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Laboratory for Molecular Pharmacology (B.H., E.B., A.B., T.W.S.), Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark; and 7TM Pharma A/S (A.H., T.W.S.), 2970 Hørsholm, Denmark
Address all correspondence and requests for reprints to: Birgitte Holst, M.D., Ph.D., Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, Blegdamsvej 3, DK-2200, Copenhagen, Denmark. E-mail: b.holst{at}molpharm.dk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), which was one of the early peptides, was shown to induce GH secretion in a dose-dependent manner, independent of the GHRH receptor and without concomitant release of other pituitary hormones. Several pharmaceutical companies initiated drug discovery projects based on this peptide and its putative receptor and series of potent and efficient peptide as well as nonpeptide compounds—so-called GH secretagogues (GHS)—were consequently described up through the mid-1990s (2, 4, 5). The first series of nonpeptide GHS—to which L692,429 (Fig. 1) belongs—turned out to have low oral bioavailability. However, members of a subsequent class of compounds, spiroindanyl-piperidines, were orally active, and from this series MK-677 (Fig. 1) was later used in clinical trials (6, 7). In the late 1990s, the receptor was cloned and eventually the endogenous ligand for this receptor, ghrelin, was identified as a 28-amino acid octanylated peptide (Fig. 1). Surprisingly, ghrelin was found to be expressed in especially large amounts in endocrine cells in the stomach, and it was soon realized that besides its function as a modulator of GH release, ghrelin mainly seems to function as a hunger signal from the gastrointestinal tract to the hypothalamus (8, 9). This has revitalized the interest in the ghrelin receptor—previously called the GHS receptor—and agonists for this receptor are now pursued especially as potential agents for the treatment of cachexia (10, 11, 12) and disorders in gastrointestinal tract motility (13), whereas antagonists (14, 15) or perhaps especially inverse agonists have been suggested as antiobesity agents (16).
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In the present study, we have characterized a series of classical nonpeptide—L692,429 and MK-677—as well as peptide agonists—GHRP-6 and ghrelin—in respect of their ability to stimulate a series of the various signal transduction pathways presented above through the ghrelin receptor (Fig. 1
). Furthermore, based on the assumption that in an in vivo, clinical setting agonist compounds will be acting in conjunction with the natural ligand on the receptor, we have tested the effect of the nonendogenous ghrelin receptor agonists on the ability of ghrelin to stimulate its receptor. It was found that the classical GHS compounds have different molecular pharmacological properties on their own as agonists and that they influence both the potency and efficacy of ghrelin signaling surprisingly different. That is, the nonendogenous ghrelin receptor agonists can act both as positive and as negative allosteric modulators of ghrelin signaling. It is proposed that a compound, which acts both as agonists and as positive allosteric modulators of the signaling of the endogenous ligand, ghrelin could be the optimal type of compound to be used in the clinic.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). In contrast, GHRP-6 competed for ghrelin binding in an apparent multicomponent manner with a dissociation constant of 260 nM and a Hill slope of –0.6. L692,429, which is structurally related to MK-677 (Fig. 1), was almost unable to compete for ghrelin binding because concentrations above 10 µM of this nonpeptide compound were required to displace 125I-ghrelin.
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). This was particularly surprising because the nonpeptide agonist MK-677 stimulated calcium mobilization with a high potency of 1.4 nM, which is slightly higher than the affinity measured for this nonpeptide compound in the binding experiments. In respect of calcium mobilization, also GHRP-6 and L692,429 showed potencies, which were superior to that of ghrelin, i.e. EC50 values of 4.5 nM and 26 nM, respectively (Fig. 3A and Table 1). Thus, the calcium dose-response curves for both GHRP-6 and especially for L692,429 were shifted several orders of magnitude to the left as compared with their dose-response curves for competition against 125I-ghrelin (Figs. 2 and 3A)
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and Table 1). Also, GHRP-6 functioned as a high-potency agonist—EC50 = 0.83 nM—in respect of IP production. In fact, GHRP-6 functioned as a super-agonist because it stimulated IP production up to a level, which was 150% of the maximal level reached by the endogenous agonist, ghrelin, and by MK-677 (Fig. 3B). L692,429 functioned as a normal full agonist in respect of stimulation of IP turnover and did so with a potency of 47 nM, which is similar to the potency observed for this compound in calcium mobilization. Thus, whereas the dose-response curves for IP mobilization were relatively similar to the dose-response curves for calcium mobilization for the nonendogenous ligands—MK-677, L692.429, and GHRP-6—the endogenous agonist ghrelin stimulated IP turnover with a potency that was approximately 100-fold better than its potency in mobilizing intracellular calcium (Fig. 3, A and B; and Table 1).
