Functional Characterization and Structural Modeling of Obesity ...

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Functional Characterization and Structural Modeling of Obesity Associated Mutations in the Melanocortin 4 Receptor Karen Tan,* Irina D. Pogozheva,* Giles S. H. Yeo, Dirk Hadaschik, Julia M. Keogh, Carrie Haskell-Leuvano, Stephen O’Rahilly, Henry I. Mosberg, and I. Sadaf Farooqi University of Cambridge Metabolic Research Laboratories (K.T., G.S.H.Y., D.H., J.M.K., S.O., I.S.F.), Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom; Department of Medicinal Chemistry (I.D.P., H.I.M.), College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065; and Departments of Medicinal Chemistry and Pharmacodynamics (C.H.-L.), University of Florida, Gainesville, Florida, 32610

Mutations in the melanocortin 4 receptor (MC4R) gene are the most common known cause of monogenic human obesity. The MC4R gene was sequenced in 2000 subjects with severe early-onset obesity. We detected seven different nonsense and 19 nonsynonymous mutations in a total of 94 probands, some of which have been reported previously by others. We functionally characterized the 11 novel obesity associated missense mutations. Seven of these mutants (L54P, E61K, I69T, S136P, M161T, T162I, and I269N) showed impaired cell surface trafficking, reduced level of maximal binding of the radioligand [125I]NDP-MSH, and reduced ability to generate cAMP in response to ligand. Four mutant MC4Rs (G55V, G55D, S136F, and A303T) displayed cell surface expression and agonist binding similar to the wild-type receptor but showed impaired cAMP production, suggesting that these residues are likely to be critical for conformational rearrangement essential for receptor activation. Homology modeling of these mutants using a model of MC4R based on the crystal structure of the ␤2-adrenoreceptor was used to provide insights into the possible structural basis for receptor dysfunction. Transmembrane (TM) domains 1, 3, 6, 7, and peripheral helix 8 appear to participate in the agonist-induced conformational rearrangement necessary for coupling of ligand binding to signaling. We conclude that G55V, G55D, S136F, and A303T mutations are likely to strengthen helix-helix interactions between TM1 and TM2, TM3 and TM6, and TM7 and helix 8, respectively, preventing relative movement of these helices during receptor activation. The combination of functional studies and structural modeling of naturally occurring pathogenic mutations in MC4R can provide valuable information regarding the molecular mechanism of MC4R activation and its dysfunction in human disease. (Endocrinology 150: 114 –125, 2009)

he melanocortin 4 receptor (MC4R) is a G protein-coupled receptor (GPCR) and is positively coupled to adenylate cyclase via the stimulatory heterotrimeric G proteins. MC4R is expressed predominantly in the brain and is known to play a role in energy homeostasis (1). Mutations in MC4R have been reported in up to 6% of patients with severe early-onset obesity (2) and are found at a frequency of approximately one of 1000 in the general United Kingdom population (3), making this one of the most common human monogenic diseases. In families carrying MC4R mutations, obesity is inherited in a codominant manner with variable expression and penetrance in heterozygous carriers

T

(2, 4, 5). To date, more than 70 missense mutations at over 60 different positions within the human MC4R have been reported (4, 6 – 8). Most naturally occurring disease-causing MC4R mutations disrupt expression and trafficking of the receptor to the cell surface (4, 6, 8). Numerous mutations are defective in agonist binding, and some also alter relative potency of antagonists (6, 8 –11). L250Q MC4R was found to display constitutive activation with presumed accelerated receptor down-regulation (12). The N-terminal domain of MC4R has been suggested to function as a tethered intramolecular partial agonist that maintains constitutive activity of the receptor (13). Mutations have also been

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2008-0721 Received May 15, 2008. Accepted September 10, 2008. First Published Online September 18, 2008 * K.T. and I.D.P. contributed equally to this work.

Abbreviations: AGRP, Agouti-related protein; BMI, body mass index; DPBS, Dulbecco’s PBS; ER, endoplasmic reticulum; EXL, extracellular loop; FACS, fluorescence-activated cell sorting; GOOS, Genetics of Obesity Study; GPCR, G protein-coupled receptor; HEK293, human embryonic kidney 293; ICL, intracellular loop; MC4R, melanocortin 4 receptor; NDP, 关Nle4, D-Phe7兴; SDS, SD score; 3D, three-dimensional; TM, transmembrane.

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described that do not appear to impair localization to the plasma membrane and primarily impair the coupling of ligand binding to signaling (6). MC4R belongs to the family A superfamily of GPCRs that also includes rhodopsin and the ␤2-adrenergic receptor, whose high resolution crystal structures have recently been obtained (14, 15). However, there is only 20% homology across GPCRs within family A, and a greater knowledge regarding the structural and functional features of each individual receptor will be needed to gain a full understanding of their specific mechanisms of ligand binding and signal transduction. Similar to other members of family A, MC4R consists of a hydrophobic core of seven transmembrane (TM) ␣-helices, three intracellular loops (ICLs), three extracellular loops (EXLs), an N terminus outside the cell, and an intracellular C terminus (16). Endogenous neuropeptide ligands ␣-MSH, ␤-MSH, and ␥-MSH bind to the cavity within the TM ␣-bundle, resulting in a conformational change and receptor activation. Activation of MC4R can be blocked by endogenous agouti-related protein (AGRP) that acts as a competitive antagonist and/or inverse agonist (17). The Genetics of Obesity Study (GOOS) cohort is a large cohort of patients with severe early-onset obesity (2). We have previously reported the prevalence, functional properties, and phenotypical correlates of MC4R mutations in 500 subjects from this cohort (2, 6). In this study we report the results of direct nucleotide sequencing of the MC4R gene in 2000 newly recruited subjects from the GOOS cohort that resulted in the detection of eleven novel missense mutations. We have functionally characterized those novel mutations and also used homology models of human MC4R to map these mutations in the context of a putative three-dimensional (3D) receptor structure, and to analyze the possible molecular mechanisms underlying receptor dysfunction.

Materials and Methods Human genetic studies Inclusion criteria for the GOOS cohort are severe obesity defined as a body mass index (BMI) SD score (SDS) of more than three, and onset of obesity before 10 yr of age. The MC4R gene was screened for mutations in 2000 subjects with severe early-onset obesity as described previously (2); all mutations were confirmed by direct nucleotide sequencing. DNA and phenotypical information was obtained from family members of the probands, and the genotype of relatives was ascertained by direct nucleotide sequencing. The prevalence of each mutation in an appropriate ethnically matched control group was determined by sequencing (2).

