that observed for the wild-type receptor. When the corresponding substitutions were performed in the GPR39 receptor, a very similar picture was found; i.e. introduction of either a Phe or a Tyr residue at position VI:16 instead of the endogenous Asn resulted in increased constitutive activity of a magnitude similar to that observed in the wild-type ghrelin and neurotensin receptors (Fig. 8). In GPR39, Ala substitution at this position decreased the observed constitutive activity but did not totally eliminate it as observed in the ghrelin receptor. Thus, it is possible to tune the constitutive activity of both the ghrelin receptor and the GPR39 receptor up and down in a relatively systematic manner by changing the physicochemical property of the inner phase of TM VI in the main ligand binding pocket at position VI:16. To probe whether the mutations affected the surface expression, ELISA assay was performed with the FLAG-tagged GPR39 mutations, which showed a very similar surface expression for all the mutations (Fig. 8B).



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  		FIG. 8.
Effect of substitutions in the aromatic cluster in TMs VI and VII of the ghrelin receptor and the GPR39 on the basal, constitutive signaling. A, inositol phosphate accumulation was determined in COS-7 cells transiently transfected with the wild-type (WT) ghrelin receptor or with mutant forms in which either PheVI:16, PheVII:06, or PheVII:09 was systematically substituted (black columns). For position VI:16, the corresponding substitutions were made in FLAG-tagged GPR39 and probed for basal, constitutive signaling in parallel (white columns). Note that GPR39 has an Asn in position VI:16 and that the [PheVI:16Asn] mutant in the ghrelin receptor has a basal signaling similar to that of wild-type GPR39 and that the [AsnVI:16Phe]GPR39 displays a constitutive signal similar to that of the wild-type ghrelin receptor. B, ELISA of the FLAG-tagged GPR39 mutations in parallel with the wild-type GPR39 showed that the expression levels were unaltered by the mutations (data not shown).

 


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  		TABLE I
Activity of ghrelin receptor mutations Basal and ghrelin-induced activity of the ghrelin receptor mutations are expressed as percentage of basal activity of the wild-type ghrelin receptor ± S.E.M. The basal activity as percentage of the ghrelin-induced maximum activity is shown in the last column.

 
The mutations of the hydrophobic pocket between TMs VI and VII modulated the signaling through CRE- and SRE-mediated transcription in a manner similar to that of the affected phospholipase C-mediated signaling. It is interesting that in CRE-mediated signaling, ghrelin was able to stimulate FVI:16A equally efficiently as the wild-type ghrelin receptor (Fig. 9A).



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  		FIG. 9.
Effect of substitutions of PheVI:16 in the ghrelin receptor in SRE- and CRE-dependent gene transcription. A, CRE-dependent and B, SRE-dependent gene transcription were performed in transiently transfected HEK293 cells in which the basal, ligand-independent signaling activities (black bars) and the signaling in the presence ghrelin (10-7 M) (white bars) were measured for wild-type (WT) ghrelin receptor and ghrelin receptor with PheVI:16 substituted with and Ala, Asn, or Tyr.

 
Because of the close proximity and, therefore, likely structural interaction between PheVI:16 and PheVII:06 and Phe VII:09 at the inner face of TM VII (Fig. 10), these two residues were also subjected to further mutational analysis in the ghrelin receptor. A similarly high constitutive activity was observed when PheVII:06 was substituted with other large residues such as Tyr, His, or Leu. However, the constitutive activity was eliminated if Ala was introduced in position VII:06. In contrast, only substitution with Tyr for PheVII:09 was allowed, giving normal high constitutive activity, whereas substitution with His, Leu, or Ala in all cases impaired the constitutive activity of the ghrelin receptor.



