Rescue of defective MC4R cell-surface expression and signaling by a novel pharmacoperone Ipsen 17

in Journal of Molecular Endocrinology
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Xiao-Hua WangKey Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Obesita and Algaegen LLC, College of Biotechnology, College of Food Engineering and Biotechnology, Ministry of Education, No. 29 13rd Road, Tianjin Economy-and-Technology Development Area, Tianjin 300457, People's Republic of China

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Hao-Meng WangKey Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Obesita and Algaegen LLC, College of Biotechnology, College of Food Engineering and Biotechnology, Ministry of Education, No. 29 13rd Road, Tianjin Economy-and-Technology Development Area, Tianjin 300457, People's Republic of China

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Bao-Lei ZhaoKey Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Obesita and Algaegen LLC, College of Biotechnology, College of Food Engineering and Biotechnology, Ministry of Education, No. 29 13rd Road, Tianjin Economy-and-Technology Development Area, Tianjin 300457, People's Republic of China

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Peng YuKey Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Obesita and Algaegen LLC, College of Biotechnology, College of Food Engineering and Biotechnology, Ministry of Education, No. 29 13rd Road, Tianjin Economy-and-Technology Development Area, Tianjin 300457, People's Republic of China

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Zhen-Chuan FanKey Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Obesita and Algaegen LLC, College of Biotechnology, College of Food Engineering and Biotechnology, Ministry of Education, No. 29 13rd Road, Tianjin Economy-and-Technology Development Area, Tianjin 300457, People's Republic of China
Key Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Obesita and Algaegen LLC, College of Biotechnology, College of Food Engineering and Biotechnology, Ministry of Education, No. 29 13rd Road, Tianjin Economy-and-Technology Development Area, Tianjin 300457, People's Republic of China

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Melanocortin 4 receptor (MC4R) is a key factor in regulating energy homeostasis, and null mutations occurring in the gene encoding MC4R cause severe early-onset morbid obesity in humans. Many obesity-causing mutations affecting MC4R clinically identified so far lead to failure of mutant receptors to shuttle to the plasma membrane. In this study, we show that a novel human MC4R antagonist, Ipsen 17, acted as an pharmacological chaperone of human MCR4. As tested with 12 obesity-causing human MC4R variants including S58C, E61K, N62S, I69T, P78L, C84R, G98R, T162I, R165W, W174C, C271Y, and P299H, Ipsen 17 was found to be the most universal pharmacological chaperone of MC4R reported so far because it can completely rescue nearly all mutant receptors (except P299H) with the highest potency (an EC50 value of approximately 10−8 M) and efficiency when compared with results for other tested pharmacological chaperones of MC4R including ML00253764, PBA, MTHP, PPPone, MPCI, DCPMP, and NBP described in the literature. Once restored to the plasma membrane, defective human MC4R variants responded to α-MSH stimulation with an EC50 value of approximately 10−8 M and displayed dramatically enhanced signaling ability (except for G98R) in a mutant-specific efficacy and potency profile. Taken together, these results indicate that Ipsen 17 represents a candidate for the development of a targeted treatment of severe early-onset morbid obesity caused by a large subset of inherited mutations in the human MC4R gene.

Abstract

Melanocortin 4 receptor (MC4R) is a key factor in regulating energy homeostasis, and null mutations occurring in the gene encoding MC4R cause severe early-onset morbid obesity in humans. Many obesity-causing mutations affecting MC4R clinically identified so far lead to failure of mutant receptors to shuttle to the plasma membrane. In this study, we show that a novel human MC4R antagonist, Ipsen 17, acted as an pharmacological chaperone of human MCR4. As tested with 12 obesity-causing human MC4R variants including S58C, E61K, N62S, I69T, P78L, C84R, G98R, T162I, R165W, W174C, C271Y, and P299H, Ipsen 17 was found to be the most universal pharmacological chaperone of MC4R reported so far because it can completely rescue nearly all mutant receptors (except P299H) with the highest potency (an EC50 value of approximately 10−8 M) and efficiency when compared with results for other tested pharmacological chaperones of MC4R including ML00253764, PBA, MTHP, PPPone, MPCI, DCPMP, and NBP described in the literature. Once restored to the plasma membrane, defective human MC4R variants responded to α-MSH stimulation with an EC50 value of approximately 10−8 M and displayed dramatically enhanced signaling ability (except for G98R) in a mutant-specific efficacy and potency profile. Taken together, these results indicate that Ipsen 17 represents a candidate for the development of a targeted treatment of severe early-onset morbid obesity caused by a large subset of inherited mutations in the human MC4R gene.

Introduction

Many human diseases such as retinitis pigmentosa, nephrogenic diabetes insipidus (NDI), hypogonadism, Hirschsprung disease, and certain kinds of severe early-onset morbid obesity can be caused by mutations in G protein-coupled receptors (GPCRs; Bernier et al. 2004a,b, Tao 2006, Conn & Ulloa-Aguirre 2010). Disease-causing mutations in GPCR genes usually result in conformational change and then improper folding, leading to intracellular retention of the receptors in endoplasmic reticulum (ER) and Golgi apparatus (Conn & Ulloa-Aguirre 2010, Granell et al. 2010, Ulloa-Aguirre & Michael Conn 2011). The mutant receptors are generally destined to be degraded by the cell's quality control system (Granell et al. 2010). To date, numerous functional studies performed on a number of mutant GPCR proteins of different classes have clearly shown that many mutations in GPCR genes occur in the receptor domain that only affects the intracellular trafficking of the mutant receptors to their functional site, the plasma membrane (Conn et al. 2007). In general, these mutations are likely to have no effect or to only show a minor effect on ligand binding or signaling directly and might be capable of binding to the ligand and signal if the mutant receptors can be targeted to the cell surface. Evidence has shown that cell-surface expression of both WT and mutant GPCR proteins can be elevated by treatment with some small molecular chemicals at a high concentration such as dimethyl sulfoxide, glycerol, and trimethylamine-N-oxide (Robben et al. 2006). Although these approaches obviously are not viable treatment options in clinical settings, they do raise the possibility that the receptor signaling function can be restored by rescuing receptor folding and cell-surface expression by treatment with pharmacologically selective compounds, termed pharmacological chaperones (Robben et al. 2007). In recent years, accumulating evidence has shown that pharmacological chaperones act as folding templates and assist defective receptor proteins of different classes to fold into their correct conformation allowing their export from ER to the cell surface where they are active functionally (Morello et al. 2000). Successful examples include rhodopsin and gonadotropin-releasing hormone receptor (GnRHR; Janovick et al. 2002, 2003, Noorwez et al. 2003, 2004, Knollman et al. 2005), μ- and δ-opioid receptors, melanin-concentrating hormone receptor 1, V1a, V1b, and V2 vasopressin receptors (Morello et al. 2000, Janovick et al. 2002, Petaja-Repo et al. 2002, Chaipatikul et al. 2003, Bernier et al. 2004c, 2006, Fan et al. 2005, Robert et al. 2005, Hawtin 2006, Robben et al. 2006, 2007), as well as melanocortin 4 receptor (MC4R; Fan & Tao 2009, Granell et al. 2010, Rene et al. 2010).

