Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The melanocortin receptors are members of the seven transmembrane (TM) G protein-coupled receptor family. There are five melanocortin receptor subtypes cloned to date, MC1 through MC5, all of which couple to Gs to stimulate adenylate cyclase. These receptors are activated by a group of peptides derived from post-translational processing of the pro-opiomelanocortin gene transcript. These peptides include -melanocyte-stimulating hormone (-MSH) and adrenocorticotropin hormone (ACTH), both of which contain the common amino acid sequence His6-Phe7-Arg8-Trp9 (Sahm et al., 1994; Haskell-Luevano et al., 1997). Melanocortin receptor activation is modulated by two endogenous antagonists/inverse agonists, agouti and agouti-related protein (AGRP) (Lu et al., 1994; Ollmann et al., 1997). AGRP is 132 amino acids in length but is thought to mimic the binding of the much smaller 13-amino acid agonist -MSH through an Arg-Phe-Phe motif. This motif is found in the C-terminal half of the protein, and commonly, a truncated portion of the protein AGRP(87-132) is used in characterization studies. AGRP(87-132) exhibits the same melanocortin receptor binding selectivity as full-length AGRP and has been reported to act as a competitive antagonist (Yang et al., 1999).

Recently, there has been great interest in the MC4 receptor, as it has been reported to be important in the regulation of feeding. MC4/ mice exhibit hyperphagia and accelerated weight gain (Huszar et al., 1997). Ectopically expressed AGRP results in an obese phenotype (Butler et al., 2001). Furthermore, the MC4 receptor agonist MTII inhibits feeding in both mice models of hyperphagia and fasted mice (Fan et al., 1997). It was also demonstrated that this effect could be blocked by the antagonist SHU9119, which when given alone was able to enhance nocturnal feeding. However, both MC3 and MC4 receptors are expressed at high levels in the brain, and MC3 receptor action in the regulation of food intake is unclear. MC3 receptor knockout mice exhibit increased fat mass but are not significantly overweight (Butler et al., 2000; Chen et al., 2000). This indicates that the MC3 and MC4 receptors serve different roles in the regulation of energy homeostasis. Potential pharmaceutical benefit can be reaped from development of ligands that can discriminate between MC3 and MC4 receptors, and therefore, it is important to identify the molecular determinants of the receptor that enable MC4/MC3 receptor discrimination. A number of MC4-selective ligands have been developed, including the agonist peptide HfRWK (Bednarek et al., 1999), the antagonist peptide M10 (Bednarek et al., 2001), and the nonpeptide agonist tetrahydroisoquinoline (THIQ) (Van der Ploeg et al., 2002) (Table 1). Conversely, -MSH and a modification of the peptide trp9 show some selectivity for the MC3 receptor over the MC4 receptor.

Earlier mutagenesis studies have already identified a number of important residues involved in ligand binding to the MC4 receptor (Yang et al., 2000; Haskell-Luevano et al., 2001). These include an ionic interaction between melanocortin peptide residue Arg8 and aspartate residues 122 and 126 in TM3 and a hydrophobic interaction between melanocortin peptide residue Phe7 with aromatic residue Phe261 in TM6. A further phenylalanine residue in TM4 of the mouse MC4 receptor has also been proposed to interact with melanocortin peptide Phe7 residue (Haskell-Luevano et al., 2001). However, the binding pocket of the melanocortin peptides and the endogenous antagonists is still not clearly defined.

