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Laboratory for Molecular Pharmacology (B.H., A.C., T.H.J., T.W.S.), The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark; 7TM Pharma A/S (B.H., T.W.S.), Fremtidsvej 3, DK-2970 Hørsholm, Denmark; and Department of Medicinal Chemistry (M.A.), Novo Nordisk A/S, DK-2760 Måløv, Denmark
Address all correspondence and requests for reprints to: Birgitte Holst, M.D., Ph.D., Laboratory for Molecular Pharmacology, Institute of Pharmacology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen, Denmark. E-mail: b.holst{at}molpharm.dk.
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ABSTRACT |
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INTRODUCTION |
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Ghrelin is a 28-amino-acid peptide, which has a unique structure among peptide hormones as it is acylated at Ser3 usually with an n-octanyl moiety (7, 8). This posttranslational modification is essential for the activity of the hormone both in vitro and in vivo (7, 9). The ghrelin receptor was initially—as expected— found on GH producing cells in the pituitary and like the GHRH receptor it stimulates pulsatile secretion of GH (5). However, ghrelin receptors were also discovered in other parts of the central nervous system especially in the hypothalamus (10). Accordingly, both intracerebroventricular and iv injection of ghrelin strongly stimulates feeding and increases body weight in rodents—independent of its effect on GH secretion (11, 12). It appears that ghrelin is involved in the initiation of food intake as the plasma level of the peptide increases significantly just before meals (13, 14). Thus, ghrelin functions not only as a GH-releasing substance but also as an orexigenic hormone and the physiological role of ghrelin appears to be a link or messenger between the stomach and the hypothalamus and the pituitary. Centrally, ghrelin acts mainly on receptors expressed on neuropeptide Y (NPY)/agouti-related peptide (AGRP) producing cells in the arcuate nucleus of the hypothalamus because antibodies and antagonists of NPY and AGRP abolish the ghrelin-induced feeding response (12, 15, 16, 17). Interestingly, the ghrelin receptor was recently found to be expressed in large amounts also on afferent vagal neurons (18, 19). In accordance with this, the effect of peripheral administration of ghrelin on c-fos expression in NPY/AGRP neurons and the effect on feeding in rats is totally dependent on an intact vagal nerve, whereas the effect on GH secretion was only partially mediated through the proposed vagal afferent pathway (18). These findings indicate that gastric vagal afferents may be the major pathway conveying ghrelins signaling from the stomach to the central nervous system. Interestingly, the closest homolog to the ghrelin receptor is the receptor for motilin (Fig. 1
), which like ghrelin is a hormone secreted from the upper part of the gastrointestinal tract and which also interacts with the autonomic nervous system (19, 20).
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). Because it previously has been shown that adenosine possibly could act as an agonist on the ghrelin receptor and because adenosine perhaps could be produced by the cells used for transfection, we pretreated the cells with adenosine deaminase (ADA). However, ADA did not affect the observed ligand-independent signaling of the ghrelin receptor (Fig. 2A) and pretreatment with the same concentration of ADA totally blocked the cAMP inhibition observed upon stimulation of the cells transfected with the adenosine receptor (data not shown). An increased production of IP was observed in cells transfected with the ghrelin receptor upon stimulation with 10-6 M ghrelin, which was most clearly observed at the higher levels of receptor expression (Fig. 2A).
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). Upon stimulation with the motilin peptide ligand, IP accumulation reached a level comparable to that observed in cells transfected with the ghrelin receptor after stimulation with the ghrelin agonist (Fig. 2B). In fact, the constitutive, ligand-independent signaling of the ghrelin receptor was comparable to that observed with one of the most well-established highly constitutively active 7TM receptors, the virally encoded ORF74 receptor (Fig. 2B) (21, 22).
Ligand Binding Analysis
A number of peptide and nonpeptides, most of which are known to be agonists on the ghrelin receptor, were selected and initially studied in binding assays (for structures see Fig. 3). The data in the literature concerning affinities of these ligands vary considerable depending on choice of tracer and assay conditions (23, 24). As shown in Table 1, ghrelin itself and the peptide agonist, ipamorelin competed against 125I-ghrelin with subnanomolar affinity. Interestingly, the two nonpeptide compounds L-692,400 and L-692,429, which previously have been shown to compete with nanomolar affinity against a structurally similar radiolabeled compound, MK677 (24, 25), were unable to compete for binding against the peptide agonist 125I-ghrelin in concentrations up to 1 uM (Table 1). In other 7TM receptor systems, differences in affinities of 100- to 10,000-fold have been observed dependent on whether it was determined in homologous or heterologous competition assays (21, 26, 27, 28). However, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P, which has been described to act as an antagonist with micromolar potency on the ghrelin receptor (23), was found to compete against 125I-ghrelin with a surprisingly high affinity, i.e. Ki = 45 nM.