The cell-permeable, peptidomimetic small molecule SR49059 originally developed as a vasopressin antagonist was capable of rescuing the function of ER-retained V2 vasopressin receptor (V2R) variants as a V2R-specific pharmacological chaperone (Bernier et al. 2004c, 2006). Treatment of NDI disease caused by mutations that render V2R transportation-defective led to a decrease in urine volume in patients, clearly showing the significantly improved kidney function of these patients (Bernier et al. 2006). This pioneer clinical test revealed that cell-permeable, small-molecule GPCR antagonists have great potential to act as pharmacological chaperones to rescue the mutant GPCR function (Robben et al. 2007, Ulloa-Aguirre & Michael Conn 2011). MC4R is a typical GPCR and plays a pivotal role in energy homeostasis as shown by both human and rodent genetic studies (Huszar et al. 1997, Chen et al. 2000, Farooqi et al. 2000). Mutations in MC4R gene cause an obese phenotype and represent the most frequent monogenetic form causing early-onset obesity (Farooqi & O'Rahilly 2006). To date, more than 150 distinct MC4R mutations spread over all domains of the receptor have been identified clinically from obese patients of various ethnic backgrounds and it was estimated that approximately 6% of the early-onset obesity was caused by the mutated MC4R genes (Spiegelman & Flier 2001, Cone 2005, Tao 2005). Functional characterization assay showed that the majority of the products of these mutant MC4R genes are defective to different extents in trafficking onto the cell surface (Cone 2005). Therefore, MC4R-specific pharmacological chaperones, by rescuing the cell-surface expression and signaling of the defective MC4R, provide a possible therapeutic option for hereditary early-onset obesity caused by mutations in the MC4R gene.

In accordance with the above notion, several cell-permeable, nonpeptidic small-molecule antagonists of MC4R have recently been examined to determine their ability to rescue the cell-surface expression and signaling of intracellularly retained defective MC4R proteins (Fan & Tao 2009, Granell et al. 2010, Rene et al. 2010, Tao 2010). The tested compounds belong to structurally different chemical classes and show various efficacies and potencies toward MC4R variants. Even for a specific compound, clear differences in its ability to rescue the receptor mutants harboring distinct mutations were documented (Granell et al. 2010, Rene et al. 2010). The existence of a large diversity of obesity-related trafficking-defective mutations in MC4R calls the ability of a single chemical compound to restore cell-surface expression and function to all mutant forms into question. As a consequence, a MC4R pharmacological chaperone has to be seriously evaluated to determine its rescue profile for distinct mutants. In this study, we tested the ability of a novel selective antagonist of MC4R, Ipsen 17 (Poitout et al. 2007), to rescue cell-surface expression and signaling activity of 12 naturally occurring variant forms of MC4R that cause obesity (Fig. 1). As expected, Ipsen 17 exerts different efficiencies and potencies on distinct mutants. Interestingly, this chemical was found to be the most universal pharmacological chaperone of MC4R recorded so far in the literature because it can rescue nearly all mutations with the highest potency and efficiency when compared with other tested compounds including ML00253764 (Fan & Tao 2009, Tao 2010), 4-phenyl butyric acid (PBA) (Granell et al. 2010), 2-(2-(2-methoxy-5-nitrobenzylthio)phenyl)-1,4,5,6-tetrahydropyrimidine (MTHP), 3-(4-(2-(4-fluorophenyl)-2-(4-methylpiperazin-1-yl)ethyl)piperazin-1-yl)-2-methyl-1-phenylpropan-1-one (PPPone), 2-(5-bromo-2-methoxyphenethyl)-N-(N-((1-ethylpiperidin-4-yl)methyl)carbamimidoyl)-3-fluorobenzamide (MPCI), N-((2R)-3(2,4-dichlorophenyl)-1-(4-(2-((1-methoxypropan-2-ylamino)methyl)phenyl)piperazin-1-yl)-1-oxopropan-2-yl)propionamide (DCPMP) and 1-(1-(4-fluorophenyl)-2-(4-(4-(naphthalene-1-yl)butyl)piperazin-1-yl)ethyl)-4-methylpiperazine (NBP) (Rene et al. 2010).

Figure 1
Figure 1

Schematic of human melanocortin 4 receptor. The positions of the engineered HA epitope tags are indicated as dark circles with white letters. The positions of mutated residues are indicated as white circles with gray letters.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-14-0005

Materials and methods

Generation of WT and mutant human MC4R constructs

Human MC4R was PCR amplified from human genomic DNA as a template and inserted into pcDNA3.1 using standard molecular techniques. A single hemagglutinin (HA) tag was attached to the N-terminus of the cloned receptors. Mutations of human MC4R used in this study were created by site-directed mutagenesis methodology as described in our previous report (Fan et al. 2008).

Compound synthesis and agonist

Compounds Ipsen 17 and ML00253764 were synthesized and purified to 98% at Tianjin University of Science and Technology according to the previously reported synthesis procedures with modifications (Vos et al. 2004, Poitout et al. 2007). The peptide hormone α-MSH (95% purity) was purchased from Qiang Yao Biological Technology Co. Ltd (Shanghai, China). Mouse FITC-conjugated HA.11 against HA was purchased from Sigma-Aldrich Co. LLC. DMEM, HEPES, and newborn calf serum were purchased from Life Technologies Co. Ltd. Penicillin, streptomycin, and G418 were the products of Solarbio Co. Ltd (Beijing, China). Other reagents and materials were purchased from various commercial suppliers.