Anthropometric measurements The clinical studies were performed after approval by the local-regional ethics committee of Cambridge. Each subject/parents in the case of children provided written informed consent. All clinical studies were conducted in accordance with the principles of the Declaration of Helsinki. Weight and height were measured barefoot in light clothing. BMI SDSs were calculated using United Kingdom reference data (18).

Generation of mutant MC4R constructs MC4R mutant receptors were generated from wild-type MC4R (6) using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla,


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CA) according to the manufacturer’s protocols. The mutant construct was verified by direct nucleotide sequencing.

Expression of MC4R mutants in human embryonic kidney 293 (HEK293) cells HEK293 cells were maintained in DMEM (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 250 ng/ml Fungizone (Life Technologies, Inc., Gaithersburg, MD). Cells were incubated at 37 C in humidified air containing 5% CO2, and transfections were performed using Lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s protocols. Cells were at 80 –90% confluence on the day of transfection.

Measurement of cAMP accumulation One hundred nanograms of wild-type or mutant MC4R were cotransfected with 100 ng internal control plasmid, Bos-␤-galactosidase, in a 96-well plate. The next day, cells were serum starved overnight, and varying concentrations of ␣MSH (Bachem Americas, Inc., Torrance, CA) were then added for 30 min. The cells were lysed, and a cAMP Biotrak enzyme immunoabsorbent assay kit (Amersham Biosciences Inc., Piscataway, NJ) was used to measure the cAMP produced according to the manufacturer’s protocol. A ␤-galactosidase assay using o-Nitrophenyl-␤-galactopyranoside as substrate was used to assess transfection efficiency. The amount of cAMP produced per well was calculated using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA), and this was normalized to the ␤-galactosidase activity to correct for transfection efficiency. The data were fitted to a sigmoidal dose-response curve using GraphPad Prism 4.0. Data are expressed as amount of cAMP produced per well, means ⫾ SE (n ⱖ 3).

Cell surface expression For fluorescence-activated cell sorting (FACS) analysis of MYCtagged wild-type and mutant MC4Rs in HEK293 cells, cells were dissociated from monolayer culture dishes using 5 mM EDTA in PBS, centrifuged at 300 ⫻ g for 3 min at 4 C, and pelletted cells were resuspended in FACS buffer (0.5% BSA/5 mM EDTA/PBS). The cells were counted, and 1 million cells were used per tube. To determine cell surface receptor expression, the cells were incubated with 0.5 ␮g anti-MYC-Alexa Fluor 488 clone 4A6 antibodies (Upstate Biotechnology Inc., Lake Placid, NY) for 1 h at 4 C, washed twice in 0.5% BSA/PBS, and resuspended in FACS buffer for analysis. To determine total cellular receptor protein expression, the cells were fixed with 2% paraformaldehyde at room temperature for 5 min, permeabilized on ice for 15 min with saponin buffer (0.05% saponin/0.5% BSA/PBS), and subsequently washed with saponin buffer. After centrifugation (300 ⫻ g, 3 min, 4 C), cell aliquots were incubated with 0.5 ␮g anti-MYC-Alexa Fluor 488 clone 4A6 antibodies for 1 h at 4 C to label the total (intracellular and surface) MYC-tagged receptors. After the antibody incubation, the labeled cells were washed twice in saponin buffer and resuspended in FACS buffer for analysis. Vector-transfected cells served as controls for the anti-MYC antibody and were used to set the background for fluorescence staining detection on BD Biosciences FACSCalibur flow cytometers (BD Biosciences, San Jose, CA). The data were collected as both stained cell percentages (either surface or total detected) from a minimum of 10,000 collected events for each sample run. The total cell receptor expression levels were determined using permeabilized cells measuring both cell surface and intracellular protein expression. The cell surface expression levels were determined using nonpermeabilized cells. Cell expression levels are presented relative to the wild-type MC4R control, means ⫾ SE of three to four independent experiments. For immunofluorescence studies, COS-7 cells were seeded onto eightwell chamber slides 24 h before transfection. Cells were transiently transfected with 200 ng N-terminal myc-tagged wild-type or mutant MC4R. The next day the cells were washed four times with Dulbecco’s PBS (DPBS) (Sigma-Aldrich) and fixed for 20 min in 4% paraformaldehyde

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nology), followed by three 10-min washes with blocking buffer. Cells were then incubated for 30 min in blocking buffer containing 1:150 dilution of antimouse IgG Alexa Fluor 594 conjugated antibody (Invitrogen). After washing twice for 10 min each with DPBS, cells were mounted with coverslips using VECTASHIELD Hardset mounting medium with 4⬘,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and viewed using a Zeiss/ AxioVision inverted fluorescence microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY).

Competitive ligand binding assay Ligand binding assays were performed on transiently transfected HEK293 cells. Forty-eight hours after transfection, the cells were washed once in binding buffer and distributed in 96-well plates. Cells were incubated at room temperature for 2 h with 0.05 ml binding buffer containing 15,000 cpm Bolton-Hunter labeled [1251]NDP-MSH (Amersham Biosciences) and the appropriate concentration of unlabeled NDP-␣MSH (Bachem Americas). Nonspecific binding was determined in the presence of 10 ␮M NDP-MSH. The cells were then washed once with 0.1 ml ice-cold binding buffer, once with 0.1 ml ice-cold DPBS, and resuspended in 0.1 ml MicroScint 20 (PerkinElmer, Waltham, MA). Radioactivity was counted using a Packard Topcount Microplate Scintillation Counter according to the manufacturer’s instructions. Data are expressed as a percentage of the maximum counts of [125I]NDPMSH binding to wild-type MC4R. The data were fitted to a one-site competition curve in GraphPad Prism 4.0.