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). As expected based on previous reports using calcium mobilization assays most of the peptide and nonpeptide ligands behaved as agonist with variable potencies and efficacies also in the functional assay measuring IP turnover. L-692,429 displayed an efficacy like ghrelin and a potency only 20-fold less than the endogenous agonist, EC50 = 3.9 nM (Fig. 3). The hexa-peptide analog ipamorelin had a potency of 13 nM and an efficacy of 65% of that of ghrelin. NN703, which in respect of GH secretion is known as a full agonist (23), surprisingly also functioned only as a partial agonist (43%) in respect of IP turnover (Fig. 3). L-692,400, a compound structurally similar to L-692,429, which has been shown to be an antagonist on the ghrelin receptor (29), was here found to be a neutral ligand as it did not change IP turnover in the ghrelin receptor-transfected cells (Fig. 3).
Inverse Agonist Modulation of the Constitutive Signaling of the Ghrelin Receptor
[D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P has previously been described as being a low potency ghrelin receptor antagonist (23). We could confirm that, as the substance P analog inhibited the ghrelin-stimulated IP accumulation with an EC50 value of 630 nM. Importantly, when [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P was applied to the ghrelin receptor in the absence of ghrelin it was—in contrast to the hexa-peptide analogs and nonpeptide ligands—found to function as a high efficacy, full inverse agonist as it inhibited the spontaneous, ligand-independent signaling in cells transfected with the ghrelin receptor down to the level observed in cells transfected with the empty expression vector (Fig. 4A). [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P had no effect on the constitutive activity of the ORF-74 receptor (data no shown). Unexpectedly, the potency of [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P as an inverse agonist on the ghrelin receptor was observed to be 5.2 nM, which is approximately 50- to 100-fold higher than the potency of the same peptide when studied as an antagonist against submaximally, ghrelin stimulated IP accumulation (Fig. 4A). Schild-type analysis also demonstrated that the ligand independent, basal signaling of the ghrelin receptor was inhibited by low doses of [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P, which did not shift the dose-response curve for ghrelin to the right (Fig. 4B). Thus [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P is a high potency, high efficacy inverse agonist for the constitutive, ligand-independent signaling of the human ghrelin receptor, which functions as a relative low potency antagonist for ghrelin-induced signaling.
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). As shown in Fig. 5, Zn(II) functioned as a full inverse agonist on the metal-ion site engineered receptor with a potency of 4.3 µM through binding to the two His residues located in an i and i+4 position at the extra-cellular end of TM-V. This demonstrates that the ligand-independent activity of the ghrelin receptor can be blocked through binding and stabilization of the extracellular end of TM-V as in the ORF-74 receptor (21).
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). Ghrelin stimulated this activity further whereas [D-Arg1,D-Phe5,D-Trp7,9,Leu11]- substance P again acted as an inverse agonist, here by decreasing the constitutive CREB-dependent transcriptional activity of the ghrelin receptor (Fig. 6A and inset). The substance P analog did not appear to be a full inverse in this assay, conceivably due the technical, timing difficulties in inhibiting a constitutive activity determined in a far downstream reporter system.
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). Like the ghrelin receptor, the virally encoded ORF-74 receptor also signaled with high ligand-independent activity through the CRE pathway with an efficacy, which was even somewhat higher than the maximal efficacy observed for the ghrelin receptor (Fig. 6C). However, as compared with both the motilin receptor (agonist-stimulated response) and the ORF-74 receptor (ligand-independent response) the gene-dose required for ghrelin receptor to stimulate CREB transcriptional activity was surprisingly almost two orders of magnitude lower. In fact, a bell-shaped stimulation was observed with the ghrelin receptor. Thus, the ghrelin receptor in a highly efficient, ligand-independent manner stimulates transcriptional activity though the CRE pathway. |
DISCUSSION |
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The fact that the ghrelin receptor is highly constitutively active raises a series of questions concerning the physiological importance of this activity and whether the receptor is regulated not only by the ghrelin agonist but also by a yet unknown endogenous inverse agonist. Moreover, knowledge of the high constitutive activity opens for novel pharmaco-therapeutic opportunities in developing inverse agonist compounds for the ghrelin receptor for the treatment of, for example obesity.