Cell culture and transfection

Human embryonic kidney (HEK) 293 cells were grown in an atmosphere of 5% CO2 in DMEM containing 10 mM HEPES, 10% newborn calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin, as described previously (Fan et al. 2008). For establishment of stably transfected cells, HEK 293 cells were seeded at a density of 5×107 cells/100-mm dish (Corning, Inc., Beijing, China) and transfected after 48 h using a calcium phosphate precipitation protocol as described previously (Chen & Okayama 1987). To create stable cell lines, 10 μg of DNA per dish were used to transfect cells and cells were selected for 4 weeks with G418 at a concentration of 250 μg/ml. The G418 medium was changed once every 3 days. A mixture of stable cells was used for further investigation. Cells were rinsed three times with saline and Ipsen 17, ML00253764, α-MSH, or other buffer solutions were added to reach a final concentration as appropriate. The cells were then cultured for an additional time period as appropriate before being assayed.

Quantification of cell surface expression of WT and mutant receptors

Cell-surface expression of WT and mutant receptors were quantified by FACS assay as described previously (Fan & Tao 2009). Briefly, stable HEK 293 cell lines expressing WT receptor, mutant receptor, or empty vector pcDNA3.1 were seeded in duplicate in six-well plates (Corning, Inc.) at a density of 1×104 cells/well and incubated for 24 h. Cells were then treated with or without compound for 12 h. On the day of the experiment, cells were transferred into 1.5 ml centrifuge tubes and fixed with 5% (v/v) paraformaldehyde in PBS for 30 min at room temperature. Cells were then washed three times with PBS before the addition of 5% (w/v) BSA/PBS for 1 h to block nonspecific binding. Anti-HA FITC-conjugated HA.11 antibody (Sigma–Aldrich) diluted to 1:1000 in 0.5% (w/v) BSA/DPBS was incubated on cells for 1 h. Cells were gently washed five times and the cell-surface expression of the receptors (mean fluorescence intensity (MFI)) were quantified by a BD FACSAria flow cytometry system (BD Biosciences, San Jose, CA, USA). For each experiment, HEK 293 cells stably expressing the empty pcDNA3.1 vector were included as negative control. The percentage of mutant receptor expressed at the cell surface is defined as ((MFImutant−MFImock)/(MFIWT−MFImock)). All experiments were performed in triplicate for each condition, and values were obtained from at least three separate experiments.

Confocal microscopy

HEK 293 cells expressing the WT MC4R, MC4R variants or empty vector pcDNA3.1 were seeded in poly-d-lysine-coated eight-well chambers (Corning, Inc.) at a density of 5×103 cells/well and incubated for 24 h. Cells were then treated with Ipsen 17 or vehicle for 12 h. At room temperature, the treated cells were then fixed with 5% (v/v) paraformaldehyde in PBS for 30 min and washed three times with PBS. Cell staining was performed as described above. Cells were washed five times with PBS before coverslips were mounted on glass slides before confocal microscopy. Confocal microscopy was performed using a Nikon A1 si confocal microscope (Nikon, Tokyo, Japan) as described previously (Fan & Tao 2009).

Agonist-induced cAMP generation

HEK 293 cells stably expressing WT receptor, mutant receptor, or empty vector pcDNA3.1 were seeded in poly-d-lysine-coated 96-well plates (Corning, Inc.) at a density of 5×102 cells/well and incubated for 24 h. Cells were treated with Ipsen 17 or vehicle 12 h before being stimulated with α-MSH. The agonist-induced accumulation of intracellular cAMP was determined by the cAMP screen 96-well cAMP immunoassay system (Applied Biosystems) and using a cAMP HiRange kit (CisBio Bioassays, Beijing, China) according to the manufacturer's recommendations. Briefly, after 24 h inoculation and treatment with drugs, cells were washed three times (for extensive wash, two more wash steps were performed) with DMEM and then incubated in DMEM containing α-MSH for 45 min. EC50 values were determined by nonlinear regression after fitting of logistic sigmoidal curves to the experimental data using GraphPad Prism5 (GraphPad Software, Inc., San Diego, CA, USA).

Agonist-induced internalization of WT and mutant receptors

HEK 293 cells stably expressing WT receptor, mutant receptor, or empty vector pcDNA3.1 were seeded in poly-d-lysine-coated 24-well plates (Corning, Inc.) at a density of 5×103 cells/well and incubated for 24 h. Cells were then treated with Ipsen 17 or vehicle. After 12 h incubation, the medium was removed from cells and was replaced with fresh DMEM before the addition of α-MSH to induce human MC4R internalization at various time intervals. Expression of the receptor remaining on the cell surface is quantified by FACS assay as described above. The percentage of mutant receptor internalized is defined as ((MFIbasal−MFImock)−(MFIstimulated−MFImock))/(MFIbasal−MFImock). All experiments were performed in triplicate for each condition, and values were obtained from at least three separate experiments.

Statistical analyses

All the data are expressed as mean±s.e.m. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc.). For comparisons of the WT and variant MC4R proteins on the cell surface and maximal signaling, a one-sample unpaired t-test was used. For comparisons of EC50, a two-sample unpaired t-test was used. Differences with a P<0.05 were considered statistically significant.

Results

Identification of Ipsen 17 as an human MC4R pharmacological chaperone and its rescue profile on mutant receptors

This and other previous studies have shown that some cell-permeable, nonpeptidic human MC4R antagonists or inverse agonists of low molecular weight act as pharmacological chaperones to restore the cell-surface expression of human MC4R proteins defective in intracellular trafficking (Fan & Tao 2009, Rene et al. 2010). Ipsen 17 is a newly synthesized nonpeptidic compound of benzimidazole backbone that was originally developed as a small-molecule human MC4R antagonist (Poitout et al. 2007; Fig. 2a). To investigate whether Ipsen 17 has a pharmacological chaperone effect on human MC4R, the WT and 12 mutant receptors including S58C (Dubern et al. 2001), E61K (Tan et al. 2009), N62S (Farooqi et al. 2000), I69T (Tan et al. 2009), P78L (Hinney et al. 1999), C84R (Hainerova et al. 2007), G98R (Kobayashi et al. 2002), T162I (Tan et al. 2009), R165W (Nijenhuis et al. 2003), W174C (Buono et al. 2005), C271Y (Farooqi et al. 2003), and P299H (Lubrano-Berthelier et al. 2006) were tested to determine its ability to rescue the cell-surface expression of the mutant receptors (Fig. 1). The selected variant human MC4R proteins were chosen because they almost completely lose cell-surface expression and have been reported to cause inherited obesity in humans of various ethnic backgrounds. In addition, these mutants reflect a wide region of conformational changes conferred on the receptor by the mutations and thus can provide us detailed information about the rescue profile of Ipsen 17 on distinct mutant receptors.