Structural modeling We previously developed 3D models of human MC4R based on the rhodopsin crystal structure and using distance geometry calculations with experimentally derived distance constraints (19, 20). The coordinates of MC4R models in the active conformation in complex with an agonist, ␣MSH, and in the inactive conformation in complex with an inverse agonist, AGRP(87–132) fragment, have been deposited into the Protein Data Bank as the entries 2iqp and 2iqv, respectively. Here, we similarly modeled human MC4R using the crystal structure of the ␤2-adrenoreceptor with the C terminus truncated and ICL3 replaced by T4 lysozyme [2rh1 PDB entry (15)] as a template. Amino acid sequence alignments FIG. 1. Mutations in MC4R. A, A schematic representation of human MC4R with the location of 11 of MC4R and other melanocortin receptors with new mutations identified in this study. B, Amino acid sequence alignment of selected melanocortin ␤2-adreneroceptor and rhodopsin are shown in Fig. receptors and two GPCRs with experimentally obtained 3D structures: bovine rhodopsin 1B. In the new MC4R model, the structure of ICL1, (RHOD_BOVIN) and human ␤2-adrenergic receptor (B2ADR_HUMAN). GenBank accession numbers ICL2, and helix 8 reproduced conformations of corof the aligned melanocortin receptors are as follows: human MC4R AAG35602, mouse MC4R responding loops in ␤2-adrenoreceptor. StructurAAI16958, chicken MC4R BAA25252, zebrafish MC4R AAO24745, human MC5R AAH95531, ally flexible ICL3 was taken from the previous rhohuman MC3R AAH69105, human MC1R AAK58525, and human MC2R AAH69074. Residues where disease-causing mutations occurred are marked by gray. In human ␤2-adrenoreceptor, gray dopsin-based MC4R model because in the template shading indicates mutated sites that cause constitutive activation of receptor uncoupling with G structure, it was substituted by a fusion protein, T4 proteins (23). Mutated sites characterized in the current study are boxed and marked by arrows. lysozyme. EXLs of MC4R were modeled, as deResidues from TM helices in crystal structures and receptor models are underlined. scribed earlier (19). EXL1 (residues 108 –114) was modeled as a strand connecting TM2 and TM3. EXL2 (residues 188 –193) represented a short conin DPBS, followed by four washes with DPBS. Cells were then incubated nection between TM4 and TM5. EXL3 (residues 271–280) was confor 5 min either with DPBS alone for nonpermeabilized staining or with strained by two disulfide bonds, one between Cys277 and Cys271, anDPBS containing 0.2% Triton X-100 for permeabilization. Cells were other between Cys279 and Cys40 from the N terminus (residues 40 – 47) incubated in blocking buffer (10% fetal bovine serum and 2% BSA in in the extended conformation, in accordance with previous mutagenesis DPBS) for 30 min, and then incubated for 1 h in blocking buffer conand computational studies (19). The active conformation of MC4R was taining 1 ␮g/ml anti-myc monoclonal antibody 4A6 (Upstate Biotech-



produced from the model of inactive receptor by rigid-body movement and counterclockwise rotation (as viewed from the extracellular side) of TM6 to satisfy a set of distance constraints between TM3, TM5, and TM6, which were obtained from previously collected data on disulfide cross-linking, spin-labeling studies and design of metal-binding clusters for different GPCRs (21). Particularly, the C␤-C␤ distances between residues from TM5 and TM6 (F216-A244 and F216-L247) were constrained at 5.5 and 7.5 Å, respectively, in accordance with the formation of disulfide bonds between corresponding cysteine residues in m3-muscarinic receptor in the presence of an agonist (22). Superposition of ␤2-adrenoreceptor-based MC4R models with previous rhodopsin-based models resulted in root mean square deviations at 2.21 Å (inactive conformations) and 2.47 (active conformations) for 233 common C␣ atoms. These deviations are mainly related to the difference in helix packing in rhodopsin and ␤2-adrenergic receptor templates. The coordinates of new models are available upon request. Here, we used mainly ␤2-adrenoreceptor-based models to analyze the localization of the mutated residues to propose possible mechanisms of impaired function for the different mutants. Upon substitutions of the corresponding residues, energy minimization of each receptor-agonist complex, with all added hydrogen atoms, was performed using the QUANTA/CHARMm simulation package (Accelrys, Inc., San Diego, CA), with ␧ ⫽ 10 and the adopted basis Newton-Raphson method (100 steps). Throughout the text Ballesteros-Weinstein numbering (45) is used as superscript to the receptor numbering. Thus, the most conserved residue in each TM helix is designated X.50, where X is the TM number, whereas other residues of that helix are numbered relative to the conserved position in ascending or descending order. In MC4R the most conserved residues are: N621.50, D902.50, R1473.50, W1744.50, M2045.50, P2606.50, and P2997.50.

TABLE 1. All MC4R mutations found in 2000 severely obese patients in this study Mutation Frameshift/nonsense Y35X;D37V A ins c.112 TACTT delc148 CTCT delc.211 GTins c279 G ins c.7 GA del c.250 Missense I125K R165Q R165W

No. of probands

Heterozygous/ homozygous

13 6 2 4 6 1 3

Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous

2 2 2 2 2

8 6 4

Heterozygous Heterozygous 3 heterozygous, 1 homozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous 2 Heterozygous, 1 homozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous Heterozygous 3 Heterozygous, 2 homozygous Heterozygous Heterozygous

2 2 7

G238D G252S C271Y P299H I316S L54P G55D

3 3 4 3 4 3 3

G55V E61K I69T S136F S136P M161T T162I

3 8 4 1 1 1 6

I269N A303T

2 3

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Results Detection of missense mutations in human MC4R in obese humans The MC4R gene was sequenced in 2000 subjects from the GOOS cohort, with severe obesity defined as a BMI SDS of more than three, and onset of obesity before 10 yr of age. Seven different nonsense and 19 nonsynonymous mutations were detected in a total of 94 probands (Table 1). The majority were found in heterozygous form, and many have been reported previously. The prevalence of such mutations (5%) in these newly recruited subjects is similar to previous reports based on this cohort (2). None of these variants was found in 200 alleles from nonobese controls. Family material was available in a subset of these probands, and, when information was available, the mutations cosegregated with obesity/overweight (supplemental Fig. 1 and supplemental Table 1, which are both published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). To analyze the conservation of residues substituted by nonsynonymous variants (Fig. 1A) between MC4Rs across species and other melanocortin receptors, we compared 257 sequences of melanocortin receptors from the GPCR database (http://www. gpcr.org/). The sequence alignment of representatives such as mouse, chicken, and zebrafish MC4R, together with sequences from the five different human melanocortin (MC1, MC2, MC3, MC4, and MC5) receptors is shown in Fig. 1B. It appeared that 10 missense mutations affected residues that are highly conserved (75–100% conservation) in the melanocortin receptor

Ref.