In this study, we have taken three approaches to further define the molecular determinants of the MC4 receptor involved in ligand interaction. In the first approach, we have used molecular modeling to identify candidate residues of the receptor involved in ligand binding. Using this method, we identified Phe284 as a potential ligand binding site. Second, given the potential importance of the MC4 receptor in treating obesity, we have fully characterized the effect of a mutation in the receptor (I137T) reported to directly lead to obesity (Gu et al., 1999). Finally, using sequence alignment and knowledge of the binding site, we have identified a residue that may account for some of the differences in the MC4 and MC3 receptor binding sites. In all cases, a large array of ligands have been used to define the effect of the mutation, allowing inferences in regard to effects on specific ligands and effects on specific residues, regions, or physical properties of the ligands.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Radiolabeled [¹²⁵I]NDP-MSH, [¹²⁵I]AGRP(87-132), and [¹²⁵I]SHU9119 were obtained from PerkinElmer Life Sciences (Boston, MA). -MSH, NDP-MSH, ACTH, and SHU9119 were obtained from Peninsula Laboratories (Belmont, CA). -MSH, MTII, and AGRP(83-132) were obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA). Peptides, M10 (Bednarek et al., 2001), HfRWK (Bednarek et al., 1999), and trp9 were synthesized by solid-phase methodology on a Beckman Coulter 990 peptide synthesizer (Fullerton, CA) using t-N-tert-butoxycarbonyl-protected amino acids, and the assembled peptide was deprotected with hydrogen fluoride. The crude peptide products were purified by preparative high-performance liquid chromatography. All primers were synthesized and desalted by Invitrogen (Carlsbad, CA). The nonpeptide agonist THIQ (Van der Ploeg et al., 2002) was synthesized by the Chemistry Department at Neurocrine. All other reagents were of the highest quality available.

Creation of MC4 and MC3 Receptor-Stable Cell Lines. HEK-293 cells were maintained in DMEM with 10% fetal calf serum. Wild-type receptor constructs in pcDNA3.1 were transfected using LipofectAMINE (Invitrogen). Stable receptor populations were generated using G418 (Invitrogen) selection (1 mg/ml).

Receptor Mutagenesis. Human MC4 or MC3 receptor cDNAs in pcDNA3.1 were used as a template for mutagenesis. Mutants were prepared using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). PCR reactions (95°C, 1 min; 52°C, 1 min; and 72°C, 16 min) were performed using Pfu polymerase and a complementary set of primers encoding the nucleotide mutation(s), resulting in the desired amino acid residue change. Template DNA was nicked with DpnI, and mutant DNA was subcloned into competent TOP10 cells. Clones were sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA), and clones containing the desired mutation were subcloned into the EcoRI/XhoI sites of a fresh pcDNA3.1 vector. Complete mutant MC4 receptor sequences were confirmed by DNA sequencing.

Transient Transfection. HEK-293 cells were maintained in DMEM with 10% fetal calf serum. Twenty-four hours prior to transfection, cells were seeded at 2 × 10⁶ cells/100-mm dish. Eight micrograms of mutant DNA were transfected using Polyfect (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Cells were either harvested or assayed for cAMP accumulation after 48 h.

Membrane Preparation. Cells were washed once with phosphate-buffered saline. Then, 5 ml of ice-cold buffer (20 mM HEPES, 1 mM MgCl₂, and 1 mM EDTA, pH 7.4) was used to disrupt the cell monolayer in each 100-mm dish. This was transferred to a Dounce glass/glass homogenizer. The cells were homogenized at 4°C by 40 strokes of the Dounce homogenizer. The homogenate was centrifuged for 10 min at 1,700g, and the supernatant was collected and centrifuged at 48,000g (4°C) for 1 h. The resulting pellet was resuspended in buffer, and aliquots were stored at 80°C. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA) using BSA as standard.

Iodinated Radioligand Saturation Binding Assays. Cell membranes (5 µg of protein) were incubated in duplicate with either [¹²⁵I]SHU9119, [¹²⁵I]AGRP(87-132), or [¹²⁵I]NDP-MSH at concentrations ranging from 0.001 to 5 nM [[¹²⁵I]SHU9119 and [¹²⁵I]AGRP(87-132)] or 0.005 to 15 nM ([¹²⁵I]NDP-MSH), in a total volume of 100 µl of buffer [25 mM HEPES, 1 mM MgCl₂, 2.5 mM CaCl₂, 0.5% BSA, and 1% Bacitracin (1 complete EDTA-free protease inhibitor tablet/50 ml; Roche Diagnostics, Indianapolis, IN), pH 7.0]. Nonspecific binding was determined by the inclusion of 1 µM SHU9119. The reaction was initiated by the addition of membranes, and the plates were incubated at 25°C for 2 h. The reaction was terminated by rapid filtration using a vacuum harvester with five 100-µl washes of ice-cold wash buffer (25 mM HEPES, 1 mM MgCl₂, 2.5 mM CaCl₂, 0.1% BSA, and 500 mM NaCl, pH 7.0). The filters were soaked in 50 µl of scintillation fluid, and the amount of radioactivity present was determined by liquid scintillation counting.