Possible Physiological Importance of the High Constitutive Activity of the Ghrelin Receptor
First, it should be emphasized that both in general and in the specific case of the ghrelin receptor, it is very difficult to determine the in vivo physiological importance of high ligand-independent signaling activity. However, in the in vitro setting the ghrelin receptor is massively constitutively active and with the homologous motilin receptor as a conveniently silent control receptor for effect of in vitro expression. In fact, the ligand-independent signaling of the ghrelin receptor is similar to that displayed by one of the most vigorously constitutively active receptors yet reported, the ORF-74 oncogene, encoded by human herpes virus 8 (21, 22). Thus, we expect that the constitutive activity of the ghrelin receptor is a physiologically important phenomenon.
It is interesting to note that GH is secreted from isolated pituitary cells in culture in a manner, which not only can be stimulated by GHRH and ghrelin, but which can be inhibited by somatostatin (34). Thus, there is a high level of GH secretion even from isolated cells, where no hormonal tonus should be present. It could be speculated that this ligand-independent GH secretion may be a consequence of the high level of constitutive signaling from the ghrelin receptor, which is known to be found on such pituitary cells. The substance P analog [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P described in the present study as an inverse agonist for the ghrelin receptor could be a useful tool to probe this hypothesis.
As presented in the introduction, ghrelin has been strongly implicated in the control of appetite and food intake acting both centrally via receptors found in the arcuate nucleus of the hypothalamus and peripherally through receptors on afferent vagal neurones (11, 12, 18). Centrally the ghrelin receptor is expressed mainly on the NPY and AGRP containing cells and antibodies and antagonists against NPY and AGRP have been shown to abolish the ghrelin induced effect on feeding (12, 15, 16). It has been hypothesized that ghrelin should increase the expression of NPY, however this has not been tested directly yet (35). Fasting induces an increase in the NPY level which appears to be mediated through an increase in CREB-dependent gene transcription as shown in transgenic mice expressing a CRE-lacZ construct (36). Both the CRE-activation and the NPY up-regulation in response to fasting were clearly attenuated by leptin. However, in view of the strong effect of the ghrelin receptor on CREB-dependent transcription observed in the present paper (Fig. 6) and the fact that ghrelin is the major chemical messenger of fasting and appetite signals could suggest that the CRE-mediated up-regulation of NPY is regulated through the ghrelin receptor. To what degree the constitutive activity of the ghrelin receptor is involved in this mechanism remains to be shown. Also in this case, inverse agonists such as the [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P could be valuable pharmacological tools.
Is There an Endogenous Inverse Agonist for the Ghrelin Receptor?
Food intake is regulated by a very intricate and redundant system of chemical messengers acting through a complicated neuronal network. The system is characterized by several layers of parallel, stimulating, and inhibitory mechanisms. However, in this system ghrelin is the only known peripherally acting orexigenic hormone. The fact that the ghrelin receptor is characterized by a high degree of constitutive activity raises the question of whether this receptor is regulated by an endogenous inverse agonist, which would act by decreasing the constitutive activity of the receptor. In this connection it should be noted that the MC-4 melanocortin receptor, which on the inhibitory side is crucially involved in the control of food intake, also is characterized by a high degree of constitutive activity (37). Importantly, the activity of the MC-4 receptor is know to be controlled not only by an endogenous agonist—
-MSH—but also by an endogenous inverse agonist—AGRP (37). Apparently, for certain systems it must be an advantage to modulate or fine-tune a high level of constitutive activity through the action of two ligands having opposite effects on the same receptor in stead of having two ligands acting as agonists on two receptors, which have opposite signaling effects. The latter arrangement is clearly a regulatory set-up used in many other systems in the organism. One interesting observation is, that the effect of the inverse agonist AGRP on the MC-4 receptor both in vitro and in vivo can be surprisingly long lasting (38, 39). The molecular/cell biological mechanism behind this phenomenon is not yet clear.
Ghrelin Receptor Inverse Agonists for the Treatment of Obesity?