2 6 2 4 2

FIG. 2. cAMP accumulation of MC4R mutants. Each point represents the mean ⫾ SE of at least three independent experiments performed in duplicate. A, Mutants that were completely unresponsive to ␣MSH. B, Mutants that displayed partial response to ␣MSH. WT, Wild type.

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family (L54, G55, E61, I69, S136, M161, T162, A303). Less conserved (57% conservation) is I269 located at the lipid-exposed face of the end of TM6. This position is usually occupied by a nonpolar side chain. Signaling properties of MC4R mutants To investigate whether the 11 novel missense mutations in MC4R identified here affect the ability of the MC4R to signal to adenylate cyclase in response to the natural ligand ␣-MSH, we introduced these mutations into a vector that expresses the human MC4R. HEK293 cells were transiently transfected with these MC4R cDNA constructs, and cAMP production was measured using a commercial enzyme immunoabsorbent assay. S136F, S136P, I269N, and A303T MC4R mutants resulted in a complete loss of function as assayed by cAMP generation (Fig. 2A), whereas other mutant MC4Rs (L54P, G55D, G55V, E61K, I69T, M161T, and T162I) showed an impaired ability to generate cAMP in response to an increased concentration of ␣-MSH (Fig. 2B). Potency of ␣-MSH to induce cAMP production was markedly reduced for G55D and G55V mutants (by 30- to 80fold), compared with the other mutants; the corresponding EC50 values are shown in Table 2. Cell surface expression of MC4R mutants Mutant MC4Rs were N-terminally MYC-tagged to allow studies of cell surface expression. To examine whether these mutations affect the trafficking of the receptor to the plasma membrane, we quantitated intracellular and plasma mem-

brane expression by FACS using a fluorescent-tagged antiMYC antibody (Fig. 3). In addition, we visualized cell surface expression in COS-7 cells by immunofluorescence microscopy (supplemental Fig. 2). Two mutants, L54P and S136P, were minimally expressed at the cell surface (⬃20% relative to the wild-type receptor), and their total expression, which accounted for receptors localized either at cell surface or intracellularly, was also largely impaired (Table 2). E61K, I69T, M161T, T162I, and I269N mutant MC4Rs exhibited reduced expression at the cell surface (⬍50% of wild-type receptor), but their total expression was less affected. Only G55V, G55D, S136F, and A303T mutants were expressed at the cell surface to an extent close to wild-type receptors (Fig. 3). Ligand binding of MC4R mutants To assess the ligand binding affinity of mutant MC4Rs, we undertook competitive displacement studies using [125I]NDPMSH. Whole HEK293 cells transiently transfected with vectors expressing wild-type or mutant MC4Rs were used in the competitive binding assays. Cells were exposed to a fixed amount of a tracer, [125I]NDP-MSH, and the ability of increasing concentrations of unlabeled NDP-MSH to displace radioligand was measured. Cells transfected with L54P, E61K, I69T, S136P, T162I, and I269N mutants bound tracer at less than 40% of wild-type receptors (Table 2). A smaller effect on maximal tracer binding was observed for M161T mutant (57% of wild-type receptor). However, the binding affinities of unlabeled NDP-MSH (IC50) were

TABLE 2. Summary of functional characterization of MC4R mutations

Mutation WT Defective expression, normal binding, normal activation (class I) L54P I69T Defective expression, normal binding, defective activation (class II) T162I Defective expression, defective binding, defective activation (class III) E61K S136P M161T I269N Normal expression, normal binding, defective activation (class IV) G55D G55V S136F A303T WT, Wild type.

Total (surface) expression (% of WT); approximate values

Maximum tracer binding (Bmax, %)

NDP-MSH binding IC50 (nM)

Basal cAMP (fmol)

␣-MSH Emax (fmol cAMP)

␣-MSH EC50 (nM)

100

100

9.6 ⫾ 3.7

42.9 ⫾ 4.5

870.7 ⫾ 84.8

3.76 ⫾ 2.9

44 (21) 100 (44)

32.5 ⫾ 31.4 24.8 ⫾ 22.8

8.8 ⫾ 2.5 8.5 ⫾ 3.5

33.1 ⫾ 28.4 89.1 ⫾ 22.4

435.5 ⫾ 65.0 409.3 ⫾ 50.0

7.69 ⫾ 6.2 6.31 ⫾ 5.1

67 (20)

7 ⫾ 13.2

9.2 ⫾ 2.3

39.7 ⫾ 15.8

267.9 ⫾ 51.9

44.0 ⫾ 36.0

72 (25) 37 (15) 95 (35) 70 (50)

39.6 ⫾ 20.4 27.2 ⫾ 20.5 57.4 ⫾ 16 30.1 ⫾ 19.3

⬎200 ⬎100 ⬎100 9.7 ⫾ 3.1

71.3 ⫾ 7.4 30.5 ⫾ 4.7 44.5 ⫾ 6.3 48.5 ⫾ 14.1

172.8 ⫾ 17.9 31.1 ⫾ 8.7 102.6 ⫾ 17.3 47.2 ⫾ 21.1

10.8 ⫾ 8.8

86 (67) 140 (138) 65 (85) 64 (72)

68.6 ⫾ 21 128.5 ⫾ 30.2 118.9 ⫾ 33.7 86.6 ⫾ 5.1

18.9 ⫾ 9.0 16.6 ⫾ 8.4 5.5 ⫾ 2 25.0 ⫾ 18.2

51.4 ⫾ 4.5 78 ⫾ 18.1 32.7 ⫾ 4.9 40.5 ⫾ 6.1

103.7 ⫾ 20.2 1014 ⫾ 95.8 32.4 ⫾ 6.4 65.5 ⫾ 22.2

20.1 ⫾ 16.0

110 ⫾ 90.0 281 ⫾ 140



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which was not included in our models. The model of MC4R in the active state further deviated from the template due to large movement of TM6 (root mean square deviation was 2.09 Å for 233 C␣ atoms).

