Abstract
Mouse gene targeting studies revealed that the melanocortin-3 receptor (MC3R) affected feeding efficiency and fat storage in mice. The functions of the MC3R in other mammalian species remain to be investigated. We are interested in exploring the functions of the porcine MC3R (pMC3R) in regulating fat storage because of the economical importance of swine industry. Although nucleotide sequences of MC3Rs from several species have been reported, pMC3R had not been cloned and sequenced. We reported herein the molecular cloning and pharmacological analysis of the pMC3R. Sequence analysis revealed that pMC3R was highly homologous (>80%) at nucleotide and amino acid sequences to human, rat, and mouse MC3Rs. With human MC3R (hMC3R) as a control, the binding and signaling properties of pMC3R were investigated using several agonists including α- and γ-melanocyte-stimulating hormone (α- and γ-MSH), d-Trp8-γ-MSH, and [Nle4-d-Phe7]-MSH (NDP-MSH) and the natural antagonist agouti-related protein (AgRP). The results showed that pMC3R bound NDP-MSH with the highest affinity followed by d-Trp8-γMSH, γ-, and α-MSH. The same ranking was also found for hMC3R, although pMC3R had two- to ninefold higher affinities for these ligands. Both pMC3R and hMC3R bound AgRP with high affinity. d-Trp8-γ-MSH was the most potent agonist to stimulate cAMP generation followed by NDP-, α-, and γ-MSH. This ranking was the same as that of hMC3R. The availability of pMC3R and its pharmacological characteristics will facilitate the investigation of pMC3R in regulating food intake and fat storage.
Introduction
Melanocortins including α-, β-, and γ-melanocyte-stimulating hormones (α-, β-, and γ-MSH) and adrenocorticotropic hormone (ACTH) are short peptide hormones derived from pro-opiomelanocortin (POMC) through tissue-specific post-translational processing (Smith & Funder 1988). The melanocortin receptors (MCRs) are members of rhodopsin-like (family A) G-protein-coupled receptors (GPCRs). They are expressed on the cell surface, predicted to have seven transmembrane domains (TMDs) connected by alternating extracellular and intracellular loops, with the N-terminus lying on the outside of the cell and the C-terminus locating on the inside of the cell. Once bound to MCRs, melanocortins induce conformational changes in MCRs facilitating their coupling to and activation of the stimulatory G-protein, Gs. Activated Gs enhances adenylyl cyclase activity resulting in increased production of the intracellular second messenger cAMP triggering downstream signal transduction pathways (Gantz & Fong 2003). Agouti and agouti-related protein (AgRP) are the endogenous antagonists of the MCRs. So far, five MCRs have been cloned, named MC1R to MC5R according to the sequence of their cloning (Gantz & Fong 2003). Of the MCRs, MC3R and MC4R are the main subtypes expressed in the brain. They are expressed in the hypothalamic paraventricular nucleus and arcuate nucleus as well as in other brain regions including the cortex, thalamus, and hippocampus (Gantz et al. 1993a,b, Roselli-Rehfuss et al. 1993, Mountjoy et al. 1994). The MC3R is also expressed in the placenta and gut (Gantz et al. 1993a) and immune cells such as macrophages (Getting et al. 1999).
Although both the MC3R and the MC4R are involved in the central control of energy homeostasis, it is clear that the MC4R primarily regulates food intake and energy expenditure (reviewed in Cone 2005). On the other hand, the MC3R does not regulate food intake. MC3R knockout mice did not have hyperphagia and obesity; they had similar or even decreased levels of food intake and normal energy expenditure as compared with their wild-type littermates (Butler et al. 2000, Chen et al. 2000). Of particular interest was the observation that these mice have increased fat mass and decreased lean mass. Therefore, the MC3R is involved in regulation of feeding efficiency and fat storage. When compared with MC3R or MC4R single gene knockout mice, mice lacking both MC3R and MC4R developed exacerbated obesity, suggesting that the two central MCRs regulate different aspects of energy homeostasis (Chen et al. 2000). However, a recent gene knockout study performed with an obesity-resistant mice strain (Black Swiss 129) indicated that MC3R single gene knockout mice developed a comparable degree of increased adiposity as the MC4R knockout mice (Zhang et al. 2005) suggesting that the MC3R might also be involved in regulating food intake and energy expenditure. A potential pathway that the MC3R might regulate food intake is by releasing the inhibitory effect of the MC3R on POMC neurons (Marks et al. 2006).