Figure 2
Figure 2

Ipsen 17-mediated cell-surface expression of the intracellularly trapped defective MC4R proteins. (a) The molecular structure of Ipsen 17. (b) HEK 293 cells stably expressing WT or defective human MC4R were treated with vehicle alone or Ipsen 17 (1 μM) for 12 h. The cell-surface expression level of the receptors was quantified by FACS assay as described in Materials and methods. Values shown are Ipsen 17-mediated cell-surface expression as a percentage of that for the WT receptor without Ipsen 17 treatment. Data are expressed as mean±s.e.m. of three independent experiments with each performed in triplicate. *P<0.05 compared with WT or each variant human MC4R without Ipsen 17 treatment using the one-sample unpaired t-test (GraphPad Prism5). (c) HEK 293 cells stably containing the empty vector pcDNA3.1 or expressing WT or defective MC4R were treated with vehicle alone or Ipsen 17 (1 μM) for 12 h. Cells were collected, fixed, and immunostained. The rescued receptor at the cell surface was detected by confocal microscopy as described in Materials and methods. + and − represent incubation with or without the presence of Ipsen 17 respectively.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-14-0005

In this study, HEK 293 cells stably expressing N-terminal HA-tagged WT or each of the variant human MC4R proteins were generated and treated with 1 μM Ipsen 17 for 12 h. By quantifying the cell-surface receptor levels of each individual stable cell by FACS assay, relative cell-surface expression of the WT and each variant receptor were assessed. As shown in Fig. 2b and Table 1, by defining the cell-surface expression level of the WT human MC4R in the absence of Ipsen 17 as 100% and using this as a reference, we determined that the defective human MC4R proteins showed a cell-surface-expression efficiency ranging from 9 to 39% in a receptor-variant-specific pattern. In the presence of Ipsen 17, however, all variant receptors except P299H showed a cell-surface expression efficiency ranging from 134 to 179%. MC4R Ipsen 17 treatment could even increase the cell-surface expression efficiency of the WT human MC4R from 100 to 189%. In agreement with this observation, immunofluorescence microscopy revealed a significant increase in the cell-surface expression of the receptors (except for P299H) when treated with Ipsen 17 (Fig. 2c). In the case of P299H, Ipsen 17 did not restore its cell-surface expression, indicating the failure of this variant to respond to Ipsen 17. As reported previously, the fact that P299H did not respond to several other MC4R pharmacological chaperones suggested that P299H represents a mutant defective in intracellular trafficking and its cell-surface targeting cannot be rescued by pharmacological chaperones (Rene et al. 2010). Other than this, Ipsen 17 is able to rescue the cell-surface expression of 11 selected intracellularly retained human MC4R variants, indicating that Ispen 17 acts as a pharmacological chaperone of human MC4R and the pharmacological chaperone effect of Ipsen 17 on human MC4R is not mutant specific in general.

Table 1

Ipsen 17-mediated recovery of cell-surface expression of human MC4 receptors. Data are expressed as mean±s.e.m. of the number of experiments indicated (n). Cell-surface expression of WT receptor without Ipsen 17 treatment was defined as 100%

Receptor Cell-surface expression (percentage of value for WT without Ipsen 17 treatment)
n Control Ipsen 17 (1 μM, 12 h)
WT human MC4R 3 100 189±11*
S58C 3 39±8 179±9*
E61K 3 21±6 145±6*
N62S 3 16±7 151±3*
I69T 3 31±8 162±8*
P78L 3 9±3 145±3*
C84R 3 12±3 139±5*
G98R 3 13±2 159±8*
T162I 3 19±5 172±8*
R165W 3 13±6 134±9*
W174C 3 12±6 142±9*
C271Y 3 25±5 174±7*
P299H 3 19±7 20±8

*Significantly different from the corresponding receptor variant without Ipsen 17 treatment as control, P<0.05.

Signaling response of WT and variant receptors following Ipsen 17-mediated cell-surface targeting

Once it had been demonstrated that Ipsen 17 restores cell-surface expression of the intracellularly retained human MC4R proteins, we then investigated if these mutant receptors were now able to signal in response to agonist stimulation. To do so, the established stable HEK 293 cells were treated with 1 μM Ipsen 17 for 12 h before stimulation with 1 μM of the natural agonist, α-MSH, to examine intracellular cAMP accumulation. As shown in Fig. 3 and Table 2, Ipsen 17 could not restore the cell-surface expression of the P299H variant. Thus, P299H did not respond to α-MSH stimulation and therefore showed a loss-of-function phonotype as expected (Rene et al. 2010). Ipsen 17 was also not capable of restoring α-MSH-stimulated cAMP response of the G98R variant despite the fact that Ipsen 17 restored its cell-surface expression to a level much higher than that for the WT receptor (see Fig. 2b and Table 1).

Figure 3
Figure 3

Functional rescue of the defective human MC4R variants following Ipsen 17 treatment. HEK 293 cells stably expressing WT or defective human MC4R were treated with or without Ipsen 17 (1 μM) for 12 h before being stimulated with α-MSH (1 μM) for 45 min. Cyclic AMP level was measured as described in Materials and methods. Values shown are determined as a percentage of the maximal cAMP accumulation of WT receptor under the same stimulation. Data are expressed as mean±s.e.m. of three independent experiments with each performed in triplicate. *P<0.05 compared with WT or each human MC4R variant without Ipsen 17 treatment using the one-sample unpaired t-test (GraphPad Prism5).