FIG. 3. Cell surface expression of MC4R mutants. FACS analysis of MYC-tagged wild-type (WT) and mutant MC4Rs. The total cell receptor expression levels were determined using permeabilized cells measuring both cell surface and intracellular protein expression. The cell surface expression levels were determined using nonpermeabilized cells. Expression levels are presented relative to the wild-type MC4R control, means ⫾ SE of three to four independent experiments.

severely affected only for three mutants, E61K, S136P, and M161T (Fig. 4 and Table 2). The reduced values of maximum tracer binding by these seven mutants were mirrored by decreases in their cell surface expression. Four other mutants with high cell surface expression, G55V, G55D, S136F, and A303T, consistently had near-normal total specific binding (Bmax) and binding affinities (Table 2 and Fig. 4). Structural modeling of MC4Rs The homology models of the active and inactive conformations of human MC4R have been previously developed using the structural template of bovine rhodopsin, then the only GPCR with known crystal structure (19, 20). However, recently, crystal structures of other GPCRs, ␤2-adrenoreceptor and squid rhodopsin, have been obtained at 2.4 Å resolution (15). The new structural template of human ␤2-adrenoreceptor appears to be preferable for modeling of MC4R because it has higher sequence homology to human MC4R in comparison to rhodopsins. Therefore, we developed new models of human MC4R using the structure of the human ␤2-adrenoreceptor. The new model of MC4R in the inactive state mostly reproduced the structure of ␤2-adrenoreceptor with kinks and distortions in TM5, TM6, and TM7, but lacked an aneurysm (an extra-residue per helix turn) in TM2. The root mean square deviation between model and template was 1.39 Å for equivalent 233 C␣-atoms from the TM ␣-bundle. The main differences between MC4R model and the ␤2-adrenoreceptor were in the shortened N-terminal end of TM1, outward movement of TM2 by approximately 1.5 Å, and shift of the extracellular part of TM7 toward TM1 by approximately 2.5 Å. Of note, the relative shift of TMs 1, 2, and 7 is clearly seen from comparison of closely related bovine and squid rhodopsin structures. This indicates the structural flexibility of GPCRs in this region, which may facilitate conformational transition in receptors upon receptor activation. In MC4R the binding pocket appears to be more open and accessible for large peptide ligands due to the minimal size of EXL2. However, it cannot be excluded that the binding cavity of MC4R may be partially covered by N-terminal fragment 1-39,

Modeling and analysis of MC4R mutants To reproduce the 11 novel mutations studied here, we made one-by-one substitutions of the corresponding residues in the models of the active conformation of the human MC4R in complex with ␣MSH, followed by energy minimization to remove any substantial hindrance caused by these substitutions. Although our models are approximate, they help to localize mutated residues in context of the receptor structure and to characterize their interactions (Table 3), allowing us to propose possible mechanisms underlying the mutational effects. Figure 5 represents the model of human MC4R in complex with ␣MSH, where 11 currently studied missense mutations are indicated. Models of mutants with decreased cell surface translocation First, we examined the models of the mutants that result in impaired translocation of receptors to the cell surface (Fig. 5, yellow, green, and blue). Our computational analysis suggested that structural alterations caused by these mutations may decrease structural stability of receptor, or disturb its interactions with accessory proteins that assist folding or trafficking. Total and cell surface expression of receptors was minimal for L54P and S136P mutants. The effect of these mutations is likely associated with the appearance of a helix kink either in the upper third of TM1 (L54P) or in the middle of TM3 (S136P), which produces the distortion and slight outward movement of extracellular parts of TM1 or TM3, respectively. This change of helix packing may decrease structural stability of the receptor. Poor cell surface expression of another substitution in TM1, I69T, may also be related to structural destabilization. Indeed, our modeling inferred that TM1 in melanocortin receptors may be shorter from the N terminus by two helix turns than in rhodopsin or ␤2-adrenoreceptor, and, thus, it has less contact with adjacent helices. MC4R models predict that the L54P mutation in addition to helix distortion removes hydrophobic contacts with I289 and C293 in TM7 (Fig. 5H), whereas I69 substitution by smaller and more polar threonine changes van der Waals interactions with Y80, I83, and C84 from TM2 and L75 from ICL1 (Fig. 5E). I69T substitution at the cytoplasmic part of receptor may not only increase flexibility of the end of TM1 but may also alter the conformation of ICL1. On the other hand, E61K mutation introduces hydrogenbonding interactions between TM1 and carbonyl of I296, which is located in TM7 one turn apart from the conserved P299 (Fig. 5I). Thus, E61K may affect packing of TM1 with the adjacent TM7. However, an alternative explanation may be also proposed to explain the effect of E61K mutation. The acidic residue E61 is located at the lipid-exposed receptor surface between TM1 and TM7, and, thus, may represent a potential proteinprotein interaction site. Substitution of E61 to a basic lysine may change electrostatic interactions of the MC4R with other, as yet uncharacterized non-MC4R proteins that may assist normal

