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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Molecular Pharmacology Research Center, Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts
Received March 20, 2007; accepted June 26, 2007.
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Abstract |
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; Lall et al., 2004; Sun et al., 2004). Targeted disruption of the ghrelin receptor gene confirmed the link between this G protein-coupled receptor (GPCR) and metabolic function. Receptor null mice show reduced food intake, decreased body weight, improved blood glucose homeostasis, as well as resistance to diet-induced obesity (Zigman et al., 2005).
The GHSR is a seven-transmembrane domain receptor coupled to Gq-mediated activation of phospholipase C, which in turn leads to an elevation of intracellular calcium (Holst et al., 2003). In addition to the functionally intact isoform of this receptor (GHSR1a), an inactive splice variant (GHSR1b) has been reported that lacks the carboxyl-terminal portion of the receptor including transmembrane domains 6 and 7 (Kojima and Kangawa, 2005). Stimulation of GHSR1a with the orexigenic hormone, ghrelin, triggers second messenger signaling. In addition, it is well established that the ghrelin receptor shows a considerable degree of constitutive activity (i.e., basal function is greater than 50% of the ghrelin-induced maximum) (Holst et al., 2003; Holst and Schwartz, 2006). It has been postulated that both ligand-induced and basal activity of this GPCR play an important role in controlling appetite and energy expenditure (Holst and Schwartz, 2006; Pantel et al., 2006). As a result, there has been much interest in developing selective nonpeptide inverse agonist drugs to inhibit ghrelin receptor function (Holst and Schwartz, 2004). A synthetic peptide, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (SP-analog), which acts as an inverse agonist at this receptor, has been utilized to pharmacologically probe the consequences of blocking GHSR activity (Holst et al., 2003).
The pharmacological properties of GPCRs can be markedly altered by missense mutations. This variability may lead to physiologic consequences and/or have pharmacogenomic implications. Four missense mutations have been documented in either the National Center for Biotechnology Information single nucleotide polymorphism database or the literature (Holst and Schwartz, 2006; Pantel et al., 2006). These naturally occurring variants of the human GHSR1a include the I134T, V160M, A204E, and F279L substitutions. The objective of the current study was to assess the pharmacological properties of these ghrelin receptor isoforms. Our results indicate that mutations in the GHSR may affect: 1) basal activity, 2) agonist-induced receptor signaling, 3) inverse agonist function, and 4) receptor expression. Given our findings, it will be of interest to examine the extent to which naturally occurring variability in the ghrelin receptor has an impact on disease susceptibility (e.g., obesity) and/or the response to drugs targeting this GPCR.
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Materials and Methods |
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).
Construction of Human GHSR Plasmids. The wild-type human GHSR cDNA (isoform 1a) was generously provided by Dr. Michael Brownstein (National Institutes of Health, Bethesda, MD). After subcloning into pcDNA1.1, missense mutations were introduced into the cDNA using oligonucleotide-directed site-specific mutagenesis as described previously (Beinborn et al., 1993; Bläker et al., 1998). To enable assessment of receptor expression, an HA epitope (YPYDYPDYA) was introduced at the N terminus following the initiator methionine of each receptor isoform. For each mutant, the presence of only the indicated amino acid change was confirmed by full sequence analysis of each construct.
Cell Culture. Human embryonic kidney (HEK) 293 and COS-7 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA; catalog no. 12100-038 for HEK293 cells and catalog 12800-058 for COS-7 cells) supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin. The cells were maintained at 37°C in a humidified environment containing 5% CO2.
Luciferase Reporter Gene Assay. Receptor-mediated signaling was assessed using a luciferase assay as described previously (Hearn et al., 2002; Mukherjee et al., 2006; Al-Fulaij et al., 2007). In brief, HEK293 cells were plated at a density of 2000 to 3000 cells/well onto 96-well Primaria plates (BD Biosciences, Bedford, MA) and grown for 2 days to 
80% confluence. Cells were then transiently transfected using Lipofectamine reagent with 1) pcDNA1.1 or the relevant receptor plasmid and 2) an SRE-luciferase construct. Twenty-four hours following transfection, cells were stimulated for 18 h with ligand in serum-free medium. Unless otherwise noted, the final concentration of ghrelin (agonist) or substance-P analog (inverse agonist) was 1 µM. Ligand potencies were determined by stimulating receptor-expressing cells with increasing concentrations of these peptides. After ligand treatment, the cells were lysed, and luciferase activity was quantified using Luclite reagent (PerkinElmer Life and Analytical Sciences, Boston, MA). Luminescence was measured using the Topcount NXT counter (PerkinElmer Life and Analytical Sciences).