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-14-0005

Table 2

Rescue of α-MSH-stimulated cAMP production of the defective human MC4 receptors by Ipsen 17. Data are expressed as mean±s.e.m. of the number of experiments indicated (n). cAMP production of WT receptor without Ipsen 17 treatment was defined as 100%

Receptor cAMP production (percentage of value for WT without Ipsen 17 treatment)
n Control Ipsen 17 (1 μM, 12 h, three washes) Ipsen 17 (1 μM, 12 h, five washes)
WT human MC4R 3 100 105±12 134±11*
S58C 3 16±4 57±8* 67±9*
E61K 3 14±3 89±9* 112±12*
N62S 3 9±2 74±9* 98±9*
I69T 3 3±1 64±7* 92±9*
P78L 3 5±1 67±6* 87±11*
C84R 3 4±1 78±9* 112±13*
G98R 3 6±2 7±8 5±2
T162I 3 9±2 90±6* 107±10*
R165W 3 6±1 82±7* 109±12*
W174C 3 7±2 98±6* 112±12*
C271Y 3 5±1 26±4* 38±4*
P299H 3 3±1 4±1 4±1

*Significantly different from the value for the corresponding receptor without Ipsen 17 treatment as control, P<0.05.

The other ten mutant receptors were able to respond to α-MSH stimulation, and dramatic cAMP accumulation was detected following Ipsen 17 treatment. Defining the signaling response of the WT human MC4R without Ipsen 17 treatment as 100% and, using it as a reference, the signaling response of the WT human MC4R treated with Ipsen 17 was increased to 105% (Fig. 3 and Table 2). In the absence of Ipsen 17, the mutant receptors responded to the agonist stimulation with a signaling response ranging from 3 to 16%. After Ipsen 17 treatment, all receptors showed a variant-dependent enhancement in signaling response ranging from 26 to 98%. It was interesting that the signaling response for both WT and defective receptors was increased to an even higher level, ranging from 38 to 112%, if an extensive wash step (five washes) was performed before α-MSH stimulation, confirming that Ipsen 17 occupied the binding site and prevented the agonist from binding the receptor (Poitout et al. 2007). Taken together, these results show that the tested human MC4R variants except for G98R contain a change that causes a defect in intracellular trafficking/maturation of the receptors but does not affect ligand binding/signaling. Once they reach the cell surface, these mutant receptors respond to the agonist stimulation and the signaling can be restored to different levels in a variant-specific manner when compared with the WT receptor.

Pharmacological characterization of Ipsen 17 as a MC4R pharmacological chaperone

To investigate the potency and efficiency of Ipsen 17 for restoring cell-surface expression of the WT and the variant human MC4R proteins, HEK 293 cells stably expressing the WT or each of ten abnormal human MC4R variants including S58C, E61K, N62S, I69T, P78L, C84R, T162I, R165W, W174C, and C271Y were treated with various concentrations of Ipsen 17 (104 to 1011 M) over a 12 h time period. This process was followed by FACS assay to quantify the cell-surface expression of the receptors. As shown in Fig. 4 and Table 3, Ipsen 17 restored the cell-surface expression of each mutant receptors in a dose-dependent manner that was maximal at an effective concentration (EC50) of approximately 10−8 M. It was also noteworthy that the EC50 of Ipsen 17 required to elicit a maximal recovery (relative to WT level without Ipsen 17 treatment) was different for each of the receptor variants (Table 3). This result demonstrates that the Ipsen 17-mediated cell-surface targeting of the defective human MC4R proteins is strictly dependent on the concentration of Ipsen 17 and the potency of Ipsen 17 for rescuing the defective human MC4R proteins are mutant dependent.

Figure 4
Figure 4

Pharmacology characterization of Ipsen 17 as a MC4R pharmacological chaperone. HEK 293 cells stably expressing WT or variant human MC4R were treated with various concentrations of Ipsen 17 (104 to 1011 M) for 12 h. At the indicated time points, cells were collected, fixed, and immunostained for quantification of the cell-surface expression of WT and mutant receptors by FACS assay as described in Materials and methods. Data are expressed as mean±s.e.m. of three independently generated experiments with each performed in triplicate.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-14-0005

Table 3

Pharmacological analysis of Ipsen 17 for rescue of cell-surface expression of defective human MC4 receptors. Data are expressed as mean±s.e.m. of the number of experiments indicated (n). EC50 is the concentration of Ipsen 17, which results in 50% maximal cell-surface expression of each mutant receptor. Cell-surface expression of WT receptor without Ipsen 17 treatment was defined as 100%

Receptor Cell-surface expression
n Concentration EC50 (nM) Maximal cell-surface expression (percentage of WT without Ipsen 17 treatment)
S58C 3 20.45±0.13 180±3
E61K 3 22.94±0.13 153±6
N62S 3 60.37±0.13 164±4
I69T 3 36.41±0.13 171±6
P78L 3 32.48±0.17 146±6
C84R 3 21.15±0.16 142±3
G98R 3 4.54±0.26 154±6
T162I 3 3.64±0.13 168±3
R165W 3 9.12±0.13 131±7
W174C 3 11.84±0.22 138±7
C271Y 3 46.42±0.21 178±8
P299H 3 NA NA

Pharmacological characterization of the mutant receptors following Ipsen 17 treatment

As Ipsen 17 efficiently rescued the largest subset of human MC4R variants, it emerges as a potential lead for the development of a therapeutic pharmacological chaperone to treat obese patients bearing defective human MC4R. To better understand the signaling properties of the variant receptors following Ipsen 17 treatment (1 μM, 12 h), the dose–response curves for α-MSH-induced accumulation of cAMP were assessed for the WT and ten variant human MC4R proteins including S58C, E61K, N62S, I69T, P78L, C84R, T162I, R165W, W174C, and C271Y. As shown in Fig. 5, signaling of the human MC4R variants genes is significantly increased after Ipsen 17 treatment. However, the curves for the WT and variant human MC4R proteins after Ipsen 17 treatment were right-shifted compared with the untreated WT receptor, with EC50 values increasing by two- to 32-fold in a variant-dependent way (Table 4). These reduced potencies were observed despite extensive wash steps (five washes, data not shown) most probably because Ipsen 17 reduces the effects of α-MSH by competing to bind to the same binding site (Poitout et al. 2007). Alternatively, the persistence of the WT and the rescued mutant receptor functionality could be reduced compared with untreated WT receptor as a consequence of an accelerated loss of cell-surface receptor upon activation.