Ligand Competition versus [¹²⁵I]SHU9119, [¹²⁵I]AGRP(87-132), and [¹²⁵I]NDP-MSH Binding. Cell membranes (2-10 µg of protein) were incubated with 0.3 nM iodinated radioligand and various concentrations of competitor ligand, in duplicate, in a total volume of 100 µl of buffer [25 mM HEPES, 1 mM MgCl₂, 2.5 mM CaCl₂, 0.5% BSA, and 1% Bacitracin (1 complete EDTA-free protease inhibitor tablet/50 ml), pH 7.0]. Nonspecific binding was determined by the inclusion of 1 µM SHU9119. The reactions were initiated, incubated, and terminated as described above. The effect of guanine nucleotides on agonist binding was assessed by the addition of 30 µM GTPS to the buffer.

cAMP Accumulation Assay. One day prior to experimentation, cells were plated at a density of 5 × 10³ cells/well in 96-well polylysine-coated plates, in DMEM lacking phenol red. On the day of experimentation, cells were preincubated with 1 mM isobutylmethylxanthine for 10 min. They were then stimulated with various concentrations of ligand (in DMEM containing 1 mM isobutylmethylxanthine) for 1 h. Cells were then lysed and cAMP quantified using the Tropix cAMP-Screen chemiluminescent enzyme-linked immunosorbent assay system (Applied Biosystems) according to the manufacturer's instructions.

Data Analysis. Data were analyzed using PRISM (GraphPad Software Inc., San Diego, CA), and statistical significance determined using Student's t tests. p < 0.05 determined statistical significance. K_i values were determined from IC₅₀ values using the method of Cheng and Prusoff (1973).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Pharmacological Characterization of the MC4 Receptor Expressed Stably in HEK-293 Cells. Changes in ligand affinity observed in site-directed mutagenesis studies are subject to different interpretation depending on the conformation (i.e., activated or inactivated) of the receptor being studied. The MC4 receptor has not previously undergone a full in vitro pharmacological characterization to determine the potential affinity states present. Therefore, we determined this before proceeding with the mutagenesis experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   The functional potency of AGRP(83-132) () and SHU9119 () at MC4 receptors. Potency was measured by cAMP accumulation as described under Materials and Methods. Data are expressed as picomole(s) of cAMP per well, and graphs are representative of four independent experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Saturation analysis of radioligand binding to MC4 receptors. Data were fitted best by a one binding site model and are representative of three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Affinity of melanocortin ligands for the MC4 receptor measured against different radioligands. Ligand binding was measured in competition studies against [125I]NDP-MSH, [125I]AGRP(87-132), and [125I]SHU9119. Ligands are represented by the following symbols: , -MSH; , NDP-MSH; , THIQ; , AGRP(83-132); and ×, SHU9119. Data are representative of three independent experiments.

Mutagenesis Studies. A number of MC4 receptor residues have been mutated in this study and are summarized in Fig. 4A. Putative ligand-receptor interactions (Fig. 4B) were derived from homology molecular modeling of the human MC4 receptor based on the 2.8Å-resolution structure of rhodopsin (Palczewski et al., 2001). This model predicted an ionic interaction between -MSH and aspartate residues 122 and 126 in TM3 and a hydrophobic interaction between -MSH and both Phe261 in TM6 and Phe284 in TM7. In addition to molecular modeling studies, further receptor mutations were systematically executed based on both previous characterization of the ligand binding site and sequence analysis looking at the differences between the MC3 and MC4 receptors.