To begin to understand what role(s) the MC3R might play in regulating fat storage and energy homeostasis in the pig, we report herein the molecular cloning and pharmacological characterization of the pMC3R. For the pharmacological characterizations, we tested several ligands including the endogenous agonists α- and γ-MSH, a superpotent agonist specific for the MC3R, d-Trp8-γ-MSH (Grieco et al. 2000), and a superpotent analog of α-MSH specific for all MCRs except MC2R (the ACTH receptor), [Nle4-d-Phe7]-MSH (NDP-MSH; Sawyer et al. 1980). We also tested the binding of the cloned receptor to the natural antagonist of the MC3/4R, AgRP.
Materials and Methods
Plasmid and peptides
Human MC3R cDNA inserted in a mammalian expression vector, pcDNA3.1, was kindly provided by Dr Ira Gantz (University of Michigan, Ann Arbor, MI, USA). α-, γ-, and NDP-MSH were purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). d-Trp8-γ-MSH and AgRP (86–132) were obtained from Peptides International Inc. (Louisville, KY, USA). 125I-iodinated NDP-MSH was purchased from Peptide Radioiodination Service Center, The University of Mississippi (University, MS, USA).
Molecular cloning of porcine MC3R
Sequence analysis performed with DNAman program (Lynnon Corp., Quebec, Canada) suggested that the putative coding region of the pMC3R gene consists of a single exon of 960 bp. Thus, the pMC3R coding region was amplified directly from porcine genomic DNA (Novagen, San Diego, CA, USA) using a primer pair (sense primer, 5′-AAGAATTCATGAATGCTTCGTGCTGC-3′ and antisense primer, 5′-CCTCTAGAGCCTCCTACCCCAGGTTC-3′) designed based on the published pig genomic DNA sequence from clone CH242-163M14 on chromosome 17 (GenBank accession no.
Homology and phylogenetic analysis of pMC3R
Homology and phylogenetic analyses at nucleotide and amino acid levels were performed between different species including porcine, human, mouse, and rat using DNAman program and MacVector 8.0 (Accelrys Software Inc., San Diego, CA, USA) respectively, according to the manufacturer's protocols.
Transient expression of plasmids
Transient expression of plasmids was performed in HEK293T cells (American Type Culture Collection, Manassas, VA, USA). HEK293T cells were cultured at 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, 10 mM HEPES, 100 units/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen). Cells were plated on gelatin-coated 35 mm six-well plates (Corning, Corning, NY, USA) or on gelatin-coated 100 mm plates. Transient transfection was performed using the calcium precipitation method (Chen & Okayama 1987). For each 35 mm well of the six-well plates or 100 mm plates, 2 ml media containing 4 μg plasmid or 10 ml media containing 20 μg plasmid were added respectively. Cells were used for measuring ligand binding and agonist-stimulated cAMP generation 48-h post-transfection as described below.
Radioligand-binding assay with agonists
The method for binding assay has been described in detail before (Tao & Segaloff 2003). Briefly, 48 h after transfection, cells were washed twice with warm Waymouth's MB752/1 media (Sigma–Aldrich) modified to contain 1 mg/ml BSA (referred herein as Waymouth/BSA). One milliliter of fresh Waymouth/BSA with or without different concentrations of unlabeled agonists including α-, γ-, NDP-, and d-Trp8-γ-MSH and 100 000 c.p.m. of 125I-NDP-MSH (50 μl) was added to each well. The final concentration of unlabeled ligands ranged from 10−10 to 10−5 M (for α-, γ-, and d-Trp8-γ-MSH) or from 10−11 to 10−6 M (for NDP-MSH). After incubation at 37 °C for 1 h, cells were washed twice with cold Hanks’ balanced salt solution (Sigma–Aldrich) modified to contain 1 mg/ml BSA (referred herein as HBSS/BSA). Then, 100 μl of 0.5 M NaOH were added to each well. Lysed cells were collected from each well using cotton swabs to plastic tubes, and ligand binding was counted in a γ-counter. All determinations were performed in duplicates.
