Functional characterization of mutations in melanocortin-4 receptor associated with human obesity

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor implicated in the regulation of body weight. Genetic studies in humans have identified two frameshift mutations of MC4R associated with a dominantly inherited form of obesity. We have generated and expressed the corresponding MC4R mutants in 293T cells and found that cells transfected with the truncation mutants failed to exhibit agonist binding or responsiveness despite retention of structural motifs potentially sufficient for binding and signaling. Immunofluorescence studies showed that the mutant proteins were expressed and localized in the intracellular compartment but absent from the plasma membrane, suggesting that these mutations disrupted the proper cellular transport of MC4R. Further studies identified a sequence in the cytoplasmic tail of MC4R necessary for the cell surface targeting. We further investigated a possible dominant-negative activity of the mutants on wild-type receptor function. Co-transfection studies showed that the mutants affected neither signaling nor cell surface expression of wild-type MC4R. We also characterized three human sequence variants of MC4R, but these exhibited identical affinities for peptide ligands and identical agonist responsiveness. Thus, unlike the obesity-associated MC4R truncation mutants, the polymorphisms of MC4R are unlikely to be contributors to human obesity.     

Functional studies on twenty novel naturally occurring melanocortin-4 receptor mutations

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critically involved in regulating energy balance. MC4R activation results in decreased food intake and increased energy expenditure. Genetic and pharmacological studies demonstrated that the MC4R regulation of energy balance is conserved from fish to mammals. In humans, more than 150 naturally occurring mutations in the MC4R gene have been identified. Functional study of mutant MC4Rs is an important component in proving the causal link between MC4R mutation and obesity as well as the basis of personalized medicine. In this article, we studied 20 MC4R mutations that were either not characterized or not fully characterized. We showed that 11 mutants had decreased or absent cell surface expression. D126Y was defective in ligand binding. Three mutants were constitutively active but had decreased cell surface expression. Eleven mutants had decreased basal signaling, with two mutants defective only in this parameter, suggesting that impaired basal signaling might also be a cause of obesity. Five mutants had normal functions. In summary, we provided detailed functional data for further studies on identifying therapeutic approaches for personalized medicine to treat patients harboring these mutations.     

A Small Molecule Agonist THIQ as a Novel Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

Although mutations in the melanocortin-4 receptor (MC4R) gene cause severe early-onset obesity, we still do not have effective approaches to correct the defects of these mutations. Several antagonists have been identified as pharmacoperones of the MC4R whereas no agonist of the MC4R has been reported. In the present study, we investigated the effect of a small molecule agonist of the MC4R, THIQ, on the cell surface expression and signaling of ten intracellularly retained MC4R mutants using different cell lines. We showed that THIQ increased the cell surface expression of three mutants (N62S, C84R, and C271Y) and two of them (N62S and C84R) had increased signaling in HEK293 cells. Interestingly, THIQ increased the signaling of two other mutants (P78L and P260Q) without increasing their cell surface expression in HEK293 cells. In neuronal cells, THIQ exhibited a more potent effect, correcting the cell surface expression and signaling of seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y). Other mutants were not rescued by THIQ. We also showed that THIQ did not rescue MC4R mutants defective in ligand binding or signaling or one intracellularly retained mutant of the melanocortin-3 receptor. In summary, we demonstrated that a small molecule agonist acted as a pharmacoperone of the MC4R rescuing the cell surface expression and signaling of some intracellularly retained MC4R mutants.     

Functional characterization of nine novel naturally occurring human melanocortin-3 receptor mutations

The melanocortin-3 receptor (MC3R) is a member of family A rhodopsin-like G protein-coupled receptors. Mouse genetic studies suggested that MC3R and the related MC4R are non-redundant regulators of energy homeostasis. Lack of Mc3r leads to higher feed efficiency and fat mass. However, until now only a few MC3R mutations have been identified in humans and the role of MC3R in the pathogenesis of obesity was unclear. In the present study, we performed detailed functional studies on nine naturally occurring MC3R mutations recently reported. We found that all nine mutants had decreased cell surface expression. A260V, M275T, and L297V had decreased total expression whereas the other six mutants had normal total expression. Mutants S69C and T280S exhibited significant defects in ligand binding and signaling. The dramatic defects of T280S might be partially caused by decreased cell surface expression. In addition, we found mutants M134I and M275T had decreased maximal binding but displayed similar signaling properties as wild-type MC3R. All the other mutants had normal binding and signaling activities. Co-expression studies showed that all mutants except L297V did not affect wild-type MC3R signaling. Multiple mutations at T280 demonstrated the necessity of Thr for cell surface expression, ligand binding, and signaling. In summary, we provided detailed data of these novel human MC3R mutations leading to a better understanding of structure-function relationship of MC3R and the role of MC3R mutation in obesity.     