Whether or not an endogenous inverse agonist exists for the ghrelin receptor, the high constitutive signaling activity of the receptor indicates that a compound acting as an efficient inverse agonist could be an interesting antiobesity agent. Today, the strong orexigenic effect of ghrelin as such clearly suggests that an antagonist of the ghrelin receptor could be a powerful regulator of appetite (15, 16). In view of the ligand-independent signaling of the ghrelin receptor, it is tempting to suggest that a neutral antagonist would in fact not be efficient in an in vivo setting and that an antagonist, which also is an inverse agonist, would be required for a ghrelin receptor ligand to be a truly efficacious antiobesity agent. As shown in the present study, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P is a potent and highly efficacious inverse agonist for the ghrelin receptor; however, it is probably not very optimal as a general pharmacological tool because it also has effects on the tachykinin NK1, i.e. the substance P receptor and at higher concentrations even affects a number of other receptors including the bombesin receptor (40). Nevertheless, dipeptide libraries based on this and similar substance P antagonists have proven to be useful starting points for the development of nonpeptide antagonists for several types of peptide receptors (41, 42). [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P in fact has a very interesting molecular pharmacological phenotype as it is a rather clean, high affinity inverse agonist with a low potency as an antagonist (Fig. 4). The in vivo efficacy of an inverse agonist for the ghrelin receptor as an antiobesity agent remains to be demonstrated. However, if the high constitutive signaling activity of the receptor observed in the present study occurs for example in the NPY/AGRP neurons in the arcuate nucleus, then this could very likely constitute the signaling "set point" against which the receptors for the many different appetite suppressing hormones and transmitters, such as leptin, insulin, melanocortin, PYY3–36, etc. (43). Thus, it could be envisioned that an inverse agonist for the ghrelin receptor would be useful to suppress appetite especially in between meals and thus would be efficient for example to prevent the craving for second order meals, desserts and snacks.
Interestingly, the ghrelin receptor belongs to a small subset of 7TM receptors for which almost exclusively agonist ligands have as yet been discovered through chemical file screening. Some other members of this subset are the motilin receptor, the C5a, the 5HT2c and to a certain degree the somatostatin and opioid receptors.
Molecular Mechanism of the Constitutive Activity in Ghrelin Receptor
Why is the ghrelin receptor so strongly constitutively active and the highly homologous motilin receptor apparently totally silent without stimulation by agonist? There is at present no clear answer to that, since mutations and substitutions at multiple places in a 7TM receptor can lead to pronounced constitutive activity. Nevertheless, one possible molecular mechanism for the constitutive activity of the ghrelin receptor could be that TM-VI is pulled toward TM-III by a salt-bridge formed between ArgVI:20 (Arg281) and GluIII:09 (Glu124) (Fig. 1). This would fit well with our observations on the apparent requirement for an inwards movement of TM-VI and -VII at the extracellular end in receptors with engineered activating metal-ion sites (31) (our unpublished observations). Moreover, both Arg281 and Glu124 have through mutagenesis previously been implicated to be involved in agonist ligand binding (44). However, ArgVI:20 and GluIII:09 are both conserved in the nonconstitutively active motilin receptor (Fig. 1). An interesting speculative possibility for the silences of the motilin receptor could be that the conspicuous, long, and Pro-rich insertion in extracellular loop 2 of this receptor (Fig. 1) could function as a tethered inverse agonist. The neurotensin 2 receptor is another 7TM receptor characterized in respect of ligand binding and functionality which has the constellation; ArgVI:20 together with GluIII:09. Importantly, the neurotensin 2 receptor was recently shown to be as highly constitutively active as the ghrelin receptor (45).
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løv, Denmark).
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, Laboratory for Molecular Pharmacology. The human ghrelin/GHS receptor (GHS-R) cDNA was cloned by PCR from a human brain cDNA library and sequenced using an ABI 310 automated sequencer and found to be identical to the GHS receptor having accession no. NP004113. The cDNA was cloned into the eukaryotic expression vector pcDNA3 (Invitrogen, Carlsbad, CA). Mutations were constructed by PCR using the overlap expression method (46). 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.
Transfections and Tissue Culture
COS-7 cells were grown in DMEM 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 previously described (47). For Fig. 2A various amounts of DNA were used, whereas for Fig. 2B-5 20 µg DNA were used per 75-cm2 plates. In Fig. 2A tranfection with 20 µg DNA per 75-cm2 plates gave a receptor expression of 126 fmol/105 cells measured by homologous competition binding curves. Assuming a linear correlation between DNA amount and protein expression, an estimation of the expression level for the plates transfected with 1, 2, 4, 6, 8, 16, and 32 µg DNA are made. HEK-293 cells were grown in DMEM 31966 with high glucose supplemented with 10% fetal calf serum, 2 mM glutamine and 0.01 mg/ml gentamicin. Cells were transfected with Lipofectamine 2000 (Life Technologies, Gaithersburg, MD).