View larger version (68K): [in this window] [in a new window]   Fig. 4.   A, two-dimensional representation of the seven-TM structure of the hMC4 receptor. The TM residues mutated in this study are denoted by gray highlighting. B, homology modeling of the hMC4 receptor based on the 2.8 Å resolution structure of rhodopsin (Palczewski et al., 2001).

Hydrophobic Interaction with TM4, TM6, and TM7. Both the Phe7 and Trp9 residues of -MSH have been reported to be important in binding to the MC4 receptor (Yang et al., 2000). To ascertain which residues of the MC4 receptor interact with -MSH, a number of phenylalanine residues were mutated. First, a F261A mutation was characterized; this exhibited similar binding parameters to those previously reported (Yang et al., 2000), with a large loss of affinity for -MSH (>100-fold) but little loss in affinity for NDP-MSH (2-fold) and AGRP(83-132) (3.5-fold) (Fig. 5 and Table 4). Additionally, we discovered that the binding affinity of SHU9119 was not significantly decreased by the F261A mutation (p > 0.05, Student's t test), but the binding of the small-molecule agonist THIQ was, with a 66-fold decrease in affinity (p < 0.05, Student's t test). Consistent with previous literature and binding data, the F261A receptor exhibited decreased agonist potency and discriminated between -MSH and NDP-MSH (Fig. 6 and Table 5). The potency of NDP-MSH was only marginally affected, but an EC₅₀ value could not be determined for -MSH. Furthermore, the small-molecule agonist THIQ showed a 55-fold decrease in potency (p < 0.05, Student's t test). The efficacies of the agonists were not significantly affected by this mutation (Table 6).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   The effect of point mutations on ligand binding to melanocortin 3 and 4 receptors. The binding of agonists was analyzed by competition assays versus [125I]SHU9119, as described under Materials and Methods. Data were fitted to a one binding site model. Graphs are representative of three to six independent experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Potency of agonists at wild-type and mutant melanocortin 3 and 4 receptors. Potency values were obtained from cAMP accumulation assays as described under Materials and Methods. Data are expressed as percentage of the maximal cAMP produced by -MSH, except D122A, which is expressed as percentage of the maximal cAMP produced by NDP-MSH, due to the reduction in activity of -MSH. Graphs are representative of three to six independent experiments.

View this table: [in this window] [in a new window]   TABLE 6 Efficacy values of melanocortin agonists at wild-type and mutant MC4 and MC3 receptors measured using cAMP accumulation Agonist-stimulation of cAMP accumulation was determined as described under Materials and Methods, and efficacy (Emax) values were determined. For both wild-type receptors and all the mutant receptors, with the exception of D122A, the maximal response of -MSH is designated as 100%. Due to the decrease in -MSH activity at D122A, the maximal response of NDP-MSH was designated as 100%. Efficacy values are expressed as Emax ± S.E.M. (n = 3-6). The asterisk (*) indicates that F157I exhibits a significantly different functional potency to the wild-type MC3 receptor (* p < 0.05, Student's t test).

Interaction with TM3. MC4 receptors have been implicated in obesity, and a number of studies have suggested that mutations in the MC4 receptor may be one of the causes of human obesity. Therefore, we decided to further characterize a TM3 mutation found in obese subjects (I137T) (Gu et al., 1999). The I137T mutation significantly decreased the binding of most ligands, with the exception of -MSH and NDP-MSH, with decreases between 1.8- and 4.8-fold compared with wild-type MC4 receptor (p < 0.05, Student's t test). The I137T mutation did not significantly affect efficacy but caused a decrease in potency of most agonists, with THIQ, HfRWK, and MTII showing significant differences of between 3.7- and 9.6-fold (p < 0.05, Student's t test).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we used site-directed mutagenesis to further define the MC4 receptor binding site and to determine which residues contribute to the selectivity between the MC3 and MC4 receptors. We examined the binding and function of a large range of peptides and a small molecule to define differences in binding between similar compounds. We also investigated some of the pharmacological properties of this receptor, in particular, the sensitivity of ligands to GTPS, to gain some insight into the process of receptor activation.