CRE Binding Protein (CREB) Controlled Transcriptional Activity
The ghrelin receptor signals mainly through the Gq pathway; nevertheless, it also activates the transcription factor CREB conceivably through kinases such as Ca2+/calmodulin kinase IV and protein kinase C (18, 20, 21). As monitored by a reporter assay using CRE-driven luciferase activity in transiently transfected human embryonic kidney (HEK)-293 cells, ghrelin and MK-677 stimulated CREB activity with similar high potencies as observed in the IP turnover assays for these compounds, i.e. with EC50 values of 0.58 nM and 0.19 nM, respectively (Fig. 3C). Interestingly, GHRP-6 stimulated the CRE pathway with an even 7.5-fold higher potency than it stimulated IP production—EC50 being 0.11 nM (Table 1). Thus, in respect of stimulating the CRE pathway ghrelin, MK-677 and GHRP-6 were very similar both in respect of relatively high potency and efficacy. In contrast, but as observed in the other assays, L692,429 was both much less potent (EC50 = 61 nM) than the other three compounds, and in respect of stimulation of CREB activity this nonpeptide compound was even just a 50% partial agonist (Fig. 3C).
SRE Controlled Transcriptional Activity
Previously, we have found that the ghrelin receptor stimulates SRE-mediated transcriptional activity mainly through a G
13 pathway (19) (and our unpublished data). As monitored by a reporter assay using SRE-driven luciferase activity in transiently transfected HEK-293 cells, the endogenous ligand, ghrelin displayed around 10-fold reduced potency—EC50 = 2.6 nM—as compared with that measured in IP production and CRE signaling (Fig. 3D and Table 1). As a consequence, MK-677 was clearly the most potent agonist in the SRE transcriptional pathway with a high potency similar to that observed in the other signal transduction assays—EC50 = 0.27 nM. Moreover, MK-677 consistently acted as a super-agonist through the SRE pathway with a maximum efficacy (Emax) of approximately 125% of that of ghrelin. GHRP-6, which was almost equipotent as compared with ghrelin and MK-677 in respect of stimulating IP production and activating the CRE pathway, surprisingly acted as a relatively low potency agonist through the SRE pathway (EC50 = 63 nM) and with a Hill coefficient of 0.65 (Fig. 3D). Also in the SRE assay, L692,429 acted with similar low potency as observed in the other functional assays and—as observed in the CREB assay—L692,429 was a partial agonist (62%) also in the SRE assay.
Arrestin Mobilization
Receptor binding of arrestin, which blocks G protein signaling but often linked to activation of various kinase cascades (22), is a phenomenon mediated by agonist binding and receptor activation in the vast majority of 7TM receptors—independently on their G protein signaling pathway (23). To directly monitor agonist-mediated association between the ghrelin receptor and arrestin, we used bioluminescence resonance energy transfer (BRET) between Renilla luciferase-labeled arrestin stably transfected into HEK-293 cells and transiently transfected green fluorescent protein (GFP)-labeled ghrelin receptor (23). In respect of arrestin translocation, ghrelin and MK-677 acted as almost equipotent, subnanomolar agonists with EC50 values of 0.32 nM and 0.45 nM, respectively (Fig. 3E and Table 1), with a tendency for MK-677 to reach a higher maximal efficacy than ghrelin as also observed in the SRE assay. As in the IP accumulation assay, the dose-response curve for GHRP-6 in respect of arrestin mobilization was only shifted approximately 5-fold to the right compared with ghrelin and MK-677, i.e. with an EC50 value of 1.3 nM. Also L-692,429 displayed a similar low potency (EC50 = 58 nM) in the arrestin translocation assay as observed in all the other signal transduction assays.