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Our models suggest that in accordance with the structure of the ␤2-adrenergic receptor, M161 represents a hydrophobic staple motif, whereas T162 acts as an N-capping residue forming an H bond with N-terminal K164 of TM4 (Fig. 5F). Both these residues stabilize ICL2 and N terminus of TM4 via hydrophobic or hydrogen-bonding interactions. We suggest that substitution of hydrophobic M161 to weakly polar threonine and substitution of T162 to isoleucine lacking hydrogenbonding capacity probably destroy these interactions. Thus, M161T may lead to the displacement of ICL2, whereas T162I may cause the unfolding of the end of TM4. These structural alterations in ICL2 may affect interactions of this loop with proteins that govern folding and transport of receptor between organelles along the exocytic pathway (26, 27). Another mutant with poor cell surface expression, I269N, is localized between the extracellular ends of TM6 and EXL3. I269 forms hydrophobic contacts with nonpolar V193 and I194 from TM5, and is partially exposed to the hydrophobic lipid environment (Fig. 5G). The I269N substitution may change the packing of the extracellular ends FIG. 4. Competitive binding of MC4R mutants (A–F). HEK293 cells transiently transfected with wildtype (WT) or mutant MC4Rs were incubated with [125I]NDP-MSH in the presence of increasing of TM6 and TM5, and, thus, may affect the concentrations of NDP-␣MSH. The ordinate is expressed as a percentage of total specific binding receptor stability, leading to misfolding and (Bmax). Curves are fitted using a nonlinear regression analysis and a one-site competition model loss-of-function. This mechanism is sup(GraphPad Prism). All curves are representative of three or four independent experiments, and each point is the mean of quadruplicate values. ported by a recent observation that the I194T mutation of a contacting residue from MC4R trafficking and/or signaling. In the case of the closely related TM5 caused impaired MC4R expression and signaling and an MC2R, a physical interaction with single-spanning melanocortin obese phenotype in mice (28). The presence of a polar residue in receptor accessory protein 2 has been essential for normal expresthe lipid-exposed surface of TM6 may also alter the orientation sion and function of MC2R (23). One possible candidate for the of the whole receptor relative to the membrane plane that would interaction with MC4R is the single-spanning attractin-like protein, affect protein-protein interaction in membranes. whose C-terminal fragment (1280 –1317) adjacent to the predicted TM ␣-helix (1228 –1254) is able to bind to the fragment 303–313 Models of mutants with reduced binding affinity for encompassing the end of TM7 and the peripheral helix 8 (24). NDP-MSH The deleterious effect of S136P mutation may be also caused We next analyzed S136P, E61K, and M16IT mutants that by the disturbance of potential hydrogen-bond interactions bedemonstrated reduced ligand binding affinities (Fig. 5, blue). The tween polar side chains buried within the TM ␣-bundle. In the MC4R models show that none of these mutated residues is diwild-type MC4R, the hydrogen-bonding network may be 1.46 1.50 rectly involved in ligand binding. Therefore, we suggested that formed by polar residues from: TM1 (S58 , N62 ); TM2 2.40 2.50 2.54 2.57 2.60 3.35 these residues may be essential for structural rearrangements that (Y80 , D90 , S94 , N97 , E100 ); TM3 (S132 , 3.39 3.49 3.50 7.45 7.46 accompany receptor activation. Indeed, the impaired agonist S136 , D146 , R147 ); and TM7 (N294 , S295 , 7.49 7.53 binding in S136P may be associated with the involvement of this D298 , Y302 ). H bonds between some of these side chains residue in the conserved hydrogen bonding network extending are actually present in our models, whereas additional H bonds from the ligand-binding pocket to the putative G-protein binding may be formed via buried water molecules. Indeed, similar hysite that likely rearranges upon receptor activation (21). The drogen-bonding interactions between conserved polar residues 1.50 2.50 7.45 7.46 effect of E61K on ligand binding may be related to the forat positions equivalent to N62 , D90 , N294 , S295 , 7.49 7.53 mation of an H bond between E61K and TM7, which may lock D298 , and Y302 together with five ordered water molereceptor in the inactive-like conformation with low binding cules were found in crystal structures of rhodopsin and ␤2-adrenoaffinity for agonist, although change in interactions with unreceptor (25). identified partner that may affect ligand binding cannot be Two other mutations associated with reduced cell-surface exexcluded in this case. pression, M161T, T162I, are located in the cytoplasmic ICL2.



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TABLE 3. Possible structural effects of MC4R mutations and mechanisms underlying loss-of-function

Location

Class

Maximal cAMP response

L54P

TM1

I

⫹⫹

⫹

⫹

G55D

TM1

IV

⫹

⫹⫹

⫹⫹

G55V

TM1

IV

⫹⫹

⫹⫹⫹

⫹⫹⫹

E61K

TM1

III

⫹

⫹

⫹

I69T

TM1

I

⫹

⫹

⫹

S136P

TM3

III

⫺

⫹

⫹

S136F

TM3

IV

⫺

⫹⫹⫹

⫹⫹⫹

M161T

ICL2

III

⫹

⫹

⫹⫹

T162I

ICL2

II

⫹

⫹

⫹

I269N

EXL3

III

⫺

⫹⫹

⫹

A303T

TM7

IV

⫺

⫹⫹⫹

⫹⫹⫹

Mutation

Cell surface expression

Maximal tracer binding

Predicted structural effect/mechanism underlying loss-of-function Shift of the receptor backbone and loss of contacts between TM1 and TM7 may cause structural instability and misfolding Formation of H bonds between TM1 and TM2 may stabilize the incorrect conformation with decreased efficiency of agonist-induced conformational changes Appearance of hydrophobic contacts between TM1 and TM2 may stabilize the inactive-like conformation with decreased efficiency of agonist-induced conformational changes Formation of an H bond between TM1 and TM7 may stabilize the incorrect conformation and prevent TM7 movement upon activation Possible alteration of interactions with accessory proteins involved in trafficking Reduced interactions between TM1 and TM2 may cause structural flexibility in ICL1, and, thus, may affect folding and trafficking Shift of the receptor backbone may cause the misfolding. Inability to form correctly the H-bond network between TM1, TM2, TM3, and TM7, and to allow H-bond rearrangement during receptor activation Appearance of hydrophobic interactions between TM3 and TM6 may stabilize the inactive-like conformation. Inability to form correctly the H-bond network between TM1, TM2, TM3, and TM7, and to allow H-bond rearrangement during receptor activation Disordering of ICL2 may impair coupling with G proteins and interaction with intracellular proteins involved in trafficking Destabilization and unfolding of N terminus of TM4 may impair coupling with G proteins and interaction with intracellular proteins involved in trafficking Appearance of polar residue in a hydrophobic environment may affect orientation of receptor in the membrane, and change the packing of extracellular ends of TM6 and TM5 that is essential for activation Steric clash between end of TM7 and helix 8 may prevent the agonist-induced movement of helix8

Class I combines mutants with loss of function that demonstrate defective expression, normal binding, and normal activation. Class II includes mutants with defective expression, normal binding, and defective activation. Class III unifies mutants with defective expression, defective binding, and defective activation. Class IV represents mutants with significant loss of function but normal expression and normal binding. ⫺, No activity; ⫹, ⬍50% activity; ⫹⫹, ⬎50% activity; ⫹⫹⫹, 100% activity and potency.