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  		FIG. 10.
Molecular model of the residues proposed to be part of the structural basis for the high constitutive activity in the ghrelin receptor. A, molecular model of the ghrelin receptor built over the inactive structure of rhodopsin (60, 61). The seven helical bundle is displayed without the loops as viewed from the extra-cellular side. Only the residues on the inner faces of TMs III, VI, and VII, which in the mutational analysis were identified to potentially be involved in the constitutive activity, are shown. The two arrows indicate the proposed inward movement of TM VI and VII toward TM III that occur during activation of 7TM receptors based on metal-ion site engineering between residues: III:08, VI:16, and VII:06. B, the extracellular ends of TMs VI and VII as viewed from between TMs IV and V, showing the close proximity of the three aromatic residues: PheVI:16, PheVII:06, and PheVII:09. These three residues were subject to further mutational analysis, as shown in Fig. 8.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the orphan receptor GPR39 was identified as a constitutively active member of the ghrelin receptor family. A parallel analysis of the signaling properties of GPR39, NT-R2, and the ghrelin receptor itself demonstrates considerable differences in the relative efficacy by which these structurally related, constitutively active receptors signal through different pathways. Thus, the ghrelin receptor and NT-R2 are more efficacious than GPR39 in Gq/phospholipase C signaling as well as in CRE signaling, whereas GPR39 clearly was the most constitutively active through the SRE pathway. The receptors also displayed major differences in their internalization properties. The ghrelin receptor internalized in a ligand-independent fashion and accumulated in intracellular vesicles in contrast to GPR39, which despite its high constitutive signaling activity remained at the cell surface. Despite their differences in signaling and internalization properties, a common structural "volume knob" or lever could be identified in this family of receptors in the form of an aromatic cluster of residues on the inner face of TMs VI and VII. Thus, both in the ghrelin receptor and in GPR39, the level of constitutive activity could, in a similar manner, gradually be turned up or down depending on the size and hydrophobic properties of the side chain of the residue in position VI:16.

Different and Similar Signaling Properties among Three Structurally Related, Constitutively Active Receptors—First, it should be emphasized that constitutive activity of a receptor is a difficult property to measure quantitatively because it is dependent on the expression level of the receptor as emphasized by the gene dosing experiments. Therefore, the structurally homologous motilin receptor serves as a highly important "negative" control in both the present and the previous studies of the ghrelin receptor (14). Not only does the motilin receptor in parallel gene dosing experiments display no sign of constitutive activity, but also administration of the agonist ligand (the peptide motilin) results in signaling through the various pathways of a magnitude comparable to that observed in the basal state with the three constitutively active receptors (Figs. 4 and 5). This strongly indicates that the expression levels of the receptors are similar and that the constitutive signaling observed with GPR39, NT-R2, and the ghrelin receptor is a physiologically important phenomenon and not an experimental artifact caused, for example, by overexpression of these receptors.

In the signaling assay, which measures activity near the receptor i.e. determination of inositol phosphate accumulation as a reflection of Gq-mediated phospholipase activity, the ghrelin receptor and NT-R2 both signal with a similar constitutive efficacy of ~50% of the maximal capacity (8, 17). GPR39 (although clearly being constitutively active) signaled through this pathway with a somewhat lower efficacy (Fig. 2). However, when more downstream signaling pathways were studied, the relative efficacy and even the rank order of constitutive signaling efficacy varied among the three related receptors. With respect to CRE-mediated transcription, the ghrelin receptor showed very strong ligand-independent activity, reaching a level 2-fold higher than the neurotensin receptor, whereas GPR39 showed only a very limited degree of constitutive activity in this signaling pathway. Because there is no indication that these receptors couple through Gs and cAMP, it is likely that the CRE activation is mediated through a Gq pathway and various down-stream kinases (29, 30). When SRE mediated transcriptional activity was measured instead, a different rank order of potency was observed among the three related receptors. GPR39, in contrast, which clearly signaled with the lowest degree of constitutive activity in the possibly Gq-mediated IP and CRE pathways, showed the highest degree of constitutive signaling in the SRE pathway. This suggests that a G-protein other than G{alpha}q might be responsible for at least the majority of the observed constitutive activation of SRE. G12/13 has previously been suggested to mediate the coupling of several other 7TM receptors to SRE activation through Rho kinase (32). The specific Rho kinase blocker Y-27632 decreased the basal SRE activity in GPR39-transfected cells by ~50%, indicating that G12/13 is involved in the GPR39-induced, ligand-independent stimulation of SRE activation. The constitutive activation of SRE observed in cells transfected with the ghrelin receptor or with NT-R2 was much lower than that observed with GPR39; this SRE activity was also inhibited only marginally by the Rho kinase blocker (Fig. 4). A close interaction between PI3K- and SRE-mediated transcription has been described previously (36). Through the use of a selective blocker of PI3K (LY-294002), we found for GPR39 that this pathway was responsible for a fraction of SRE activation similar to that of Rho. However, many different signal transduction pathways have been suggested as links between 7TM receptors and PI3K activation, including G{beta}{gamma},Gq, and G13. SRE activation can also be mediated through Gi (33), but the lack of effect of PTX would tend to exclude this pathway for at least GPR39 and the ghrelin receptor, whereas the NT-R2 coupling to SRE seemed to be at least partly dependent on Gi/o due to the effect of PTX.