Figure 5
Figure 5

Pharmacology of WT and variant human MC4R proteins following Ipsen 17 treatment. HEK 293 cells stably expressing WT or variant human MC4R were treated with Ipsen 17 (1 μM) for 12 h before being stimulated with various concentrations of α-MSH (10−5 to 10−12 M) for 45 min. At the indicated time points, cAMP was measured as described in Materials and methods. Values shown are determined as a percentage of the maximal cAMP accumulation of the WT receptor under the same hormonal stimulation. Data are expressed as mean±s.e.m. of three independently generated experiments with each performed in triplicate.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-14-0005

Table 4

Pharmacology of WT and variant human MC4R proteins following Ipsen 17 treatment. Data are expressed as mean±s.e.m. of the number of experiments indicated (n). The Emax of WT human MC4R without Ipsen 17 treatment was defined as 100% (control) and was calculated from nine experiments (n). EC50 is the concentration of α-MSH required to result in 50% stimulation of the maximal response

Receptor cAMP accumulation (1 μM Ipsen 17, 12 h)
n Emax (percentage of WT control) EC50 (nM)
WT (control) 9 100 1.79±0.08
WT 3 128±5 3.18±0.15*
S58C 3 63±4* 11.85±0.21*
E61K 3 88±3 12.28±0.09*
N62S 3 66±2* 11.77±0.15*
I69T 3 88±6 11.99±0.21*
P78L 3 85±6 12.83±0.23*
C84R 3 108±6 7.71±0.17*
T162I 3 104±6 22.67±0.18*
R165W 3 110±6 55.49±0.15*
W174C 3 107±6 13.55±0.18*
C271Y 3 38±2* 19.78±0.20*

*Significantly different from the WT receptor without Ipsen 17 treatment as control, P<0.05.

To compare the kinetics of cell-surface residency of the treated WT receptor and the rescued receptors S58C, E61K, N62S, I69T, P78L, C84R, T162I, R165W, W174C, and C271Y with that of the untreated WT receptor, we determined their kinetics of agonist-promoted endocytosis after a 12 h pretreatment with 1 μM Ipsen 17. As shown in Fig. 6, in response to α-MSH stimulation, both the WT and the defective human MC4R proteins were able to internalize following pretreatment with Ipsen 17, albeit with a variant-dependent reduced maximum level (Imax) and rate compared with the WT receptor without receiving Ipsen 17 treatment (Table 5). This result demonstrates that, following Ipsen 17 treatment, the rescued mutant receptors and the WT receptor are able to internalize with similar kinetics, indicating that the dynamics of mutant receptors once at the cell surface are identical to those of the WT receptor. It is therefore unlikely that the difference in the apparent potency of Ipsen 17 results from distinct kinetics of cell-surface residency of the rescued receptor. However, it was also obvious that the efficiency of α-MSH to mediate this effect is compromised by a reduced potency due to the incomplete removal of Ipsen 17 from the receptor.

Figure 6
Figure 6

Internalization of WT and variant human MC4R proteins following Ipsen 17 treatment. HEK 293 cells stably expressing WT or variant human MC4R were stimulated with α-MSH (1 μM) for 45 min following treatment with Ipsen 17 (1 μM) for 12 h. Cells were collected, fixed, and immunostained for quantification of the cell-surface expression of WT and mutant receptors by FACS assay as described in Materials and methods. Values are expressed as a percentage of the cell-surface expression of WT receptor with Ipsen 17 treatment. Data are expressed as mean±s.e.m. of three independent experiments with each performed in triplicate.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-14-0005

Table 5

Internalization of WT and variant human MC4R proteins stimulated by α-MSH following Ipsen 17 treatment. Data are expressed as mean±s.e.m. of the number of experiments indicated (n). The Imax is the maximal internalization, representing the percentage of the receptor that was internalized upon α-MSH stimulation. Time T1/2 refers to the time that is required for 50% of the cell-surface receptor to be internalized. WT (control) receptor was treated with Ipsen 17 before α-MSH stimulation and the initial cell-surface expression level was defined as 100%

Receptor α-MSH-stimulated human MC4R internalization (1 μM)
n Imax (percentage of initial cell-surface expression level) Time T1/2 (min)
WT (control) 9 42±6 3.47±0.85
S58C 3 48±7 4.93±1.14
E61K 3 55±5 4.34±1.03
N62S 3 54±8 4.76±0.92
I69T 3 59±4 6.30±1.24
P78L 3 49±5 4.07±0.91
C84R 3 40±4 7.26±0.98
T162I 3 56±9 6.20±1.22
R165W 3 36±5 6.67±1.22
W174C 3 49±6 6.64±1.21
C271Y 3 58±7 4.03±0.64

Discussion

Over the past 4 years, seven synthesized small-molecule antagonists or inverse agonists of MC4R, including ML00253764, APB, MTHP, PPPone, MPCI, DCPMP, and NBP, have been reported to act as pharmacological chaperones to rescue the cell-surface targeting of obesity-causing MC4R proteins defective in intracellular trafficking (Fan & Tao 2009, Granell et al. 2010, Rene et al. 2010, Dong & Fan 2013). Pharmacological characterization of these chemicals revealed that they either show limited rescue ability or have a narrow rescue profile on intracellularly retained MC4R variants, suggesting that they may not be suitable to act directly as a therapeutic option to treat morbid obesity caused by MC4R deficiency (Fan & Tao 2009, Granell et al. 2010, Rene et al. 2010, Dong & Fan 2013). For instance, in the presence of ML00253764 (10 μM), the cell-surface-expression efficiency of the C84R and W174C receptor variants was only increased to 35% of that of the untreated WT human MC4R (Fan & Tao 2009). In this study, we investigated the ability of ML00253764 to mediate this increase by treating the mutant receptors C84R and W174C with various concentrations of ML00253764 (10−3 to 1010 M) over a 12 h time period. As shown in Fig. 7a, ML00253764 was able to increase the cell-surface expression of each mutant in a dose-dependent manner, which was maximal at a concentration of 10−4 M, when incubated with ML00253764, and the maximal cell-surface expression of the mutant receptors was only increased to approximately 65% of that of the untreated WT human MC4R, showing that ML00253764 rescues the cell-surface expression of the defective MC4R variants with a much lower efficiency than Ipsen 17 (Fig. 7a). Our current study identified the nonpeptidic high-affinity MC4R-selective antagonist Ipsen 17 (Poitout et al. 2007) as a novel MC4R pharmacological chaperone. As tested on as many as 12 distinct mutant receptors defective in intracellular trafficking, Ipsen 17 rescued the cell-surface expression of 11 mutant receptors. Incubation of these mutant receptors with Ipsen 17 rescued their cell-surface expression to a level almost superior to that observed for untreated WT receptor, revealing that Ipsen 17 has a broad rescue spectrum and is very efficient for rescuing the cell-surface expression of mutant receptors. As for the D299H variant, this receptor did not respond to several agents including MTHP, PPPone, MPCI, DCPMP, NBP (Rene et al. 2010), and Ipsen 17 (this study), indicating that the replacement of an aspartic acid residue with a histidine residue at amino acid position 299 of human MC4R in transmembrane domain 7 (Fig. 1) probably disrupted the correct folding process of the receptor and made P299H resistant to pharmacological chaperone rescue. More importantly, our data showed that Ipsen 17 rescued the cell-surface expression of these mutant receptors at an EC50 value of approximately 10−8 M, 100-fold lower than that of ML00253764 as determined in this study (Fig. 7a). It was obvious that Ipsen 17 is one of the most potent of the pharmacological chaperone compounds of the human MC4R tested so far and described in the literature (Fan & Tao 2009, Granell et al. 2010, Rene et al. 2010, Dong & Fan 2013).