Models of mutants with decreased activation properties Particularly interesting are G55V, G55D, S136F, and A303T mutants (Fig. 5, red), which show impaired cAMP production but display near-normal ligand binding and cell surface expression. Our computational analysis suggested that these mutants may be locked in the inactive-like conformation by interhelical hydrophobic or hydrogen-bonding interactions. In the G55V mutant, the introduction of a branched valine side chain at the interface between TM1 and TM2 may stabilize helix-helix interactions in the inactive-like conformation due to the appearance of new contacts with G98 in TM2 (Fig. 5C). On

the other hand, introduction of a polar residue in G55D mutant allows hydrogen-bond formation between D55 and polar residues from TM2 (N97 and T101) (Fig. 5B). However, the formation of these H bonds requires a slight shift of the extracellular end of TM2 and reorientation of N97, which likely participates in structural rearrangement of the receptor. Thus, stabilization of an inactive or incorrect conformation of these mutant MC4Rs would decrease the efficiency of the agonist-induced conformational changes. The absence of signaling in the S136F mutant likely reflects the inability of S136 substitutes to form correctly the H-bond

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demonstrated decreased cAMP production in response to natural agonist, ␣MSH. Further studies to measure the effects of specific mutants on G-protein activation itself would be of interest. Seven mutants (L54P, E61K, I69T, S136P, M161T, T162I, and I269N) were poorly expressed at the cell surface, whereas expression of other four mutants (G55V, G55D, S136F, and A303T) was near normal (⬎70% of the wild-type receptor). Thus, a significant number of MC4R mutants studied here were likely retained in the endoplasmic reticulum (ER) for degradation. Our observations are consistent with previous studies in which the majority of obesity associated mutations in human MC4R result in receptors that are intracellularly retained (4, 6, 8). During the course of these studies, a single Austrian patient with the S136F mutation was reported. In this paper the mutant MC4R also completely failed to generate cAMP in vitro FIG. 5. Positioning of 11 mutated residues on the models of human MC4R. A, Model of the active (30). conformation of MC4R in complex with natural agonist ␣-MSH. Mutated residues from class I are Notably, for L54P, I69T, and T162I muyellow, from class II are green, from class III are blue, and from class IV are red. Position of hydrophobic tants, the loss of activation of adenylate cymembrane boundaries was shown in accordance with Orientations of Proteins in Membranes database (http://opm.phar.umich.edu). B–I, Packing interactions around mutated residues in the inactive clase was mirrored by the decrease of their conformation are shown for the following MC4R mutants: G55D (B), G55V (C), S136F (D), I69T (E), translocation to the cell surface, whereas for M161T and T162I (F), I269N (G), L54P (H), and E61K and A303T (I). Figures were prepared by PyMOL other mutants, the loss of activation was much (http://www.pymol.org). larger than the reduction of cell surface expression (Table 2). This result indicates that mechanisms undernetwork between TM1, TM2, TM3, and TM7, and to allow lining the loss-of-functions of studied mutants are different. BeH-bond rearrangement during receptor activation. On the other cause a large number of MC4R mutations have been reported to hand, bulky S136F3.39 may also form favorable hydrophobic date and because the understanding of the molecular mechacontacts with nonpolar residues from TM2 (L862.46), TM3 nisms involved has evolved, we considered it would be beneficial (L1403.43), and TM6 (L2506.40, F2546.44) (Fig. 5D). Formation to derive a classification system to identify common mechanisms of a hydrophobic lock formed by tightly packed residues would of receptor dysfunction. Such a system might usefully be used to stabilize the inactive-like conformation and prevent structural identify subgroups of mutations that are associated with a hurearrangements during receptor activation. man disease phenotype through a shared molecular mechanism. Furthermore, the loss of activation in the A303T mutant may Previously, Tao (11) proposed the following system: MC4R mube related to the appearance of steric clash between A303T and tants with defective protein synthesis (class I), intracellularly F310 from C-terminal helix 8 (Fig. 5I) that may prevent the trapped mutants (class II), binding defective mutants (class III), agonist-induced outward movement of the helix 8. Indeed, susignaling defective mutants (class IV), and variants with normal perposition of several available crystal structures of bovine and function (class V). Another classification distinguished MC4R squid rhodopsins and ␤2-adrenergic receptor indicates that the mutants with intracellular retention (class 1), mutants with good position of helix 8 may slightly move (by 2.5 Å) with respect to cell surface expression, but decreased constitutive activity (class cytoplasmic ends of TM1 and TM7. The proposed outward 2B), decreased response to agonist (class 2C), or both (class 2A) movement of helix 8 would increase its accessibility to protein(Govaerts et al., 2005). However, there are shortcomings with protein interactions. This is consistent with the implication that any classification because some mutants may belong to several helix 8 of MC4R can be phosphorylated at T312, and is involved classes at once. Therefore, we propose an alternative classificain desensitization and internalization through protein kinase A-, tion into four categories, which is based on a combination of G protein-coupled receptor kinase-, ␤-arrestin-, and dynaminproperties of the in vitro expressed mutants, such as total and cell dependent processes (29). surface receptor expression, agonist binding, and agonist-induced receptor activation (Table 3). In our classification, class I combines mutants with loss-of-function that demonstrate defecDiscussion tive expression, normal binding, and normal activation. Class II includes mutants with defective expression, normal binding, and We have undertaken functional characterization of 11 novel misdefective activation. Class III unifies mutants with defective exsense mutations in MC4R associated with dominantly inherited pression, defective binding, and defective activation. Class IV human obesity (summarized in Tables 1–3). All mutants studied