Modulation of Ghrelin Signaling by Nonendogenous Ligands
The fact that L-692,429 signals with a potency that is around 1000-fold higher than the apparent affinity with which it competes for ghrelin binding, indicates that this nonpeptide compound could be an allosteric modulator of ghrelin function (Table 1). Thus, the effect of L-692,429—or MK-677 as a control—on ghrelin-mediated signal transduction was monitored in a Schild-type analysis. As shown in Fig. 4, L-692,429, acted as a positive modulator or enhancer of ghrelin signaling, as doses of 10–7.5 M and 10–7 M of L-692,429, which stimulated the basal IP production up to 15 and 70% of the maximal ghrelin response, shifted the dose-response curve for ghrelin approximately 2- and 4-fold to the left, respectively (Fig. 4, A and B). Moreover, an additive effect was observed as a higher maximal efficacy for ghrelin in the presence of the L-692,429 than in the absence (Fig. 4). MK-677 on the other hand, in doses of 10–9.5 M and 10–9 M, which increased basal IP production, up to 35% and 75% of the maximal response to ghrelin did not affect the potency of ghrelin because the dose-response curves for ghrelin gave EC50 values similar to that observed in the absence of MK-677 (Fig. 4, C and D). However, as observed with L-692,429 the maximal ghrelin-induced IP response was increased to 119% and 144% in the presence of 10–9.5 and 10–9 M MK-677, respectively (Fig. 4D). Similar patterns of effects—with L-692,429 being a positive modulator and MK-677 being neutral—was observed for the ghrelin-stimulated SRE activity, but with a more pronounced effect of L-692,429 (Fig. 5). Thus, L-692,429 in concentrations of 10–7.5 M and 10–7 M shifted the ghrelin dose-response curves 3- and 10-fold to the left, respectively. Also in this case, an additive effect was observed with both of the nonpeptide compounds and ghrelin (Fig. 5).
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). Nevertheless, as was the case both for MK-677, which did not shift the ghrelin dose-response curves, and for L-692,429, which acted as a positive modulator, an additive effect was observed also for GHRP-6 because the maximal ghrelin-induced IP response was increased to 160% in the presence of GHRP-6—despite the fact that it acted as a negative modulator of ghrelin action (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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MK-677
MK-677 is a nonpeptide compound that acts as a high-potency, full agonist in all signal transduction pathways studied. MK-677 has the most simple agonist phenotype because it displays very similar potencies (from 0.19–1.4 nM) in all the signaling pathways, which we monitored. Furthermore, the potency of MK-677 is within one order of magnitude of its apparent affinity measured in competition against radiolabeled ghrelin, indicating that it is a classical competitive agonist (Table 1). Accordingly, MK-677 acts neither as a positive nor as a negative modulator of ghrelin activity in respect of effects on the potency of ghrelin. However, coadministration of MK-677 did increase the Emax of the endogenous ligand as monitored for example in the IP accumulation assay (see below concerning molecular mechanisms of action).
L-692,429
L-692,429, the other nonpeptide compound tested in the present study, acts as a relatively low potency agonist on the ghrelin receptor in all signal transduction pathways tested and—like MK-677—L-692,429 displays an almost identical potency in all these assays (from 26–63 nM). However, surprisingly, L-692,429 functions as an allosteric enhancer or positive modulator of the signaling of the endogenous agonist ghrelin. The allosteric binding mode is suggested by the fact that L-692,429 competes for ghrelin binding with an apparent affinity, which is approximately 1000-fold lower than its potency measured in the various signal transduction assays. And the enhancer function of L-692,429 is illustrated by the fact that the compound in concentrations corresponding to those which in themselves stimulate signaling, shifts the dose-response curves for ghrelin to the left (Figs. 4 and 5). As observed with MK-677, L-692,429 also has an additive effect when administered together with ghrelin as higher maximal levels of signal transduction are reached.