In agreement with previous literature (Haskell-Luevano and Monck, 2001; Nijenhuis et al., 2001), AGRP(83-132) was an inverse agonist at the MC4 receptor. NDP-MSH was a full agonist, and SHU9119 was a very weak partial agonist. The MC4 receptor is largely guanine nucleotide insensitive. In [¹²⁵I]SHU9119 displacement assays, a one binding site model was preferred in both the absence and presence of GTPS, and agonists only displayed a minimal decrease in affinity in the presence of GTPS. In particular, NDP-MSH binding was not detectably affected by GTPS. This is consistent with the saturation data; the B_max of [¹²⁵I]NDP-MSH was equivalent to that of [¹²⁵I]SHU9119, indicating that NDP-MSH does not discriminate between G protein-coupled and -uncoupled forms of the receptor. Similar observations have been made for the somatostatin 5 receptor (Siehler et al., 1999) and the D₂ dopamine receptor (Cordeaux et al., 2001); at both of these receptors, agonists differ in their sensitivity to guanine nucleotides. It has been proposed that guanine nucleotide-insensitive agonists are able to stabilize a conformation of the receptor, which is close to the conformation of the active receptor/G protein complex, so that there is little energy gain in coupling to the G protein (Strange, 1999). Overall, it is concluded from these data that a single activated receptor population is being labeled. This simplifies interpretation of the site-directed mutagenesis binding data, as changes in ligand affinity likely affect receptor/ligand interaction, be it directly or indirectly, rather than the receptor state.

The B_max of [¹²⁵I]SHU9119 and [¹²⁵I]NDP-MSH binding was twice that of [¹²⁵I]AGRP(87-132) binding. These data may indicate that AGRP binds to an MC4 receptor dimer. Previous evidence for MC4 receptor dimerization is seen in a report demonstrating that palmitoylated peptides derived from the third intracellular loop of the MC4 receptor acted as agonists in cells expressing the MC4 receptor (Covic et al., 2002). Furthermore, the high-resolution NMR structure of AGRP implies that there may be more than one site that interacts with the receptor (McNulty et al., 2001). Following this assumption, the complete inhibition of [¹²⁵I]SHU9119 binding by AGRP(83-132) may imply that the receptors are dimerized. Nevertheless, little evidence for this phenomenon was observed in ligand binding. NDP-MSH, SHU9119, and AGRP all acted competitively against each other, and ligands displayed similar affinity in competition studies against the different radioligands, which is consistent with all three radioligands binding to the same site.

Analysis of the expression levels of the MC4 receptor mutants revealed large variation. However, an increase in receptor expression level did not correlate with an increase in agonist affinity or potency, which may have been predicted. It is considered unlikely that the mutations themselves have affected this correlation, as the F184M, F261A, and D122A mutant receptors did not display increased agonist potencies or binding affinities despite their higher expression levels. In addition, other mutations in the MC4 receptor (S190A, S191A, and F267L) showed no appreciable change in agonist affinity or potency relative to the wild-type MC4 receptor, despite the fact that the S190A mutant was expressed at a 10-fold lower level (data not shown). Furthermore, studies in our laboratory have shown that increasing the expression level of the MC4 receptor does not lead to an increase in agonist potency (M. Cismowski, unpublished data). It is, therefore, believed that the differences in expression level do not invalidate the comparisons between wild-type and mutated receptors.