Assessment of Receptor Expression Using ELISA. The expression levels of the GHSR variants were determined using a procedure described by Shinyama et al. (2003). In brief, HEK293 cells grown in 96-well plates were transiently transfected with either pcDNA1.1 or a plasmid encoding an HA-tagged wild-type (WT) or mutant ghrelin receptor. Forty-eight hours post-transfection, cells were washed once with phosphate-buffered saline (PBS), pH 7.4, and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. For assessment of total receptor expression, cells were permeabilized with 0.1% Triton-X100 in PBS for 2 min after fixation. For the measurement of surface expression, cells were not permeabilized. After washing with 100 mM glycine in PBS, the cells were incubated for 30 min in blocking solution (PBS containing 20% bovine serum). A horseradish peroxidase-conjugated monoclonal antibody (3F10) directed against the HA-epitope (1:500 dilution in blocking solution) was then added to the cells. After 1 h, the cells were washed five times with PBS, and BM-blue solution (50 µl per well) was added. After incubation for 30 min at room temperature, conversion of this substrate by antibody-linked horseradish peroxidase was terminated by adding 2.0 M sulfuric acid (50 µl/well). Converted substrate (indicating the amount of receptor-bound antibody) was assessed by measuring light absorbance at 450 nm using a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA).
Measurement of Inositol Phosphate Production. Ligand-induced inositol phosphate (IP) formation in COS-7 cells expressing either the WT or a mutant GHSR was assayed as described previously (Beinborn et al., 1998). COS-7 cells were grown for 24 h (1,000,000 per 10-cm plate). Using the DEAE-dextran method, cells were transfected with 5 µg of either pcDNA1.1 or a receptor-encoding plasmid. Twenty-four hours post-transfection, cells were split into 24-well plates (Falcon Primaria; BD Biosciences) at a density of 200,000 cells/well. After an overnight incubation with 3 µCi/ml myo-[3H]inositol in serum-free Dulbecco's modified Eagle's medium, labeled cells were incubated with increasing concentrations of ligand in PBS containing 10 mM LiCl. After an additional 1-h incubation at 37°C, the cells were lysed and extracted with methanol/chloroform. The upper aqueous phase was analyzed for IP content by strong anion exchange chromatography. IP production was expressed as a percentage of the total cellular tritium activity that had been incorporated into the cells during overnight exposure to myo-[3H]inositol.
Data Analysis. GraphPad Prism software version 3.0 (GraphPad, San Diego, CA) was used for sigmoidal curve fitting of ligand concentration response curves. The same program was utilized to calculate half-maximal effective concentrations (EC50 values) as an index of ligand potency. Results are presented as mean ± S.E.M. GraphPad INSTAT software was used to perform statistical data analysis as specified in the figure legends.
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Results |
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GHSR Variants Show Alterations in Basal and/or Ligand-Induced Signaling. Using an SRE-luciferase reporter gene assay, we confirmed that the WT GHSR has a high level of constitutive activity (Fig. 2) as has been previously reported (Holst et al., 2003). Ligand-independent signaling of GHSR is highlighted by comparison with the WT cholecystokinin receptor type 2, a Gq-coupled GPCR that is not constitutively active (included as a negative control). The V160M, A204E, and F279L variants of the GHSR each show a significant decrease in basal activity relative to the wild-type protein. With the exception of the A204E variant that lacks constitutive activity, each of these mutants retained some degree of ligand-independent signaling. The I134T variant shows constitutive activity comparable with the WT value.
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Stimulation of the WT receptor with ghrelin (1 µM) results in a significant elevation (2-fold) in signaling relative to basal. The I134T mutant, despite its similar level of constitutive activity compared with WT, fails to respond to ghrelin stimulation. With each of the other variants, ghrelin shows significant agonist function.
In addition to agonist-induced modulation of basal activity, it is well established that the SP-analog is an efficacious inverse agonist at the WT ghrelin receptor (Holst et al., 2003). The effect of this ligand (1 µM) was explored on each of the GHSR variants. As anticipated with a receptor lacking constitutive activity, there was no effect of the SP-analog on the A204E variant. Each of the other receptors, despite variability in the level of basal signaling, showed a significant decrease in constitutive activity after treatment with the SP-analog.