GHRP-6
GHRP-6 is a classical GHS oligo-peptide. This hexa-peptide has the most complex mode of action of the compounds tested in the present study because its potency varies around 600-fold depending on the signal transduction assay. In the IP accumulation and calcium mobilization assays, which are just downstream of Gq, as well as in arrestin mobilization the EC50 for GHRP-6 only varies between 0.8 and 4.5 nM; however, in CREB-mediated transcriptional activity, the EC50 for GHRP-6 is 0.1 nM, whereas in SRE-mediated transcriptional activity it is 61 nM (Table 1). It should be noted that this cannot easily be explained by trivial differences in cellular context or inherent differences in potencies in different types of assays, as the potencies of for example MK-677 and L-692,429 are very similar across assays (Table 1). Just like L-692,429, GHRP-6 appears to have an allosteric mode of action because it competes for ghrelin binding with an apparent affinity that is 500- to 1000-fold lower than its highest potency measured in the CRE signaling assay. However, as opposed to L-692,429, GHRP-6 does not act as an enhancer but rather as a negative modulator of ghrelin signaling, at least as measured in the IP accumulation assay (Fig. 6). However, despite the fact that GHRP-6 shifts the dose-response curves for ghrelin to the right, it has—like the other GHS compounds—an additive effect on the efficacy of the ghrelin signaling.
Positive and Negative Allosteric Modulation of Ghrelin Signaling
Among 7TM receptors allosteric modulators have previously only been characterized for relatively few receptor systems such as the adrenergic, muscarinic, adenosine, and melanocortin receptors from class-I, rhodopsin-like receptors (24, 25, 26, 27, 28) and for the calcium sensors and the glutaminergic and 
-amino butyric acid (GABA)B receptors from class-III (29, 30, 31). An allosteric modulator is in principle able to bind to the receptor at the same time as the natural agonist and consequently it does not—or only very poorly—compete for binding against the natural ligand. Instead, the allosteric modulator either increases or decreases the sensitivity of the receptor for the natural ligand, i.e. it displays either positive or negative cooperativity. In the muscarinic receptor system, enhancers have been characterized in great detail and for example shown to improve the potency of agonists usually 2- to 3-fold as measured in IP turnover, calcium mobilization, or [35S]-GTP-sulfur binding (25, 26, 32). These compounds have no agonist activity by themselves on the muscarinic receptor, and they do not affect the maximally achievable response to the endogenous ligand (25). However, for both the melanocortin receptors and for the GABAB receptor, positive allosteric modulators have recently been described, which function also as partial agonists and—importantly—coadministration of such compounds results in an increased maximal signaling response for the natural ligand (28, 29). On the ghrelin receptor, L-692,429 appears to belong to this class of ligands—i.e. being both an agonist and a positive modulator. Although L-692,429 is a partial agonist in the reporter assays for SRE- and CRE-induced transcription, it appears to be a full agonist in respect of IP turnover and arrestin mobilization. Because it increases the potency of ghrelin 4- and 10-fold, L-692,429 acts as a positive allosteric modulator both in stimulation of Gq-mediated IP turnover and in the presumably G12/13-mediated SRE transcription. In both coupling assays, L-692,429 increases the maximal response to ghrelin while shifting its dose-response curve to the left—most pronounced in the SRE assay (Figs. 4 and 5). The effects we observe in the present study for L-692,429 on the ghrelin receptor in respect of leftwards shifts in potency as well as increase in maximal response for the natural ligand, are strikingly similar in magnitude, etc. to those we previously have observed for Zn(II) in the melanocortin receptors (Fig. 5 in Ref.28) and to those observed by Pin and co-workers for CGP7930 on the GABAB receptor (Fig. 2 in Ref.29)—except that L-692,429 is approximately 100-fold more potent than any of those compounds. Classically, enhancers or positive modulators have been considered not to be agonists by themselves and the enhancers are normally believed only to shift the potency of the natural ligand, not to increase the maximal efficacy of the receptor (25, 34). This concept is clearly changing because more and more examples of positive modulators having such properties are being reported (Figs. 4 and 5) (28, 29).