Radioligand-binding assay with AgRP
For AgRP-binding assay, transient transfection of plasmids was performed in 100 mm dishes as described above. Twenty-four hrs after transfection, cells were trypsinized, split into 24-well plates, and incubated for an additional 24 h. Cells were washed twice with warm Waymouth/BSA. Fresh Waymouth/BSA containing 20 μl of 125I-NDP-MSH (40 000 c.p.m.) and 20 μl different concentrations (including zero) of unlabeled antagonist AgRP were added to each well to a total volume of 200 μl per well. The final concentration of the unlabeled AgRP ranged from 10−11 to 10−6 M. After incubation at 37 °C for 1 h, cells were washed twice with cold HBSS/BSA. Then, 50 μl of 0.5 M NaOH were added to each well. Cell lysate collection and ligand binding were performed as described above. All determinations were performed in triplicates.
Intracellular cAMP generation
HEK293T cells were plated and transfected as described above. Forty-eight hours after transfection, cells were washed twice with warm Waymouth/BSA. Then, 1 ml fresh Waymouth/BSA containing 0.5 mM isobutylmethylxanthine (Sigma–Aldrich) was added to each well. After incubation at 37 °C for 15 min, either buffer alone or different concentrations of ligands (final concentrations of 10−11–10−5 M for α- and γ-MSH, and 10−12–10−6 M for NDP-MSH and d-Trp8-γ-MSH) were added, and the incubation was continued for an additional 1 h. Cells were then placed on ice, media were aspirated, and intracellular cAMPs were extracted by the addition of 1.5 ml fresh 0.5 M perchloric acid containing 180 μg/ml theophylline, and measured using RIA. All determinations were performed in triplicate.
Statistical analysis
Maximal binding (Bmax), IC50, maximal responses (Rmax), and EC50 were calculated using Prism 4.0 (GraphPad, San Diego, CA, USA). Data were presented as the mean and s.e.m. All statistical analyses were done using Prism 4.0.
Results
Nucleotide and deduced amino acid sequences of the putative pMC3R
Civanova et al. (2004) reported the partial sequence of the pMC3R and mapped the pMC3R gene to porcine chromosome 17. Recently, the complete nucleotide sequence of porcine chromosome 17 was deposited in GenBank. We performed nucleotide sequence alignment among the partial nucleotide sequence of pMC3R gene (GenBank accession no.
Deduced nucleotide and amino acid sequences of pMC3R and comparison with MC3Rs from other mammal. (A) Sequence of the full-length pMC3R cDNA with in silico translation is shown. Arrows indicate the position and direction of the primers used for amplification of the cDNA by PCR. (B) Homology and (C) phylogenetic analysis of MC3R nucleotide sequences from several species.
Citation: Journal of Endocrinology 196, 1; 10.1677/JOE-07-0403
Comparison of amino acid sequences between pMC3R and MC3Rs from other species. Alignment of the amino acid sequences between pMC3R and human, mouse and rat MC3Rs. Positions of different regions of the receptor are indicated above the sequences and labeled as follows: transmembrane domains as TMD 1–7, extracellular loops as EL 1–3, intracellular loops as IL 1–3, amino and carboxyl termini as extracellular amino terminus and cytoplasmic tail respectively. The conserved residues are shaded in gray. The transmembrane residues are in bold. (B) Homology and (C) phylogenetic analysis of MC3R amino acid sequences from several species.
Citation: Journal of Endocrinology 196, 1; 10.1677/JOE-07-0403
Expression and functional analysis of the cloned pMC3R in HEK293T cells
Based on the sequence analysis described above, primers were designed and the 960 bp of putative pMC3R was directly amplified from porcine genomic DNA by PCR and cloned into mammalian expression vector pcDNA3.1 for functional analysis. To demonstrate whether the cloned pMC3R is capable of producing a biologically active receptor protein, we transfected the pMC3R construct obtained into HEK293T cells and analyzed its ligand binding and signaling properties using the natural agonist for MC3R, γ-MSH, and ligand-binding property with the natural antagonist for MC3R, AgRP. Human MC3R was used for comparison. As shown in Fig. 3 and Table 1, γ-MSH bound the cloned pMC3R with an IC50 of 129.07 nM and hMC3R with an IC50 of 293.00 nM. Both the cloned pMC3R and hMC3R bound AgRP with similar high affinity: the cloned pMC3R with an IC50 of 0.67 nM and hMC3R with an IC50 of 2.35 nM. Furthermore, γ-MSH also induced dose-dependent accumulations of intracellular cAMP with EC50 of 79.16 and 111.13 nM for the cloned pMC3R and hMC3R respectively. These results suggested that the cloned pMC3R encoded a functional MC3R protein.