GHSR Missense Mutations Show Altered Receptor Expression. Cell surface expression of the GHSR variants was compared with that of the WT receptor after HEK293 cells had been transfected with increasing concentrations of corresponding cDNAs (0–8 ng per well) (Fig. 3A). For each receptor, transfection of increasing amounts of cDNA led to an elevation in cell surface expression. When compared at equal cDNA amounts, each mutant is expressed at 40 to 60% of the corresponding WT level. To explore whether the mutation-induced alterations in expression levels correlated with observed differences in basal signaling, we performed a parallel series of experiments where constitutive activity of the GHSR variants was also assessed with increasing concentrations of transfected cDNA. Figure 3B illustrates that both the WT receptor and the I134T mutant display a similar cDNA dose-dependent increase in constitutive activity. In contrast, the A204E variant failed to show appreciable basal signaling at any amount of transfected cDNA, consistent with the premise that this variant lacks constitutive activity. The other two variants, V160M and F279L, showed a minor elevation in constitutive activity with increasing expression; corresponding levels of basal signaling were markedly lower than those of the WT receptor.
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To complement our studies of agonist induced activity using a luciferase reporter gene construct, we also assessed GHSR-mediated inositol phosphate production (Fig. 5). In response to ghrelin, the wild-type GHSR showed a concentration dependent increase in IP production (EC50 = 13.4 nM). Ghrelin potencies at the A204E and F279L variants were similar to WT. In contrast, the V160M mutant showed a markedly higher potency for ghrelin-induced IP formation (EC50 = 1.2 nM), whereas the I134T variant was unresponsive to this agonist. Comparison of ghrelin-induced IP formation at the mutant versus wild-type receptors resulted in findings that were consistent with those observed using luciferase activity as a functional readout.
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GHSR Missense Mutations Alter Inverse Agonist Potency. Further comparison of wild-type and mutant GHSR isoforms focused on the response of these receptors to an inverse agonist, the SP-analog. When assessed by luciferase reporter gene assay, this compound inhibits the basal activity of the wild-type, V160M, and F279L receptors with comparable potency (Fig. 6; Table 1). In contrast, the SP-analog has no appreciable effect on the A204E mutant, which lacks basal activity. It is noteworthy that although the constitutively active I134T variant cannot be further stimulated by ghrelin, this mutant shows a significant decrease in basal signaling when treated with the SP-analog (Fig. 6). Despite conserved inverse agonist function, both the potency and efficacy of this compound are reduced.
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In a complementary series of experiments, inverse agonist effects on wild-type and mutant GHSR were also examined using IP production as an alternative readout of receptor-mediated function. Again, mutation-induced alterations in the response to the SP-analog were consistent when assessed by either luciferase reporter gene assay (described above) or by measurement of IP production (Fig. 7). With the latter readout, mutation-induced changes in inverse agonist function at the wild-type versus mutant receptors were marked by 1) a detectable yet attenuated ability of the SP-analog to inhibit basal signaling of the I134T variant and 2) no appreciable effect of the compound on A204E activity.
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). In the current study, the effects of the SP-analog on both GHSR cell surface and total expression were examined. After exposure to 1 µM SP-analog for 18 h, surface expression of the wild-type receptor as well as the I134T and F279L variants was markedly increased as assessed by ELISA. In contrast, the SP-analog only slightly increased surface expression of the V160M variant and had no significant effect on the A204E isoform. For each of the receptor variants, total GHSR expression showed no significant change after stimulation with the SP-analog (Fig. 9).
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Immunocytochemical analysis qualitatively confirmed that cell surface receptor density of each of the mutants was reduced relative to WT (Supplemental Fig. 1). Also consistent with results obtained by ELISA, immunocytochemistry revealed a readily apparent increase in surface receptor expression of the WT, I134T, and F279L receptors after treatment with the SP-analog.
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Discussion |
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GHSR Missense Mutations Alter Receptor Expression. Each of the missense mutations in the GHSR that were characterized in this study results in a decrease in receptor expression compared with the WT value. However, these mutations do not significantly affect the ratio between total and cell surface receptor densities, suggesting that despite decreased expression, each of the variants is efficiently transported to the plasma membrane. This observation contrasts with reports on other GPCR mutants that show decreased expression. Lubrano-Berthelier et al. (2003) reported that the majority of naturally occurring MC4R mutations that result in loss of function (an alteration that is associated with childhood obesity) lead to intracellular retention of the receptor. A similar defect has been reported with the characterization of mutant human vasopressin type 2 receptors. These naturally occurring variants show defective processing to the cell membrane (Oksche and Rosenthal, 1998). The absence of ligand-induced signaling results in insufficient tubular concentration of the renal filtrate, causing nephrogenic diabetes insipidus. Although reduced surface expression of mutant ghrelin receptors occurs by a different mechanism (i.e., without intracellular receptor trapping), individuals who express these variants may also show diminished receptor-mediated function.