Molecular Mechanism of Action of Ghrelin Receptor Agonists and Allosteric Modulators
It has—for many reasons—generally been believed that positive modulators acted on the same receptor molecule as the natural ligand. Accordingly, it was not part of the theoretical concept that such compounds could be agonist by themselves—because they were supposed to bind at a site distant from the agonist site of the natural ligand. However, today there is very strong evidence in favor of the notion that especially class-III 7TM receptors function not as single receptor units, but as dimers (35, 36). In the case of the GABAB receptor, the endogenous ligand GABA binds in the so-called R1 subunit, whereas no endogenous ligand is believed to bind in the R2 subunit, which, however, is required for the R1 subunit to efficiently reach the cell surface and for the signaling of the heterodimeric receptor complex. Pin and co-workers (29) have very recently provided evidence that the allosteric enhancer, CGP7930, acts both as a partial agonist and as a positive modulator of GABA signaling through binding not in the subunit to which GABA binds, but instead through binding in the seven helical domain of the R2 subunit. Although the evidence is not as strong as for class III receptors, rhodopsin-like receptors are also believed to function as dimers (37). In these cases, it is still unclear whether a homodimeric receptor normally binds a single molecule of endogenous ligand—in analogy with the heterodimeric GABAB receptor—or whether both subunits are occupied during activation. There is, however, in rhodopsin as such strong evidence that only a single receptor molecule needs to be activated—i.e. by a single photon—to activate signal transduction (38). Moreover, Pin and co-workers (39) have clearly demonstrated in the metabotropic glutamate receptors—i.e. class-III receptors that form homodimers—that occupancy of one receptor subunit is sufficient to activate the receptor but that occupancy of both subunits with agonist will increase the maximal signaling efficacy.
Thus, a possible model for the ghrelin receptor could be that it functions as a homodimer, where ghrelin binding normally occurs only in one subunit for example due to negative cooperativity preventing another 28-amino acid ghrelin molecule from binding in the other subunit (Fig. 7A). However, the structural basis for the negative cooperativity, which prevents two normal ghrelin molecules from binding at the same time, may not prevent a small nonpeptide agonist from binding in the other subunit. Initial mutational analysis and subsequent molecular models of the pharmacophores suggest that classical nonpeptide agonists interact with residues in the main ligand-binding pocket of the ghrelin receptor that very likely also are interaction points for ghrelin—making it difficult for both types of ligands to bind to a single receptor molecule at the same time (40, 41). In analogy with the observations in the metabotropic glutamate receptor (39), occupancy of both receptor subunits should result in a signaling efficacy above what is observed with occupancy of only one subunit (Fig. 7B). The small nonpeptide compound may then for example exert a positive cooperative effect on the subunit in which ghrelin is bound—as in the case of L-692,429 (Fig. 7C, left panel)—i.e. shifting the dose-response curve for ghrelin to the left (Figs. 4 and 5). Another possibility is that the small nonpeptide agonist that is allowed to bind in the other receptor subunit may not cause any positive cooperative effect—as is the case for MK-677 (Fig. 7C, middle panel)—but through its occupancy of the other receptor subunit still will provide an increase in the maximal signaling capacity (Figs. 4 and 5). In such a receptor model, the hexapeptide agonist, GHRP-6 will like the nonpeptide compounds be allowed to bind, but—perhaps due to its larger size—it may exert a negative cooperative effect on ghrelin (Fig. 7, right panel), as observed in the shift to the right in ghrelin dose-response curves (Fig. 6). However, like the other small molecule agonists through occupancy of the other receptor subunit, GHRP-6 will still provide an increased maximal signaling efficacy. In such a model, when ghrelin is not present the small molecule agonists will be able to bind in both receptor subunits and under certain circumstances function as super-agonists by themselves. This was, in fact, observed for MK-677 and GHRP-6 in several cases, although the double, theoretical maximal signaling efficacy was not reached (Fig. 3).
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Clinical Significance of the Molecular Properties of Nonendogenous Ghrelin Receptor Agonists?