represents mutants with significant loss-of function but normal expression and normal binding. All classifications are somewhat arbitrary, but they may be helpful to understand the key molecular mechanism of functional defects and may be relevant to the design of potential therapies. Our homology models of MC4R are helpful to dissect the possible molecular mechanisms underlying the effects of these mutations (Fig. 5). We suggested that decreased cell surface expression of mutants from classes I–III may be related to changes in packing interactions observed in models of these mutants (Table 3) that could kinetically impair receptor folding. In particular, structural destabilization caused by helical distortions, loss of contacts, or formation of improper long-range interactions within TM ␣-bundle observed in models of L54P, I69T, E61K, and S136P mutants may increase the number of protein molecules that fail to compete folding promptly, are retained in the ER and eventually targeted for degradation. On the other hand, our results indicate that loop conformation may also be important for folding and trafficking of MC4R. The impaired cell surface expression of mutants M161T, T162I, and I269N was originally unexpected because these substitutions are localized in conformationally flexible loops. However, we similarly found poor cell surface expression for previously identified R165W and R165Q mutations from the intracellular end of TM4 (6). In addition, decreased cell surface expression has been shown for R151C and R160W mutants from ICL2 of the MC1R associated with red hair color (31). Numerous mutations of MC2R causing glucocorticoid deficiency syndrome were also localized in ICL2: R137W (32), H139Y, V142L (33), and R146H (34). Y157S mutation from ICL2 of MC4R was previously related to childhood obesity (11). The results obtained highlight that the proper structure of ICL2 may be essential for folding, maturation, or trafficking of melanocortin receptors to the cell surface. Indeed, site-directed mutagenesis and truncation studies of GPCRs from family A (e.g. adrenergic and adenosine receptors) suggest that the signals required for appropriate targeting reside within TM or membrane-adjacent ICLs (35). It is possible that specific residue types and particular conformations of ICL2 are required for interaction of MC4R with different proteins (such as Rab, Sar guanosine triphosphatase, chaperones, or adaptor proteins) that are involved in the folding, processing, or cellular transport of GPCRs through the exocytic pathway (26). The decreased cell surface expression of I269N mutant may be also related to alteration of interactions with chaperones from the ER lumen that assist receptor folding. In our study, cell surface expression was near normal only in four mutants from class IV (G55V, G55D, S136F, and A303T). However, for these mutants potency and/or efficiency of ␣MSHinduced cAMP production was reduced. We suggest that the effect of these mutations is associated with the structural stabilization of the receptor conformation that is unable to undergo conformational changes associated with activation. In the case of G55V and G55D mutants, substitutions in TM1 may stabilize the inactive conformation through hydrophobic or hydrogenbonding interhelical contacts, and prevent the relative movement of TM1 and TM2 upon activation, whereas steric clash between A303T from TM7 and F310 from surface helix 8 may hinder


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agonist-induced movement of the helix 8. Furthermore, we propose that S136F mutant from TM3 could lock the receptor in the inactive conformation through reinforcement of interhelical hydrophobic contacts between TM3 (S136F3.39, L1403.43) and TM6 (L2506.40, F2546.44). Therefore, our functional and computational studies suggest that the structure of TM1, TM3, TM7, and ICL2, as well as the relative helix movement in pairs TM1-TM2, TM3-TM6, and TM7-helix 8 are essential for conformational changes during receptor activation. The impaired activation of S136F and S136P mutants may be also related to the participation of conserved S1363.39 in the hydrogen-bonding network between polar residues from TM1, TM2, TM3, and TM7 that likely rearranges upon activation. Indeed, the functional role of a conserved S1363.39 has been demonstrated for different GPCRs from family A (36). Many previously identified obesity associated mutations in MC4R with impaired activation (S58C1.46, N62S1.50, D90N2.50, S94R2.54, N97D2.57, E100A2.60, D294N7.45, S295A7.46, D298N7.49, and P299H7.50) (4, 6, 7, 10, 37) affect residues that are involved in or may be added to this network. Interestingly, N62S1.50 substitution has a much larger negative effect on the binding of an agonist (NDP-MSH) than on the binding of an antagonist (AGRP) (6). This supports the notion that agonist and antagonist-bound forms of the receptor may differ in the relative arrangements of polar residues from this network. In addition, several mutations of residues from this conserved polar cluster within the TM ␣-bundle were implicated in different human diseases. Among them are D85N2.50 mutation of V2R associated with nephrogenic diabetes insipidus (38), E90K2.54 mutation in GnRH receptor that was reported from patients with idiopathic hypogonadotropic hypogonadism (39), MC2R variants S74I2.54, R128C3.50, P273H7.50 (34) that are related to familial glucocorticoid deficiency syndrome, and numerous MC1R polymorphisms associated with red hair color, fair skin, poor ability to tan, and increased susceptibility to melanoma and other skin cancers: D86E2.50, N91D2.57, D294H7.49, R142C3.50, Y298H7.53, and R142H3.50 (40). The large number of uncoupling mutations that has been found in ␤2-adrenergic receptor also involved conserved polar residues from this cluster: D792.50, N3187.45, N3227.49, and Y3267.53 (41). All these data suggest that the proposed rearrangements of H bonds between polar residues within TM ␣-bundle may be essential during agonistinduced structural transitions. Understanding the molecular mechanisms underlying lossof-function of mutants from classes I–III may be relevant for the development of membrane-permeable ligands or “pharmacological chaperones” that specifically bind to the MC4R in the ER and may rescue misfolding-prone mutants. Compelling evidence has been obtained that “pharmacological chaperones” can increase the cell surface expression of several mutant GPCRs and, therefore, may be applied to human diseases that result from misfolding, such as nephrogenic diabetes insipidus or retinitis pigmentosa (42). For example, selective nonpeptidic antagonists of vasopressin receptors (V2R) dramatically increased cell surface expression and restored the function of eight mutant V2Rs associated with nephrogenic diabetes insipidus, presumably by binding to and stabilizing partially folded mu-

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tants (43). Furthermore, several membrane-permeable ligands of ␦-opioid receptors have increased the efficiency of wild-type receptor maturation and trafficking to the cell surface (42). Thus, pharmacological chaperones may be able to rescue the expression and function of different MC4R mutants that belong to classes I–III. In our studies MC4R mutants from class IV fail to activate adenylate cyclase in response to natural peptide agonist ␣MSH at submicromolar concentrations. However, it cannot be excluded that some existing or newly developed synthetic ligands may possess subnanomolar agonist potency at these mutants, as has been found for some previously characterized human MC4R mutations (44). In conclusion, functional characterization of human MC4R mutations can provide insights into which residues are important for trafficking to the cell surface, binding and activation, whereas structural modeling can reveal structural mechanisms underlying the functional effects of mutations and allow us to propose potential approaches for drug development in obesity.

Acknowledgments We thank Emma Lank for technical assistance. Address all correspondence and requests for reprints to: I. Sadaf Farooqi, University of Cambridge Metabolic Research Laboratories, Institute of Metabolic Science, Addenbrooke’s Hospital, Box 289, Hills Road, Cambridge CB2 2QQ, United Kingdom. E-mail: [email protected]. This work was supported by the Wellcome Trust, the Medical Research Council and the National Institute for Health Research Biomedical Research Centre (to I.S.F. and S.O.), and the National Institutes of Health, DA03910 (to H.I.M. and I.D.P.). Disclosure Statement: The authors have nothing to declare.

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