Data obtained in this mutagenesis study provide evidence of a direct interaction between the Phe7 residue of -MSH with both Phe261 and Phe284. The F261A mutant data agreed with previous literature (Yang et al., 2000; Haskell-Luevano et al., 2001), with dramatic decreases in -MSH, but not NDP-MSH, binding affinity and potency. Data obtained at F284A followed this trend, with -MSH and ACTH, but not NDP-MSH, exhibiting large decreases in binding affinity and potency. NDP-MSH primarily differs from -MSH and ACTH by the stereochemistry of the Phe7 residue, indicating that it is this residue that causes the selective decrease in affinity for -MSH compared with NDP-MSH. The binding affinities of cyclic peptides that either contain D-Phe or D-Nal in the Phe7 position were also decreased by the Phe284 mutation, although not to the extent of the linear phenylalanine-containing peptides. These results demonstrate how subtle differences in ligand structure may lead to binding in slightly different orientations. We also established that the binding affinity and potency of the small-molecule agonist THIQ were both decreased by the F284A and F261A mutations. Combining this with the D122A data demonstrates that this small molecule is binding in a similar orientation to -MSH. The binding affinity of the antagonist AGRP(83-132) was also decreased by the F284A mutation, indicating that it shares an overlapping binding pocket with the melanocortin peptides. The greater changes in agonist binding affinity versus potency at the F284A mutant receptor, and the lack of effect of the F261A mutation on antagonist binding, support the conclusion that this region is not important in determining receptor activation. This is in accordance with the recent findings of Yang et al. (2002), who report that Leu133 (TM3) of the MC4 receptor is critical for the antagonist activity of SHU9119.

The TM3 mutation I137T, which is found in obese subjects (Gu et al., 1999), displayed decreased affinity and potency for most MC4 receptor ligands. Because this residue is positioned deep within the TM3 helix, we suggest that the effect of this mutation may be a consequence of nonspecific alteration of the receptor tertiary structure. Consistent with this notion, the binding of the more rigid cyclic peptides was affected, whereas that of the more flexible linear peptides was not.

It has previously been proposed that there is an ionic interaction between the Arg8 of melanocortin peptides and the aspartate residues in TM3 (Yang et al., 2000; Haskell-Luevano et al., 2001). The Asp122 mutation made in this study significantly affected the binding of all compounds except NDP-MSH, which is consistent with the above hypothesis. This study also demonstrated that the spacing between the two aspartate residues plays a small role in determining MC3/MC4 receptor selectivity. Asp122 and Asp126 theoretically both face into the binding pocket, since three to four amino acids is approximately one full turn in the presumed -helical structure of the TM domains. One would anticipate that the difference in bulk of the isoleucine (MC4) versus phenylalanine (MC3) separating these residues would lead to a slightly different orientation of the aspartate residues in the binding pocket. Accordingly, the MC4 receptor mutant I125F and the MC3 receptor mutant F157I displayed a respective decrease or increase in affinity and potency for most MC4 receptor-selective ligands, although the small shifts in affinity and potency values indicate that this locus is of minimal importance in determining receptor selectivity.

Overall, the data herein highlight potentially important differences between the ligand binding site of the human MC4 receptor and that of other melanocortin receptor subtypes and species variants of the MC4 receptor. Specifically, a mutation analogous to F284A in the hMC1 receptor had no effect on binding or function (Yang et al., 1997). The data presented in this study show that ligand binding and function was generally unaltered by the F184M mutation, which would disrupt any hydrophobic interactions between the phenylalanine and the melanocortin peptides. Conversely, mutation of an analogous residue in the mouse MC4 receptor to a serine drastically decreased peptide binding and agonist function (Haskell-Luevano et al., 2001), leading to the hypothesis that the hydrophobic binding pocket for Phe7 was formed between TMs 6 and 4 rather than TMs 6 and 7, as indicated by our data. It is considered that a F184S mutation repulses the aromatic residues in the melanocortin ligands; in accordance with this reasoning, an analogous mouse F184L mutation did not produce such a drastic decrease in ligand affinity (Haskell-Luevano et al., 2001).

In summary, this study demonstrates some unique pharmacological properties of the MC4 receptor. Agonists exhibit little sensitivity to guanine nucleotides, and the agonist NDP-MSH appears to label all forms of the receptor with the same affinity. We have demonstrated that the MC4 receptor residue Ile125 has a role in determining ligand selectivity between the MC3 and MC4 receptors. This leads to the hypothesis that conserved residues in the melanocortin receptors are involved in ligand binding, but differences in their positioning due to differences in receptor structure lead to ligand selectivity for one receptor over another. We have also determined that Phe284 in TM7 forms a hydrophobic pocket with Phe261 in TM6, in which the Phe7 residue of -MSH binds.