A number of nonpeptide agonists for the ghrelin receptor have over the years been developed initially for the treatment of dwarfism but later for treatment of frail elderly to improve functional capacity and recently for treatment of cachexia, for example cancer cachexia (10, 12). Although some of the desired beneficial effects often have been observed in the clinical trials, these compounds have in general not met the end points (6, 43). In the present study, we have found that small molecule ghrelin receptor agonists may at the same time function either as positive (L-692,429) or as negative (GHRP-6) allosteric modulators of the endogenous ghrelin signaling—or they may be neutral in this respect (MK-677). It would appear obvious that a compound that is both an agonist and that potentiates the action of the endogenous ligand, ghrelin would be superior in the in vivo clinical setting. L-692,429, which was one of the initially discovered ghrelin receptor agonists, in fact has both of these properties—but it is a relatively low potency compound with poor oral bioavailability. In the 1990s, L-692,429 served as one of the main leads for the development of subsequent generations of drug candidates such as MK-677 (7). However, in this process the aim was mainly to obtain simple receptor activation as monitored by, for example calcium mobilization. On the basis of the present study, it could now be argued—for agonists in general—that the property of enhancing the potency and efficacy of the endogenous ligand should perhaps be used as a additional, alternative parameters during the drug discovery process to obtain high-potency compounds having also this property, which obviously are particularly important in the in vivo setting, where the endogenous agonist also is present.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 with 40 µg/175 cm2 ghrelin receptor cDNA in pcDNA3 using calcium phosphate precipitation method with chloroquine addition as previously described (44). HEK-293 cells were grown in D-MEM, 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 (Invitrogen Life Technologies, Carlsbad, CA).
Competition Binding Assays
Transfected COS-7 cells were transferred to culture plates 1 d after transfection at a density of 35 x 103 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 µg/ml bacitracin. Nonspecific binding was determined as the binding in the presence of 1 µM 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.
Calcium Mobilization
The assay was performed as previously described in more details (33). The day before the assay, cells were seeded in 96-well plates at 30,000 cells/well (100 µl). Cells were grown overnight in a humidified incubator at 37 C and 5% CO2. On the day of the experiment, the cells were removed from the incubator and 100 µl loading buffer, containing the calcium sensitive flourophore, was added (FLIPR calcium assay kit was purchased from Molecular Devices Corp., Sunnyvale, CA). The cells were then incubated for 1 h at 37 C and 1 h at 25 C before use. The loaded cells were placed in the cell plate position in the NovoStar [purchased from BMG Labtechnologies (Offenburg, Germany)], and the sample plate was placed in the sample plate position. The assay was done at room temperature using the following parameters: Excitation filter is 485 nM and emission filter is 520 nM. Injection speed for a 20-µl injection, using a fixed dispenser the injection speed, was 420 µl/sec.
Phosphatidylinositol Turnover
One day after transfection COS-7 cells were incubated for 24 h with 5 µCi of [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. 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 (Hercules, CA) AG 1-X8 anion-exchange resin as described. Determinations were made in duplicates.
CRE and SRE Reporter Assay
HEK-293 cells (30,000 cells/well) seeded in 96-well plates were transiently transfected. In case of the CRE reporter assay, the cells were transfected with a mixture of pFA2-CREB and pFR-Luc reporter plasmid [PathDetect CREB trans-Reporting System; Stratagene (La Jolla, CA)]. or SRE-Luc (PathDetect SRE Cis-Reporting System; 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 medium for 5 h. The assay was terminated by washing the cells twice with PBS and addition of 100 µl luciferase assay reagent (LucLite; Packard Bioscience, Johannesburg, South Africa). Luminescence was measured in a TopCounter (Top Count NXT, Packard) for 5 sec. Luminescence values are given as relative light units.
Arrestin Translocation
Human ß-arrestin2 N-terminally tagged with GFP2 (GFP2/ß-arr2) and a GFP2-Rluc construct was purchased from PerkinElmer (Boston, MA). The C-terminally tagged Rluc-receptor construct ghrelin receptor/Rluc was made using standard molecular biology techniques. Mutations Arg393,395
Glu were introduced individually in human GFP2/ß-arr2 using QuikChange site-directed mutagenesis kit from Stratagene. A HEK-293 cell line stably expressing the Arg393,395 Glu ß-arr2 mutant was established using standard cell culture techniques. Transient transfection was performed in 70% confluent 175-cm2 flasks using 7.5 µg ghrelin receptor/Rluc DNA and Lipofectamine. Using trypsin, cells were harvested 48 h after transfection and then washed twice in PBS. Cells were resuspended in Dulbecco’s PBS supplemented with glucose to a density 1 x 106 cells/ml. All the tissue culture media and reagents were purchased from GIBCO Invitrogen Corp. (Breda, The Netherlands), unless otherwise specified. The BRET2 assay has previously been described (12). Briefly; the assay was run using the Mithras LB 940 plate reader equipped with four injectors (Berthold Technologies, Bad Woldbad, Germany). After harvesting, 180 µl of resuspended cells containing approximately 200,000 cells were distributed to wells in 96-well microplates (white Optiplate; PerkinElmer) together with 10 µl agonist and incubated for 5 min. The plate was then transferred to the Mithras LB 940, where injector 1 then inject 10 µl of the substrate DeepBlueC (final concentration 5 mM; PerkinElmer) to each well. Readings were collected 2 sec after injections. The signals detected at 395 nm and 515 nm were measured sequentially, and the 515/395 ratios calculated and expressed as a BRET ratio x 1000.