Pharmacological chaperones increase the cell-surface expression of intracellularly retained mutants of the melanocortin 4 receptor with unique rescuing efficacy profiles

Mutated versions of membrane proteins often fail to express at the plasma membrane, but instead are trapped in the secretory pathway, resulting in disease. The retention of these mutant proteins is thought to result from local misfolding, which prevents export from the ER (endoplasmic reticulum), targeting the receptor for degradation via the ER-associated quality control system. The rhodopsin-like G-protein-coupled MC4R (melanocortin 4 receptor) is an example of such a membrane protein. Over 100 natural MC4R mutations are linked with an obese phenotype and to date represent the most common monogenic cause of severe early-onset obesity. More than 80% of these mutations result in a substantial proportion of MC4R being retained intracellularly. If these receptors were expressed at the plasma membrane, many could be functional, as mutations often occur in regions distinct from those associated with ligand or G-protein binding. Our aim is to show proof of concept that selective compounds can rescue the function of MC4R mutants by increasing their cell-surface expression, and further to this, examine whether the rescue profile differs between mutants. Whole-cell ELISA and 96-well fluorescence-based assays with N-terminally HA (haemagglutinin)-tagged and C-terminally mCherry-tagged mutant MC4Rs were used to screen a number of novel MC4R-selective compounds. A total of four related compounds increased the cell-surface expression of wild-type and three intracellularly retained mutant MC4Rs, thus acting as pharmacological chaperones. There appears to be a unique rescue efficacy profile for each compound that does not correlate with potency, suggesting distinct receptor conformations induced by the different mutations. A degree of functionality of V50M and S58C was also rescued following relocation to the cell surface.     

AgRP(83-132) acts as an inverse agonist on the human-melanocortin-4 receptor

The central melanocortin (MC) system has been demonstrated to act downstream of leptin in the regulation of body weight. The system comprises alpha-MSH, which acts as agonist, and agouti-related protein (AgRP), which acts as antagonist at the MC3 and MC4 receptors (MC3R and MC4R). This property suggests that MCR activity is tightly regulated and that opposing signals are integrated at the receptor level. We here propose another level of regulation within the melanocortin system by showing that the human (h) MC4R displays constitutive activity in vitro as assayed by adenylyl cyclase (AC) activity. Furthermore, human AgRP(83-132) acts as an inverse agonist for the hMC4R since it was able to suppress constitutive activity of the hMC4R both in intact B16/G4F melanoma cells and membrane preparations. The effect of AgRP(83-132) on the hMC4R was blocked by the MC4R ligand SHU9119. Also the hMC3R and the mouse(m)MC5R were shown to be constitutively active. AgRP(83-132) acted as an inverse agonist on the hMC3R but not on the mMC5R. Thus, AgRP is able to regulate MCR activity independently of alpha-MSH. These findings form a basis to further investigate the relevance of constitutive activity of the MC4R and of inverse agonism of AgRP for the regulation of body weight.     

Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds alpha-melanocyte-stimulating hormone (alpha-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of alpha-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toalpha-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that alpha-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.     

Agouti-related protein has an inhibitory paracrine role in the rat adrenal gland

alpha-Melanocyte-stimulating-hormone (alpha-MSH) is an agonist at the melanocortin 3 receptor (MC3-R) and melanocortin 4 receptor (MC4-R). alpha-MSH stimulates corticosterone release from rat adrenal glomerulosa cells in vitro. Agouti-related protein (AgRP) an endogenous antagonist at the MC3-R and MC4-R, is expressed in the adrenal gland. We investigated the expression of the MC3-R and MC4-R and the role of AgRP in the adrenal gland. MC3-R and MC4-R expression was detected in rat adrenal gland using RT-PCR. The effect of AgRP on alpha-MSH-induced corticosterone release was investigated using dispersed rat adrenal glomerulosa cells. AgRP administered alone did not affect corticosterone release, but co-administration of AgRP and alpha-MSH attenuated alpha-MSH-induced corticosterone release. To investigate glucocorticoid feedback, adrenal AgRP expression was compared in rats treated with dexamethasone to controls. AgRP mRNA was increased in rats treated with dexamethasone treatment compared to controls. Our findings demonstrate that adrenal AgRP mRNA is regulated by glucocorticoids. AgRP acting via the MC3-R or MC4-R may have an inhibitory paracrine role, blocking alpha-MSH-induced corticosterone secretion.     