Expression and functional analysis of the cloned pMC3R in HEK293T cells. HEK293T cells were transiently transfected with pMC3R and hMC3R, and binding and signaling assays were performed as described in Materials and Methods. For ligand binding, different concentrations of unlabeled (A) γ-MSH or (B) AgRP were used to displace the binding of 125I-NDP-MSH to pMC3R or hMC3R on intact cells. Results shown are expressed as the percentage±s.e.m. of the maximal binding from duplicate determinations within one experiment. All experiments were performed at least thrice. For measurement of cAMP accumulation, the transfected cells were stimulated with various concentrations of γ-MSH, and intracellular cAMP levels were measured as described in Materials and Methods. Results are expressed as the mean±s.e.m. of triplicate determinations within one experiment, and all experiments were performed at least thrice.
Citation: Journal of Endocrinology 196, 1; 10.1677/JOE-07-0403
Ligand binding and agonist-stimulated cAMP response of porcine melanocortin-3 receptor (pMC3R) and human MC3R (hMC3R). Data shown are the mean±s.e.m. of the indicated number of experiments
| Ligand binding | Ligand-stimulated cAMP response | |||||
|---|---|---|---|---|---|---|
| MC3R | n | IC50 (nM) | n | EC50 (nM) | Rmax (pmol/106 cells) | |
| Ligands | ||||||
| α-MSH | Porcine | 3 | 674.90±83.76 | 4 | 59.03±7.51 | 2854±96 |
| Human | 3 | 1151.33±85.54 | 4 | 70.27±11.97 | 2765±238 | |
| NDP-MSH | Porcine | 5 | 4.36±0.43 | 4 | 2.24±0.24 | 1286±160 |
| Human | 5 | 21.71±2.64 | 4 | 5.01±0.87 | 2438±333 | |
| γ-MSH | Porcine | 3 | 129.07±3.28 | 4 | 79.16±6.05 | 2354±147 |
| Human | 3 | 293.00±21.64 | 4 | 111.13±11.78 | 2623±185 | |
| d-Trp8-γ-MSH | Porcine | 3 | 10.65±3.31 | 4 | 1.24±0.58 | 1894±256 |
| Human | 3 | 90.74±12.46 | 4 | 1.95±0.64 | 1972±258 | |
| AgRP | Porcine | 3 | 0.67±0.06 | NA | NA | NA |
| Human | 3 | 2.35±0.42 | NA | NA | NA | |
IC50 is the concentration of ligand that is needed to cause 50% inhibition in the binding assay. EC50 is the concentration of agonist that results in 50% stimulation of the maximal response. NA, not applicable.
Binding and signaling of pMC3R to three other MC3R agonists
For in vivo investigation of the role that pMC3R plays in pigs, the most potent and selective agonist for pMC3R must be selected from the pool of known MC3R agonists. Herein, we tested the pharmacological properties of three MC3R agonists on the cloned pMC3R. These agonists include the natural agonist of MC3R, α-MSH; a superpotent analog of α-MSH for MC3R and MC4R, NDP-MSH; and a superpotent analog specific for MC3R, d-Trp8-γ-MSH. As shown in Fig. 4, ligand-binding assays revealed that pMC3R bound NDP-MSH with the highest affinity (IC50 of 4.36 nM) followed by d-Trp8-γ-MSH (IC50 of 10.65 nM) and α-MSH (IC50 of 674.90 nM). The same ranking was also observed for hMC3R with an IC50 of 21.71 nM for NDP-MSH, 90.74 nM for d-Trp8-γ-MSH, and 1151.33 nM for α-MSH. It is interesting to note that pMC3R always had two- to ninefold higher affinities for these ligands than the hMC3R (Table 1). The data from the signaling assays demonstrated that d-Trp8-γ-MSH was the most potent agonist to stimulate dose-dependent cAMP generation with an EC50 of 1.24 nM, followed by NDP-MSH (EC50 of 2.24 nM) and α-MSH (EC50 of 59.03 nM; Table 1). Human MC3R has the same ranking with an EC50 of 1.95 nM for d-Trp8-γ-MSH, 5.01 nM for NDP-MSH, and 70.27 nM for α-MSH (Table 1). Taken together, these data demonstrated that d-Trp8-γ-MSH was the most potent agonist in eliciting pMC3R signaling.