Figure 7
Figure 7

ML00253764-mediated cell-surface expression and signaling of the intracellularly trapped MC4R proteins C84R and W174C. (a) HEK 293 cells stably expressing the WT, C84R, or W174C human MC4R were treated with various concentrations of ML00253764 (103 to 1010 M) for 12 h. At the indicated time points, cells were collected, fixed, and immunostained for quantification of the cell-surface expression of WT and variant receptors by FACS assay as described in Materials and methods. Data are expressed as mean±s.e.m. of three independent experiments with each performed in triplicate. (b) HEK 293 cells stably expressing the WT, C84R, or W174C human MC4R were treated with ML00253764 (10 μM) for 12 h before being stimulated with various concentrations of α-MSH (10−5 to 10−12 M) for 45 min. At the indicated time points, cAMP was measured as described in Materials and methods. Values shown were determined as a percentage of the maximal cAMP accumulation of WT receptor under the same hormonal stimulation. Data are expressed as mean±s.e.m. of three independently generated experiments with each performed in triplicate.

Citation: Journal of Molecular Endocrinology 53, 1; 10.1530/JME-14-0005

Following Ipsen 17 treatment, all the receptor variants except G98R displayed a dramatically increased signaling capacity upon α-MSH stimulation as a direct consequence of their increased surface expression (Fig. 3), confirming that rescued receptors were in a conformation that can respond to agonist stimulation and transduce signals. Differences in the extent of cAMP response were also observed. For instance, N62S, I69T, and P78L showed maximal cAMP production at a level similar to that of the untreated WT receptor. E61K, C84R, T162I, R165W, and W174C had maximal cAMP production levels superior to that of the untreated WT receptor. S58C and C271Y, however, were not fully rescued functionally. In the case of G98R, Ipsen 17 could not restore the α-MSH-stimulated cAMP response, indicating that the replacement of a glycine with an arginine at the amino acid 98 position of human MC4R in transmembrane domain 2 (Fig. 1) not only disrupted the correct folding process of the receptor but also caused this mutant form of the receptor to be resistant to agonist stimulation. It was also noteworthy that, although Ipsen 17 is very efficient for rescuing the cAMP production of these mutants, it only restored the majority of the mutants at an EC50 value of approximately 10 nM (e.g., R165W has an EC50 of 55 nM), several-fold higher than that of untreated WT receptor in a variant-specific pattern (Fig. 4 and Table 4). This most probably results from persistent Ipsen 17 binding because of its high affinity at a nanomolar level (Poitout et al. 2007), which prevents subsequent stimulation by α-MSH. This hypothesis is also supported by the observation in this study that treatment of the WT human MC4R with ML00253764 resulted in an EC50 value that is 10-fold to 20-fold higher than that of the untreated WT and mutant receptors C84R and W174C upon α-MSH stimulation (Fig. 7b). Despite that, the variant human MC4R proteins were able to internalize almost as normal, although a reduced level of internalized receptor was detected for both WT and the abnormal variants with Ipsen 17 treatment.

Our data also indicated that incubation with Ipsen 17 increased the cell-surface expression of the WT receptor to a level almost twofold higher than that observed for the untreated WT receptor (Fig. 2b and Table 1). Incubation of the WT receptor with Ipsen 17 caused α-MSH-stimulated cAMP production at a level 1.3-fold higher than that of the untreated WT receptor. The signaling potency (EC50) was determined to be 3.18 nM, only twofold higher than that of the untreated WT receptor (Fig. 5 and Table 4). It was thus concluded that Ipsen 17 has a sufficiently high affinity (also see Poitout et al. (2007)) for the receptor but a relatively rapid dissociation rate when compared with ML00253764 (Fig. 7b) that allows washout and competition by the endogenous ligand. In addition, Ipsen 17 is sufficiently lipophilic to penetrate cell membranes and as potent and efficacious as possible on the largest subset of receptor mutant forms. Considering these characteristics of Ipsen 17 and the current modestly effective strategies for controlling obesity, this chemical represents an interesting lead compound for the development of a therapeutically useful pharmacological chaperone that targets human MC4R. Nevertheless, our data clearly showed that, although having a broad efficacy toward many mutants, Ipsen 17 could better rescue some mutations (e.g., E61K, N62S, C84R, T162I, R165W, and W174C in this study), indicating that this chemical may play an important role in the establishment of pharmacological chaperones as personalized therapeutics for treating obese patients bearing a specific variant of human MC4R.

The currently examined pharmacological chaperones including ML00253764, MTHP, PPPone, MPCI, DCPMP, and NBP are all MC4R antagonists/inverse agonists (Poitout et al. 2007, Rene et al. 2010). These compounds compete with the natural agonist of the receptor for binding (Rene et al. 2010, Dong & Fan 2013), thus affecting the receptor signaling to different extents and calling into question their potential for clinical application. Given that allosteric pharmacological chaperones can rescue the cell-surface expression and function of the mutant receptors but do not compete with the natural receptor agonist to induce signaling, pharmacological chaperone compounds of this kind should be taken into consideration for future investigation. In addition, a recent study has shown that different inverse agonists are responsible for the activation of distinct signaling pathways (Mo & Tao 2013). It will be interesting to monitor the action of various pharmacological chaperones on the ability of abnormal human MC4R proteins to activate signaling effectors other than the canonical Gs–adenylyl cyclase signaling pathway.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was jointly supported by the Natural Science Foundation of Tianjin City (#13JCYBJC41900), Tianjin University of Science and Technology (#20130420), China, International Cooperation Project (#2013DFA31160), The Innovative Team Project (#IRT1166), and Obesita and Algaegen LLC, USA.