The cytosolic chaperone Hsc70 promotes traffic to the cell surface of intracellular retained melanocortin-4 receptor mutants

Inherited modifications in protein structure frequently cause a loss-of-function by interfering with protein synthesis, transport, or stability. For the obesity-linked melanocortin-4 receptor (MC4R) and other G protein-coupled receptors, many mutants are intracellular retained. The biogenesis and trafficking of G protein-coupled receptors are regulated by multiple factors, including molecular chaperone networks. Here, we have investigated the ability of the cytosolic cognate 70-kDa heat-shock protein (Hsc70) chaperone system to modulate cell surface expression of MC4R. Clinically occurring MC4R mutants S58C, P78L, and D90N were demonstrated to have reduced trafficking to the plasma membrane and to be retained at the endoplasmic reticulum (ER). Analyses by fluorescence recovery after photobleaching revealed that the mobility of MC4R mutant protein at the ER was reduced, implying protein misfolding. In cells expressing MC4R, overexpression of Hsc70 resulted in increased levels of wild-type and mutant receptors at the cell surface. MC4R and Hsc70 coimmunoprecipitated, and fluorescence recovery after photobleaching analyses showed that increasing cellular levels of Hsc70 promoted the mobility of ER retained MC4R. Moreover, expression of HSJ1b, a cochaperone that enhances degradation of Hsc70 clients, reduced cellular levels of MC4R. Hsp70 and Hsp90 chaperone systems collaborate in the cellular processing of clients. For MC4R, inhibition of endogenous Hsp90 by geldanamycin reduced receptor levels. By contrast, expression of the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase) increased cellular levels of MC4R. Finally, we demonstrate that signaling of intracellular retained MC4R mutants is increased in cells overexpressing Hsc70. These data indicate that cytosolic chaperone systems can facilitate rescue of intracellular retained MC4R by improving folding. They also support proteostasis networks as a potential target for MC4R-linked obesity.     

Functional characterization of novel melanocortin-3 receptor mutations identified from obese subjects

It is controversial whether mutation in the melancortin-3 receptor (MC3R) gene is a cause for monogenic obesity in humans. Three novel mutations in the MC3R, A293T, I335S, and X361S, were identified from morbidly obese subjects. We investigated whether these mutations caused loss-of-function and the molecular defects if any. Ligand binding, signaling, and cell surface expression of the mutant MC3Rs were studied. I335S resulted in a complete loss of ligand binding and signaling due to intracellular retention. A293T and X361S MC3Rs had normal ligand binding and signaling as wild type MC3R. Co-expression studies showed that the mutants did not affect wild type MC3R signaling. Hence the I335S variant previously identified from obese patients is not expressed at the cell surface when expressed in vitro, suggesting that it might contribute to obesity in carriers of this variant. Whether A293T and X361S cause obesity remains to be investigated. Additional mutations at I335 showed that I335, part of the highly conserved N/DPxxY motif, was critical for multiple aspects of the MC3R function, including cell surface expression, ligand binding, and signaling.     

Activation of MAPK by inverse agonists in six naturally occurring constitutively active mutant human melanocortin-4 receptors

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor that plays an essential role in regulating energy homeostasis. Defects in MC4R are the most common monogenic form of obesity, with about 170 distinct mutations identified in human. In addition to the conventional G_s-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2). Herein, we investigated the potential of four MC4R ligands that are inverse agonists at the G_s-cAMP signaling pathway, including agouti-related peptide (AgRP), MCL0020, Ipsen 5i and ML00253764, to regulate ERK1/2 activation (pERK1/2) in wild type and six naturally occurring constitutively active mutant (CAM) MC4Rs. We showed that these four inverse agonists acted as agonists for the ERK1/2 signaling cascade in wild type and CAM MC4Rs. Three mutants (P230L, L250Q and F280L) had significantly increased pERK1/2 level upon stimulation with all four inverse agonists, with maximal induction ranging from 1.6 to 4.2-fold. D146N had significantly increased pERK1/2 level upon stimulation with AgRP, MCL0020 or ML00253764, but not Ipsen 5i. The pERK1/2 levels of H76R and S127L were significantly increased only upon stimulation with AgRP or MCL0020. In summary, our studies demonstrated for the first time that MC4R inverse agonists at the G_s-cAMP pathway could serve as agonists in the MAPK pathway. These results suggested that there were multiple activation states of MC4R with ligand-specific and/or mutant-specific conformations capable of differentially coupling the MC4R to distinct signaling pathways.     