We would have expected that the constitutive activity of the ghrelin-like receptors could be detected in MAP kinase activation as well, as observed for many virally encoded receptors (37). However, all three ghrelin-like receptors, otherwise constitutively active, were almost silent in respect of ligand-independent signaling through at least ERK1/2 phosphorylation. This is surprising, especially because the ghrelin receptor itself showed a clear ligand-mediated activation of this MAP kinase (Fig. 5). We cannot at present explain this discrepancy. However, it illustrates the notion that there is more than one active conformation of a 7TM receptor; although constitutive activity may to a certain degree reflect the repertoire of the agonist induced signaling, it may not cover all the signaling pathways (38). This could be an important point, because we are here dealing not with virally encoded "exogenous receptors" but with endogenous receptors, which probably have evolved a high degree of constitutive activity for a particular physiological purpose and in which a continuous high signaling through other pathways could be problematic in leading, for example, to cell proliferation and cancer development.

NT-R2 is an atypical neurotensin receptor because the peptide only binds with relatively low potency to this receptor and because neurotensin has almost no effect on receptor signaling, as observed both in this study and by Richard et al. (17). It is interesting that a small non-peptide compound, SR48.692, which is a high-affinity antagonist for the normal neurotensin receptor NT-R1, in fact functions as a high-potency, high-efficacy agonist for NT-R2 (17).

Different Internalization Pattern for the Ghrelin Receptor and GPR39 —7TM receptors are generally targeted to the cell surface; upon agonist activation, they are internalized as part of a desensitization process. Thereafter, they are either recycled to the membrane or, in certain cases, sorted to the lysosomal pathway for degradation. It is generally assumed that constitutive activity would lead to constitutive phosphorylation and constitutive internalization. In the present study, we find by antibody feeding experiments that the ghrelin receptor is constitutively internalized, whereas GPR39, which in certain signaling assays is even more constitutively active, remains at the cell surface (Fig. 6). However, GPR39 was clearly not as constitutively active as the ghrelin receptor in the Gq-mediated IP signaling. However, even the mutant form of GPR39 ([QVI: 16F]GPR39), which through the IP pathway signaled with a degree of constitutive efficacy similar to that of the ghrelin receptor, showed no sign of constitutive internalization. This is in complete agreement with observations in both constitutively active, virally encoded receptors and in constitutively active mutant forms of endogenous receptors, such as the C5a receptor (38, 39). There simply is no direct correlation between constitutive signaling and constitutive internalization. In the case of the virally encoded US28 receptor, it could even be demonstrated that the structural basis for the constitutive internalization was located in the C-terminal tail and was totally independent upon the constitutive signaling (40).