Binding and signaling of pMC3R to three other MC3R agonists. HEK293T cells were transiently transfected with pMC3R or hMC3R. Binding assays were performed as described in Materials and methods. Different concentrations of unlabeled (A) α-MSH, (B) NDP-MSH, or (C) d-Trp8-γ-MSH were used to displace the binding of 125I-NDP-MSH to pMC3R or hMC3R on intact cells. To measure accumulation of intracellular cAMP, the transfected cells were stimulated with various concentrations of (A) α-MSH, (B) NDP-MSH, or (C) d-Trp8-γ-MSH, and intracellular cAMP levels were measured as described in Materials and methods. Results are expressed as the mean±s.e.m. of triplicate determinations within one experiment, and all experiments were performed at least thrice.
Citation: Journal of Endocrinology 196, 1; 10.1677/JOE-07-0403
As can be seen from Table 1, the maximal responses to α-, γ-, and d-Trp8-γ-MSH are similar between the pMC3R and hMC3R. However, the hMC3R has significantly larger maximal response to NDP-MSH than pMC3R. One reason might be that hMC3R is expressed at slightly higher level (148±8% of pMC3R, n=12). However, the maximal responses to the other three agonists are similar between the two MC3Rs. We suggest that the NDP-MSH binding to hMC3R induces a conformation with higher coupling efficiency than the pMC3R (Ballesteros et al. 1998).
Discussion
Previous pharmacological and genetic studies in rodents suggested that the MC3R was not involved in food intake regulation. Administration of MC3R-specific agonist such as γ-MSH did not inhibit food intake (Kask et al. 2000). In fact, a recent study showed that peripheral administration of d-Trp8-γ-MSH stimulated food intake in rats by releasing the inhibitory effect of the MC3R on POMC neurons (Marks et al. 2006). Changes in energy balance such as food restriction or diet-induced obesity did not change MC3R expression level in the brain, whereas the MC4R density was modulated (Harrold et al. 1999). Finally, MC3R knockout mice did not have increased food intake as did the MC4R knockout mice (Huszar et al. 1997, Butler et al. 2000, Chen et al. 2000). Mice lacking the MC3R have increased fat contents but are normal in terms of food intake and body weight (Butler et al. 2000, Chen et al. 2000), suggesting that the MC3R pathway modulates feeding efficiency and fat storage. The recent gene targeting study by Zhang et al. (2005) showed that Black Swiss 129 mice lacking MC3R gene had a comparable level of adiposity as mice lacking the MC4R gene. In humans, only a few naturally occurring mutations in the MC3R gene have been identified (Lee et al. 2002, 2007, Tao 2007). These mutations were found to cause defects in signaling (Rached et al. 2004, Tao & Segaloff 2004, Lee et al. 2007, Tao 2007). In contrast, in the MC4R, numerous mutations have been identified from various patient populations (reviewed in Tao 2005, 2006). Therefore, the roles of the MC3R in energy homeostasis are much less understood.
Pork is an important protein source in our diet. About 60% of the saturated fat comes from animal products in typical American diet. Leaner meat will decrease intake of saturated fat and decrease blood cholesterol levels. To increase the economic return for hog farmers and reduce the obesity epidemic, it is of great interest to decrease the fat content in pork. Therefore, a better understanding of fat storage in pigs is of great economic interest.
In the present study, as the first step towards elucidating the role(s) of the MC3R in pig energy homeostasis, we describe the molecular cloning and pharmacological characterization of the porcine MC3R. Through bioinformatics analysis and progress in pig genome sequencing, we were able to identify the putative full-length coding region of pMC3R. Primers designed based on this sequence were used to amplify the coding region from pig genomic DNA by taking advantage of the fact that the mammalian MCRs, including the putative pMC3R, are intronless. This amplified fragment was inserted into pcDNA3.1 and the sequence verified.
This construct was used for detailed pharmacological analysis using a series of ligands. From ligand-binding studies, we showed that of the four agonists tested, the rank order in terms of affinity is: NDP-MSH>d-Trp8-γ-MSH>γ-MSH>α-MSH. Human MC3R has the same rank order for these ligands. From signaling experiments, we showed that the rank order in terms of potency for these four ligands is: d-Trp8-γ-MSH>NDP-MSH>α-MSH>γ-MSH. Again, hMC3R has the same rank order for these ligands. Both pMC3R and hMC3R bind to AgRP with high affinity, with IC50 of 0.67 and 2.35 nM respectively.