Competition Binding Assays
Transfected COS-7 cells were transferred to culture plates 1 d after transfection at a density of 1 x 105 cells per well aiming at 5–8% binding of the radioactive ligand. Two days after transfection, competition binding experiments were performed for 3 h at 4 C using 25 pM of 125I-ghrelin (Amersham, Little Chalfont, UK). Binding assays were performed in 0.5 ml of a 50 mM HEPES buffer (pH 7.4), supplemented with 1 mM CaCl2, 5 mM MgCl2, and 0.1% (wt/vol) BSA, 40 g/ml bacitracin. Nonspecific binding was determined as the binding in the presence of 1 M of unlabeled ghrelin. Cells were washed twice in 0.5 ml of ice-cold buffer and 0.5–1 ml of lysis buffer (8 M urea, 2% Nonidet P-40 in 3 M acetic acid) was added and the bound radioactivity was counted. Determinations were made in duplicate. Initial experiments showed that steady-state binding was reached with the radioactive ligand under these conditions.
Phosphatidylinositol Turnover
One day after transfection, COS-7 cells were incubated for 24 h with 5 Ci [3H]-myo-inositol (Amersham, PT6–271) in 1 ml 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, 0.05% (wt/vol) bovine serum; and were incubated in 0.5 ml buffer supplemented with 10 mM LiCl at 37 C for 30 min. The indicated curves were furthermore incubated with adenosine deaminase ADA (200 U/mg, Roche Molecular Biochemicals, Mannheim, Germany) for 30 min in a concentration of 1 U/ml.
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 potassium hydroxide in HEPES buffer, and the generated [3H]-inositol phosphate was purified on Bio-Rad (Hercules, CA) AG 1-X8 anion-exchange resin as described. Determinations were made in duplicates.
CRE Reporter Assay
HEK-293 cells (30,000 cells/well) seeded in 96-well plates were transiently transfected with a mixture of pFA2-CREB and pFR-Luc reporter plasmid (PathDetect CREB trans- Reporting System, Stratagene) and the indicated amounts of receptor DNA. One day after transfection, cells were treated with the respective ligands in an assay volume of 100 µl medium for 5 h. When treated with the ligands cells were maintained in low serum (2.5%) throughout the experiments. The assay was terminated by washing the cells twice with PBS and addition of 100 µl luciferase assay reagent (LucLite, Packard, Meriden, CT). Luminescence was measured in a TopCounter (Top Count NXT, Packard) for 5 sec. Luminescence values are given as relative light units.
Calculations
IC50 and EC50 values were determined by nonlinear regression using the Prism 3.0 software (GraphPad Software, San Diego, CA). Values of the dissociation and inhibition constants (Kd and Ki) were estimated from competition binding experiments using the equations 
Maximal binding capacity (Bmax) values were estimated from competition binding experiments using the equation 
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ADA, Adenosine deaminase; AGRP, agouti-related peptide; CRE, cAMP-responsive element; CREB, CRE binding protein; GHS, GH secretagogue; HEK, human embryonic kidney; IP, inositol phosphate; Kd, dissociation constant; Ki, affinity constant; MC, melanocortin; NPY, neuropeptide Y; ORF-74, open reading frame 74; SP-analog/SP-a, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P; 7TM, seven transmembrane.
Received for publication March 3, 2003. Accepted for publication June 27, 2003.