The observation that a relatively short treatment with the inverse agonist for the ghrelin receptor is able to trap the vast majority of the receptors at the cell surface indicates that the internalization of this receptor is associated with signaling and postsignaling events and that the receptor is probably recycled relatively rapidly to the cell surface. More detailed cell biological studies are required to determine this; recently, however, this phenomenon was characterized in detail in the constitutively active CB1 receptor, where an inverse agonist also traps the constitutively internalized receptor (41). Trapping of constitutively active receptor mutants at the cell surface by inverse agonists has been shown in several systems (42-44).

It should be noted that a different group has recently reported that a green fluorescent protein-tagged ghrelin receptor is surface expressed in Chinese hamster ovary cells and internalizes upon ligand stimulation (45). The discrepancy may be explained by the large GFP tag, by the cell type, or by the fact that only antibody feeding experiments are more sensitive in detecting internalization of cell surface receptors (45).

A Molecular Switch Modulating the Level of Constitutive Activity—In the present study, we identified an aromatic cluster on the inner face of TMs VI and VII that seems to be at least part of the structural basis for the constitutive activity of the ghrelin-like receptors.

The molecular activation mechanism for 7TM receptors is not understood in details. However, a series of biophysical studies, including EPR studies with site-directed spin labeling and studies with various fluorescent probes, all indicate that activation of rhodopsin and {beta}-adrenergic receptors is associated with a movement of especially TM VI out and away from TM III at the intracellular face of the receptor (46-48). The corresponding movements occurring at the extracellular, agonist-binding part of the receptor have as yet not been characterized through similar biophysics methods. Nevertheless, construction of activating metal-ion sites in the main ligand-binding pocket indicates that activation of 7TM receptors involves an inward movement of TMs VI and VII toward each other and toward TM III within the main ligand binding pocket (25).2 Agonists are according to this model believed to act through binding to and stabilization of an active conformation in which the extracellular ends of TMs III, VI, and VII are "docked" onto each other. However, the receptor is assumed to be in a dynamic equilibrium between inactive and active conformations even in the absence of the agonist, which is responsible for the constitutive activity of these receptors.

Through a systematic mutational approach, we identified three positions on the inner face of the ghrelin receptor that seemed to be responsible for stabilizing the active conformation of the receptor in the absence of agonist ligand: PheVI:16, PheVII:06, and PheVII:09 (Figs. 7 and 8). Most convincingly, the constitutive signaling activity of both the ghrelin receptor and GPR39 could be raised or lowered through variation of the size and hydrophobicity or aromaticity of the residue located in position VI:16. In the model described above for 7TM receptor activation, the interpretation would be that a Phe or Tyr in position VI:16 in the context of a Phe in position VII:09 and a large hydrophobic residue in position VII:06, will ensure a favorable "docking" of the extracellular end of TM VI on TM VII through the formation of a hydrophobic core between these helices.

Although Phe and Tyr are very common residues at position VI:16 (found in 66% of 7TM receptors) the residue at position VII:09 is usually a small residue such as Ala, Gly, or Ser (found in 73% of receptors) (49). It should be noted that our mutational analysis in the ghrelin receptor, with a Phe at position VI:16, showed that the high constitutive activity was observed only with a Tyr or a Phe in position VII:09 (Fig. 8). Not even a His in position VII:09 would give the high constitutive signaling.

Mutations at two other positions in the ghrelin receptor also eliminated the constitutive activity (i.e. GlnIII:05 and ArgVI: 20), which are both located just above the described aromatic cluster (Fig. 7). It is likely that these two residues may also be involved in creating the structural basis for the high constitutive activity of this receptor through a polar interaction across the main ligand binding pocket for example hydrogen bond formation with or without water molecules. However, because ghrelin activation was also eliminated by these mutations, we cannot at present confirm this notion.