Previously, the MC3Rs have been cloned from a number of species, including human (Gantz et al. 1993a), rat (Roselli-Rehfuss et al. 1993), mouse (Desarnaud et al. 1994), and elephant (Rompler et al. 2006). In lower vertebrates, the MC3Rs have been cloned from the chicken (Takeuchi & Takahashi 1999) and zebrafish (Logan et al. 2003) but was found to be absent in some other fishes such as the pufferfishes (Klovins et al. 2004a). The numbers we reported herein for pMC3R are roughly in line with previous studies. For example, with NDP-MSH, the most commonly used analog for MCR studies, the IC50 reported here for pMC3R is 4.36 nM. Previously, the Kd for rat MC3R was reported to be 3.78 nM (Kim et al. 2002), and the Ki for human MC3R was 5.6 nM (Chen et al. 2006), and for the dogfish MC3R was 0.93 nM (Klovins et al. 2004b).
In the original publication describing the discovery of d-Trp8-γ-MSH as a potent and selective MC3R agonist (Grieco et al. 2000), an IC50 of 6.7 nM and EC50 of 0.33 nM were reported for hMC3R. Our data for hMC3R are somewhat higher than those reported before (IC50 of 90.74 nM and EC50 of 1.95 nM). These differences might be due to the use of different cell lines and different methods in the two studies. For example, for binding studies, Grieco et al. used membranes prepared from L cells stably expressing hMC3R, whereas we used live HEK293T cells transiently expressing hMC3R (only cell surface receptor binding is detected). However, our data for pMC3R (IC50 of 10.65 nM and EC50 of 1.24 nM) are very close to the numbers reported for hMC3R by Grieco et al. (2000).
It should be pointed out that the ranking in terms of affinity is not the same as that in terms of potency. For example, for pMC3R, NDP-MSH has higher binding affinity than d-Trp8-γ-MSH. However, it is not as potent in eliciting activation of signaling pathways as the latter. This may be due to the difference in their relative efficacies. Both affinity and efficacy can independently affect the potency of a particular ligand (Besse & Furchgott 1976).
The data presented herein suggest that d-Trp8-γ-MSH is a superpotent agonist for the pMC3R. Since it does not activate the MC4R efficiently (Grieco et al. 2000), it is an excellent tool for in vivo studies directed towards dissecting the role(s) of the MC3R in regulating pig energy balance. However, the in vitro data described herein should only be used as the starting point for further in vivo studies. Previous studies showed that pMC4Rs respond to the natural (AgRP) and artificial (SHU9119) antagonists normally in vitro. However, in vivo, these antagonists do not increase food intake as observed in rodents (Barb et al. 2004). It was suggested that the MC4R might not play a critical role in regulating feed intake in the pig (Barb et al. 2004). Similarly, two naturally occurring mutations (R236H and D298N) in the pMC4R were identified in different strains of pigs that are associated with differences in performance characteristics such as average daily gain and average daily intake as well as carcass fat (Kim et al. 2000, Meidtner et al. 2006). In the case of D298N, not all studies could confirm the original associations (Park et al. 2002, Stachowiak et al. 2006). Similarly, our experiments in vitro could not replicate the original functional studies (Kim et al. 2004, Fan et al. 2007). Therefore, the data on the naturally occurring mutations in the pMC4R are also much less convincing than those on human MC4R (reviewed in Tao (2005, 2006)). All these point to the importance of using caution when extrapolating in vitro data as well as the potential difference of the relevance of the melanocortin system in regulating energy homeostasis in the pig.
In summary, we have cloned the pMC3R. Expression of the cloned pMC3R in HEK293T cells revealed that indeed it was functional. Pharmacological characterizations using a series of ligands revealed that the pMC3R and hMC3R have the same rank orders for these ligands. We showed that d-Trp8-γ-MSH was the most potent ligand (and selective for the MC3R), therefore, best suited for further in vivo studies to elucidate the functional importance of the MC3R in energy homeostasis in the pig.
Acknowledgements
We thank Dr Ira Gantz (previously at University of Michigan, Ann Arbor, MI, USA) for generously providing us the human MC3R cDNA. This study was supported by a Foundation Grant from Alabama Agricultural Experiment Station (ALA080-031). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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