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 H D. Falls, B. D Dayton, D. G Fry, C. A Ogiela, V. G Schaefer, S. Brodjian, R. M Reilly, C. A Collins, and W. Kaszubska Characterization of ghrelin receptor activity in a rat pituitary cell line RC-4B/C. J. Mol. Endocrinol., August 1, 2006; 37(1): 51 - 62. [Abstract] [Full Text] [PDF]
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C. E. Elling, T. M. Frimurer, L.-O. Gerlach, R. Jorgensen, B. Holst, and T. W. Schwartz Metal Ion Site Engineering Indicates a Global Toggle Switch Model for Seven-transmembrane Receptor Activation J. Biol. Chem., June 23, 2006; 281(25): 17337 - 17346. [Abstract] [Full Text] [PDF]
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M. M. Rosenkilde, T. Benned-Jensen, H. Andersen, P. J. Holst, T. N. Kledal, H. R. Luttichau, J. K. Larsen, J. P. Christensen, and T. W. Schwartz Molecular Pharmacological Phenotyping of EBI2: AN ORPHAN SEVEN-TRANSMEMBRANE RECEPTOR WITH CONSTITUTIVE ACTIVITY J. Biol. Chem., May 12, 2006; 281(19): 13199 - 13208. [Abstract] [Full Text] [PDF]
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 N. A. Tritos and E. G. Kokkotou The Physiology and Potential Clinical Applications of Ghrelin, a Novel Peptide Hormone Mayo Clin. Proc., May 1, 2006; 81(5): 653 - 660. [Abstract] [Full Text] [PDF]
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P J D Delhanty, B C J van der Eerden, M van der Velde, C Gauna, H A P Pols, H Jahr, H Chiba, A J van der Lely, and J P T M van Leeuwen Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a J. Endocrinol., January 1, 2006; 188(1): 37 - 47. [Abstract] [Full Text] [PDF]
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B. Holst, E. Brandt, A. Bach, A. Heding, and T. W. Schwartz Nonpeptide and Peptide Growth Hormone Secretagogues Act Both as Ghrelin Receptor Agonist and as Positive or Negative Allosteric Modulators of Ghrelin Signaling Mol. Endocrinol., September 1, 2005; 19(9): 2400 - 2411. [Abstract] [Full Text] [PDF]
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P. Zizzari, H. Halem, J. Taylor, J. Z. Dong, R. Datta, M. D. Culler, J. Epelbaum, and M. T. Bluet-Pajot Endogenous Ghrelin Regulates Episodic Growth Hormone (GH) Secretion by Amplifying GH Pulse Amplitude: Evidence from Antagonism of the GH Secretagogue-R1a Receptor Endocrinology, September 1, 2005; 146(9): 3836 - 3842. [Abstract] [Full Text] [PDF]
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A. Baessler, M. J. Hasinoff, M. Fischer, W. Reinhard, G. E. Sonnenberg, M. Olivier, J. Erdmann, H. Schunkert, A. Doering, H. J. Jacob, et al. Genetic Linkage and Association of the Growth Hormone Secretagogue Receptor (Ghrelin Receptor) Gene in Human Obesity Diabetes, January 1, 2005; 54(1): 259 - 267. [Abstract] [Full Text] [PDF]
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B. Holst, N. D. Holliday, A. Bach, C. E. Elling, H. M. Cox, and T. W. Schwartz Common Structural Basis for Constitutive Activity of the Ghrelin Receptor Family J. Biol. Chem., December 17, 2004; 279(51): 53806 - 53817. [Abstract] [Full Text] [PDF]
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R. Nogueiras, S. Tovar, S. E. Mitchell, D. V. Rayner, Z. A. Archer, C. Dieguez, and L. M. Williams Regulation of Growth Hormone Secretagogue Receptor Gene Expression in the Arcuate Nuclei of the Rat by Leptin and Ghrelin Diabetes, October 1, 2004; 53(10): 2552 - 2558. [Abstract] [Full Text] [PDF]
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J. Kamegai, H. Tamura, T. Shimizu, S. Ishii, A. Tatsuguchi, H. Sugihara, S. Oikawa, and R. D. Kineman The Role of Pituitary Ghrelin in Growth Hormone (GH) Secretion: GH-Releasing Hormone-Dependent Regulation of Pituitary Ghrelin Gene Expression and Peptide Content Endocrinology, August 1, 2004; 145(8): 3731 - 3738. [Abstract] [Full Text] [PDF]
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M. M. Rosenkilde, K. A. McLean, P. J. Holst, and T. W. Schwartz The CXC Chemokine Receptor Encoded by Herpesvirus saimiri, ECRF3, Shows Ligand-regulated Signaling through Gi, Gq, and G12/13 Proteins but Constitutive Signaling Only through Gi and G12/13 Proteins J. Biol. Chem., July 30, 2004; 279(31): 32524 - 32533. [Abstract] [Full Text] [PDF]
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S. Lall, N. Balthasar, D. Carmignac, C. Magoulas, A. Sesay, P. Houston, K. Mathers, and I. Robinson Physiological Studies of Transgenic Mice Overexpressing Growth Hormone (GH) Secretagogue Receptor 1A in GH-Releasing Hormone Neurons Endocrinology, April 1, 2004; 145(4): 1602 - 1611. [Abstract] [Full Text] [PDF]
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