Biological Significance of High Constitutive Receptor Activity—The level of constitutive activity displayed by the three ghrelin-like receptors (~50% or more of their maximal capacity) is highly unusual among 7TM receptors. We would suggest that this property of the receptors is an important part of their physiological repertoire in vivo. Cells in which such receptors are expressed will be provided with a signaling tone against which other receptors may act. Thus, it has been suggested that the ghrelin receptor, which is the only stimulatory receptor expressed on the important sensory neuropeptide Y/agouti-related peptide neurons in the arcuate nucleus of the hypothalamus, provides a positive signaling tone (perhaps most significantly exemplified in the CRE activity) against which the receptors for all the appetite inhibitory hormones and transmitters act, such as leptin, insulin, PYY3-36, etc. (14). A high constitutive signaling also means that regulation of the expression of the receptor as such has direct consequences on the signaling activity independent of the hormone. It is interesting to note, therefore, that the ghrelin receptor has been described as being up-regulated by 8-fold in the hypothalamus during fasting (50). According to our model, this would provide a ligand-independent increase in ghrelin receptor signaling (i.e. stimulation of appetite) (14). The cannabinoid CB1 receptor is another highly constitutively active receptor that stimulates food intake. The CB1 receptor is expressed, for example, in vagal afferent neurons, where its expression is also highly up-regulated during fasting, and the expression returns rapidly to normal when the animals are fed again (51).

Ligands binding to a receptor that displays high constitutive signaling activity can either regulate its signaling up (agonists) or down (inverse agonists). Although only a couple of endogenous inverse agonists have been described so far (i.e. agouti and agouti gene related peptide for the melanocortin receptors), it is likely that others will be identified in the future, especially because the only reason that agouti was discovered was the skin color produced by a natural genetic variant of the protein (52). Thus, it could be interesting to search for endogenous inverse agonists for the ghrelin-like receptors. It has been suggested that inverse agonists against the ghrelin receptor could be particularly attractive antiobesity agents (14).

The orphan receptor GPR39 is widely expressed in the central nervous system, and it could be of great importance that this receptor signals constitutively with very high efficacy through SRE, possibly mediated to a large degree through G{alpha}12/13 and RhoA. The activity of G12/13 and RhoA has recently been associated with cellular plasticity in neurons, particularly with cytoskeletal contractions that prevent neural outgrowth (53). Hence, the expression of GPR39 and the following constitutive signaling through G12/13 may decrease the sensitivity of neurons for factors that induce sprouting (53, 54). Similar effects and signaling pathways could perhaps be involved in the recently demonstrated rapid rewiring of the arcuate nucleus feeding circuits in response to leptin and ghrelin, which have opposite effects in this case (55). Thus, it seems that the ghrelin receptor, and perhaps also GPR39, regulates neuronal activity not only through altering transmitter expression and release but also through altering neuronal plasticity (56). It has also recently been shown that SRE-induced transcription is an important factor for cell survival, including neuronal survival through inhibition of the complex apoptosis cascade (57). The strong increase in SRE activity induced by ghrelin through its receptor (Fig. 5) could perhaps be involved in the antiapoptotic effects observed for ghrelin on for example adipocytes (58).


   FOOTNOTES
 
* This study was supported in part by grants from The Novo Nordisk Foundation and the Danish Medical Research Council (to B. H.), a 7TM Biotech Competence Center grant from the Danish Medical Research Council (to T. W. S.), and by the Kimmel Cancer Foundation (to H. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 45-3532-7602; Fax: 45-3532-7610; E-mail: b.holst{at}molpharm.dk.

1 The abbreviations used are: 7TM, seven transmembrane; IP, inositol (1,4,5)-triphosphate; NMU, neuromedin U; NT-R2, neurotensin receptor 1; HEK, human embryonic kidney; SRE, serum-responsive element; PBS, phosphate-buffered saline; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; ELISA, enzyme-linked immunosorbent assay; CRE, cAMP-responsive element; PTX, pertussis toxin; PI3K, phosphoinositide 3-kinase; TM, transmembrane domain. Back

2 B. Holst and T. W. Schwartz, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Bente Friis and Elisabeth Ringvard for expert technical help. We are grateful to Professor Roger Morris and Dr. Angela Jen for advice and use of their immunofluorescence microscope.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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