Basal Activity of the GHSR Variants. Our study, as well as reports by others (Holst et al., 2003), have demonstrated that the WT ghrelin receptor has a high level of ligand-independent signaling. Basal activity exceeds 50% of the ghrelin-induced maximum. Of the four mutant receptors characterized in this study, the V160M, A204E, and F279L variants show reduced or completely abolished basal function. It is possible that the observed decrease in ligand-independent signaling may, at least in part, reflect reduced expression levels of the mutant receptors. The latter mechanism, however, cannot fully explain the loss of basal activity. This is suggested by the observation that transfection of 4 ng/well mutant receptor cDNA results in expression levels of corresponding GHSR variants similar to those observed with transfection of 1 ng/well WT receptor cDNA (Fig. 3). Despite comparable expression under these experimental conditions, basal activities of the variants versus the WT receptor are markedly different. It is possible that in addition to expression levels, ligand-independent signaling of the mutant receptors is affected by a structural change that shifts the putative equilibrium between active and inactive receptor conformations (Lefkowitz et al., 1993). For the F279L mutant, a structural basis for this shift in receptor equilibrium is suggested by the observation that this amino acid substitution falls within a cluster of GHSR residues that is known to be critical for the receptor to adopt an active conformation (Holst et al., 2004). Introduction of the F279L mutation may partially disrupt this structural domain, thus contributing to reduced basal signaling. A similar mechanism may apply to the V160M and A204E mutations, although the underlying structural correlate is not yet known. Conversely, mutations in GHSR may shift the receptor toward a conformation that is more active than wild type. This scenario may apply to the I134T variant, which despite decreased expression retains constitutive activity comparable with the WT receptor. The enhanced basal function of this mutant (relative to cell surface expression) may reflect a shift toward a more active receptor state (Lefkowitz et al., 1993), thereby compensating for decreased receptor expression.
It is increasingly appreciated that constitutive GPCR activity has physiologic significance. For ghrelin receptors, the high level of basal activity is postulated to play a role in regulating both food intake and growth (Holst and Schwartz, 2006; Pantel et al., 2006). On this basis, efforts are in progress to develop drugs that inhibit GHSR basal signaling (i.e., inverse agonists) as a treatment for obesity (Holst and Schwartz, 2004). A well defined pathophysiological link between basal signaling and obesity has been established for the MC4R, another GPCR that is naturally constitutively active. Mutations that decrease ligand-independent signaling of the MC4R are the most prevalent monogenic cause of obesity (Vaisse et al., 2000; Santini et al., 2004; Srinivasan et al., 2004). Illustrating the clinical importance of basal receptor signaling, obesity has been linked to a mutation of Arg18 in the MC4R that selectively affects constitutive activity without altering either agonist function or receptor surface expression (Srinivasan et al., 2004). Based on precedent with the MC4R, it will be of interest to examine the extent to which ghrelin receptor variants with alterations in basal activity occur in selected populations (i.e., those prone to obesity or anorexia).
Alterations in Agonist Potency. In addition to affecting expression and basal activity, selected GHSR receptor mutations also altered the response to ghrelin, the endogenous agonist. The V160M mutation increased ghrelin potency by 20-fold despite a decrease in basal signaling. According to the extended ternary model of GPCR function, a mutation-induced shift toward the inactive receptor state (as suggested by reduced basal activity) typically results in decreased ligand potency. The paradoxical increase in ghrelin potency suggests that the V160M substitution may alter ghrelin interaction with the GHSR, independent of affecting basal receptor activity. This explanation is consistent with prior observations on ligand interaction with other GPCRs, which suggest that two factors (i.e., the level of basal signaling and ligand-receptor interaction) together determine ligand efficacy and potency (Beinborn et al., 2004).