Calculations
IC50 and EC50 values were determined by nonlinear regression using the Prism 3.0 software (GraphPad Software, San Diego). Values of the dissociation and inhibition constants (Kd and Ki) were estimated from competition binding experiments using the equations Kd = IC50 – L and Ki = IC50/(-1 + L/Kd), where L is the concentration of radioactive ligand.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online May 19, 2005
Abbreviations: BRET, Bioluminescence resonance energy transfer; CRE, cAMP-responsive element; CREB, CRE binding protein; Emax, maximum efficacy; GABA, -amino butyric acid; GFP, green fluorescent protein; GHRP, GH-releasing peptide; GHS, GH secretagogues; HEK, human embryonic kidney; IP, inositol phosphate; SRE, serum-responsive element; 7TM, seven transmembrane.
Received for publication January 25, 2005. Accepted for publication May 9, 2005.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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G. L. Fraser, H. R. Hoveyda, and G. S. Tannenbaum Pharmacological Demarcation of the Growth Hormone, Gut Motility and Feeding Effects of Ghrelin Using a Novel Ghrelin Receptor Agonist Endocrinology, December 1, 2008; 149(12): 6280 - 6288. [Abstract] [Full Text] [PDF]
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 P. C. Jensen, S. Thiele, T. Ulven, T. W. Schwartz, and M. M. Rosenkilde Positive Versus Negative Modulation of Different Endogenous Chemokines for CC-chemokine Receptor 1 by Small Molecule Agonists through Allosteric Versus Orthosteric Binding J. Biol. Chem., August 22, 2008; 283(34): 23121 - 23128. [Abstract] [Full Text] [PDF]
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J.-N. Ma, H. H. Schiffer, A. E. Knapp, J. Wang, K. K. Wong, E. A. Currier, M. Owens, N. R. Nash, L. R. Gardell, M. R. Brann, et al. Identification of the Atypical L-Type Ca2+ Channel Blocker Diltiazem and Its Metabolites As Ghrelin Receptor Agonists Mol. Pharmacol., August 1, 2007; 72(2): 380 - 386. [Abstract] [Full Text] [PDF]
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B. Holst, J. Mokrosinski, M. Lang, E. Brandt, R. Nygaard, T. M. Frimurer, A. G. Beck-Sickinger, and T. W. Schwartz Identification of an Efficacy Switch Region in the Ghrelin Receptor Responsible for Interchange between Agonism and Inverse Agonism J. Biol. Chem., May 25, 2007; 282(21): 15799 - 15811. [Abstract] [Full Text] [PDF]
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B. Holst, M. Lang, E. Brandt, A. Bach, A. Howard, T. M. Frimurer, A. Beck-Sickinger, and T. W. Schwartz Ghrelin Receptor Inverse Agonists: Identification of an Active Peptide Core and Its Interaction Epitopes on the Receptor Mol. Pharmacol., September 1, 2006; 70(3): 936 - 946. [Abstract] [Full Text] [PDF]
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 R M Luque, J R Peinado, F Gracia-Navarro, F Broglio, E Ghigo, R D Kineman, M M Malagon, and J P Castano Cortistatin mimics somatostatin by inducing a dual, dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotropes. J. Mol. Endocrinol., June 1, 2006; 36(3): 547 - 556. [Abstract] [Full Text] [PDF]
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