Author contribution statement

Z-C F designed the research study. H-M W and P Y designed and synthesized the Ipsen 17. X-H W, B-L Z, and Z-C F performed the research and analyzed the data. Z-C F wrote the paper.

Acknowledgements

We sincerely thank Dr Hua Sun (Tianjin University of Science and Technology, Tianjin, CHhina) and Dr Zhaohui Wang (Shenzhen Institutes of Advanced Technology, Chinese Academy of Science, Shenzhen, Guangdong, China) for their useful discussion during the preparation of the manuscript.

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  • Ulloa-Aguirre A & Michael Conn P 2011 Pharmacoperones: a new therapeutic approach for diseases caused by misfolded G protein-coupled receptors. Recent Patents on Endocrine, Metabolic & Immune Drug Discovery 5 1324. (doi:10.2174/187221411794351851).

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  • Vos TJ, Caracoti A, Che JL, Dai M, Farrer CA, Forsyth NE, Drabic SV, Horlick RA, Lamppu D & Yowe DL et al. 2004 Identification of 2-[2-[2-(5-bromo-2- methoxyphenyl)-ethyl]-3-fluorophenyl]-4,5-dihydro-1H-imidazole (ML00253764), a small molecule melanocortin 4 receptor antagonist that effectively reduces tumor-induced weight loss in a mouse model. Journal of Medicinal Chemistry 47 16021604. (doi:10.1021/jm034244g).

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*

(X-H Wang and H-M Wang contributed equally to this work)

 

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    Schematic of human melanocortin 4 receptor. The positions of the engineered HA epitope tags are indicated as dark circles with white letters. The positions of mutated residues are indicated as white circles with gray letters.

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    Ipsen 17-mediated cell-surface expression of the intracellularly trapped defective MC4R proteins. (a) The molecular structure of Ipsen 17. (b) HEK 293 cells stably expressing WT or defective human MC4R were treated with vehicle alone or Ipsen 17 (1 μM) for 12 h. The cell-surface expression level of the receptors was quantified by FACS assay as described in Materials and methods. Values shown are Ipsen 17-mediated cell-surface expression as a percentage of that for the WT receptor without Ipsen 17 treatment. Data are expressed as mean±s.e.m. of three independent experiments with each performed in triplicate. *P<0.05 compared with WT or each variant human MC4R without Ipsen 17 treatment using the one-sample unpaired t-test (GraphPad Prism5). (c) HEK 293 cells stably containing the empty vector pcDNA3.1 or expressing WT or defective MC4R were treated with vehicle alone or Ipsen 17 (1 μM) for 12 h. Cells were collected, fixed, and immunostained. The rescued receptor at the cell surface was detected by confocal microscopy as described in Materials and methods. + and − represent incubation with or without the presence of Ipsen 17 respectively.

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    Functional rescue of the defective human MC4R variants following Ipsen 17 treatment. HEK 293 cells stably expressing WT or defective human MC4R were treated with or without Ipsen 17 (1 μM) for 12 h before being stimulated with α-MSH (1 μM) for 45 min. Cyclic AMP level was measured as described in Materials and methods. Values shown are determined as a percentage of the maximal cAMP accumulation of WT receptor under the same stimulation. Data are expressed as mean±s.e.m. of three independent experiments with each performed in triplicate. *P<0.05 compared with WT or each human MC4R variant without Ipsen 17 treatment using the one-sample unpaired t-test (GraphPad Prism5).

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    Pharmacology characterization of Ipsen 17 as a MC4R pharmacological chaperone. HEK 293 cells stably expressing WT or variant human MC4R were treated with various concentrations of Ipsen 17 (104 to 1011 M) for 12 h. At the indicated time points, cells were collected, fixed, and immunostained for quantification of the cell-surface expression of WT and mutant receptors by FACS assay as described in Materials and methods. Data are expressed as mean±s.e.m. of three independently generated experiments with each performed in triplicate.

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    Pharmacology of WT and variant human MC4R proteins following Ipsen 17 treatment. HEK 293 cells stably expressing WT or variant human MC4R were treated with Ipsen 17 (1 μM) for 12 h before being stimulated with various concentrations of α-MSH (10−5 to 10−12 M) for 45 min. At the indicated time points, cAMP was measured as described in Materials and methods. Values shown are determined as a percentage of the maximal cAMP accumulation of the WT receptor under the same hormonal stimulation. Data are expressed as mean±s.e.m. of three independently generated experiments with each performed in triplicate.

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    Internalization of WT and variant human MC4R proteins following Ipsen 17 treatment. HEK 293 cells stably expressing WT or variant human MC4R were stimulated with α-MSH (1 μM) for 45 min following treatment with Ipsen 17 (1 μM) for 12 h. Cells were collected, fixed, and immunostained for quantification of the cell-surface expression of WT and mutant receptors by FACS assay as described in Materials and methods. Values are expressed as a percentage of the cell-surface expression of WT receptor with Ipsen 17 treatment. Data are expressed as mean±s.e.m. of three independent experiments with each performed in triplicate.

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    ML00253764-mediated cell-surface expression and signaling of the intracellularly trapped MC4R proteins C84R and W174C. (a) HEK 293 cells stably expressing the WT, C84R, or W174C human MC4R were treated with various concentrations of ML00253764 (103 to 1010 M) for 12 h. At the indicated time points, cells were collected, fixed, and immunostained for quantification of the cell-surface expression of WT and variant receptors by FACS assay as described in Materials and methods. Data are expressed as mean±s.e.m. of three independent experiments with each performed in triplicate. (b) HEK 293 cells stably expressing the WT, C84R, or W174C human MC4R were treated with ML00253764 (10 μM) for 12 h before being stimulated with various concentrations of α-MSH (10−5 to 10−12 M) for 45 min. At the indicated time points, cAMP was measured as described in Materials and methods. Values shown were determined as a percentage of the maximal cAMP accumulation of WT receptor under the same hormonal stimulation. Data are expressed as mean±s.e.m. of three independently generated experiments with each performed in triplicate.

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    • Export Citation