Missense Mutation-Induced Alterations in Inverse Agonist Function. Our findings reveal that in addition to mutation-induced changes in agonist activity, amino acid substitutions in the ghrelin receptor can alter inverse agonist function. It has been previously shown that [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P is an inverse agonist at the WT ghrelin receptor (Holst et al., 2003, 2006). Although the SP-analog has inhibitory activity at the V160M and F279L variants that is comparable with that observed at the WT receptor, this compound only partially attenuates ligand-independent signaling of the I134T mutant. It is noteworthy that the SP-analog acts as an inverse agonist (albeit with reduced efficacy), despite the observation that this variant is unresponsive to agonist. The ligand-selective alterations in function with the I134T substitution suggest that this mutation may alter the conformation of the receptor binding pocket. Such a structural shift could eliminate the signaling properties of one compound (ghrelin) while partially conserving the function of a structurally distinct ligand (i.e., the SP-analog). Consistent with the above explanation, the I134T mutation is located on TM III, a domain that together with residues from TM VI and TM VII is known to comprise the ghrelin receptor binding pocket (Holst et al., 2006).
In addition to inhibiting basal receptor signaling, inverse agonists may also up-regulate receptor expression (MacEwan and Milligan, 1996; Shinyama et al., 2003). Different mechanisms, such as the inhibition of constitutive receptor endocytosis or stabilization of the protein, have been proposed to explain the cell surface accumulation of receptors in response to inverse agonists (Li et al., 2001). Cell-permeant nonpeptide ligands have also been shown to increase plasma membrane expression by modulating the folding of nascent receptors in the secretory pathway (Morello et al., 2000). Our data demonstrate that the SP-analog markedly increases surface expression of the WT GHSR as well as the I134T and F279L mutants. The peptidic structure of the SP-analog suggests that this ligand cannot penetrate the plasma membrane and rather acts at the cell surface to stabilize receptors (Morello et al., 2000). In addition, the observation that receptors displaying the highest levels of basal activity (WT GHSR, I134T, and F279L) respond to the SP-analog treatment with the strongest increase in surface expression suggests that their up-regulation is related to the ability of this ligand to induce a shift in the protein conformation toward an inactive state. Variants V160M and A204E, which show reduced basal activity, seem to be affected to a much lesser degree by the SP-analog. One may speculate that the SP-analog reduces constitutive receptor internalization and in addition enhances protein stability by inducing a conformational change. Regardless of the molecular mechanism underlying the ligand-induced increase in receptor surface expression, variations in this pharmacologic parameter resulting from missense mutations may alter the therapeutic response to inverse agonists acting on the GHSR.
The endogenous agonist for the GHSR is ghrelin, a well established orexigenic hormone (Kojima and Kangawa, 2005; Hosoda et al., 2006). Inverse agonists have the potential to block both ghrelin-induced signaling as well as basal receptor activity. Such compounds targeting GHSR are thus considered promising drugs for the treatment of obesity. Despite evidence supporting this strategy, it should be noted that the therapeutic consequences of altering basal activity remain difficult to predict. It is now appreciated that inverse agonists can either enhance or diminish GPCR-mediated signaling acting through two different mechanisms. Such drugs can directly attenuate basal activity while at the same time increasing receptor expression, an effect that enhances ligand-independent signaling. The net effect of an inverse agonist on the WT receptor is thus defined by an interplay of these properties. The possibility that naturally occurring GHSR mutations may alter both inverse agonist-mediated inhibition of constitutive activity as well as receptor expression adds yet another layer of complexity to the development of such drugs as a treatment for obesity.
Clinical Implications of GHSR Missense Mutations. The physiological consequences of amino acid substitutions in the GHSR are not fully understood. In a recent screen, the A204E and F279L mutations were found in children with obesity and short stature, respectively (Wang et al., 2004). Subsequently, mutation A204E was also reported in two Moroccan families with short stature (Pantel et al., 2006). These reports have raised additional speculation regarding the physiologic role of the GHSR as a determinant of height and body weight (Holst and Schwartz, 2006). Further clinical studies are needed to better define the frequency and the etiologic role of GHSR isoforms in populations with variability in these traits.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GHSR, growth hormone secretagogue receptor; GPCR, G protein-coupled receptor; SP-analog, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P; HA, hemagglutinin; BM-blue, 3.3'-5,5'-tetramethylbenzidine; SRE, serum response element; HEK, human embryonic kidney; ELISA, enzyme-linked immunosorbent assay; WT, wild type; PBS, phosphate-buffered saline; IP, inositol phosphate; TM, transmembrane domain; MC4R, melanocortin-4 receptor.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Alan S. Kopin, 750 Washington Street, P.O. Box 7703, Boston, MA 02111. E-mail: akopin{at}tufts-nemc.org
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