Point mutations in the melanocortin-4 receptor cause variable obesity in mice

Mutations in the melanocortin-4 receptor (MC4R) are associated with early-onset obesity in humans. Furthermore, a null Mc4r allele in mice leads to severe obesity due to hyperphagia and decreased energy expenditure. As part of independent N-ethyl- N-nitrosourea (ENU) mutagenesis screens, two obesity mutants, Fatboy and Southbeach, were isolated. Mapping revealed linkage to the melanocortin-4 receptor (Mc4r) and sequencing found single amino acid changes in Mc4r for each line. Expression of the mutant receptors in HEK 293 cells revealed defects in receptor signaling. The mutated Fatboy receptor (I194T) shows an increase in the effective concentration necessary for 50% of maximal signaling (EC₅₀) when stimulated with α-MSH. Based on competitive binding, I194T is expressed on the cell surface at lower levels than the nonmutated receptor. In contrast, Southbeach (L300P) displays minimal receptor signaling when stimulated with the natural ligand α-MSH or the synthetic agonist NDP-α-MSH. Cell surface binding is absent, which usually indicates a lack of cell surface expression. However, antibody binding to Flag-tagged receptors by flow cytometry analysis and immunofluorescence demonstrates that L300P is translocated to the plasma membrane at a level comparable to the wild-type receptor. These results indicate a correlation with remaining receptor activity and the severity of the obesity in the mice homozygous for the mutations. Southbeach has less receptor activity and becomes more obese. These mutants will serve as good models for the variability in phenotype in humans carrying mutations in the MC4R gene.     

Enterostatin inhibition of dietary fat intake is modulated through the melanocortin system

Enterostatin injected into the amygdala selectively reduces dietary fat intake by an action that involves a serotonergic component in the paraventricular nucleus. We have investigated the role of melanocortin signaling in the response to enterostatin by studies in melanocortin 4 receptor (MC4R) knock out mice and by the use of the MC4R and MC3R antagonist SHU9119, and by neurochemical phenotyping of enterostatin activated cells. We also determined the effect of enterostatin in vivo on the expression of AgRP in the hypothalamus and amygdala of rats and in culture on a GT1-7 neuronal cell line. Enterostatin had no effect on food intake in MC4R knock out mice. SHU9119 i.c.v. blocked the feeding response to amygdala enterostatin in rats. Amygdala enterostatin induced fos activation in alpha-melanocyte stimulating hormone (alpha-MSH) neurons in the arcuate nucleus. Enterostatin also reduced the expression of AgRP in the hypothalamus and amygdala and in GT1-7 cells. These data suggest enterostatin inhibits dietary fat intake through a melanocortin signaling pathway.     

Signal pathway analysis of selected obesity-associated melanocortin-4 receptor class V mutants

Mutations in the melanocortin-4 receptor (MC4R) in humans are the single most common cause of rare monogenic 1severe obesity, and polymorphisms in this gene are also associated with obesity in the general population. The MC4R is a G-protein coupled receptor, and in vitro analysis suggests that MC4R can signal through several different G-protein subtypes. In vivo studies show complex outcomes, with different G-proteins in different cells responsible for different physiological responses linked to obesity. There is an emerging consensus that Gαq-linked signals in the paraventricular nucleus of the hypothalamus are essential for normal satiety and the control of feeding behavior. Many MC4R mutations have been analyzed for the molecular defect underlying their association with obesity, which has revealed a group – referred to as class V mutants – with no measurable change in receptor function. However, Gαq-linked signaling leading to Ca²⁺ release has only been examined for a few MC4R mutations. In this study, we have examined seven MC4R class V mutants, as well as two other well-characterized signal-defective mutants as controls, with respect to G-protein signaling coupled to cAMP production, mitogen-activated protein kinase (MAPK) activation, and Ca²⁺ release. These data confirm, with one exception (E308K), the expected pattern of cAMP and MAPK signaling for wild type and mutant MC4R. Our results also demonstrate normal MSH-induced Ca²⁺ signals for wild type as well as all the class V mutants, but not the signal-defective controls. Thus, the means by which class V MC4R mutations lead to obesity remains an open question.     

Role of third intracellular loop of the melanocortin 4 receptor in the regulation of constitutive activity

The melanocortin 4 receptor (MC4R) has been reported to display constitutive activity, which is probably relevant to the maintenance of a normal energy balance. Among the clinically reported mutants of MC4R in human obesity patients, we investigated the functional characteristics of seven mutants characterized by mutations in the third intracellular (i3) loop of MC4R. Via a CRE (cAMP responsive element)-mediated luciferase reporter gene assay, we show that most of these mutants displayed significantly reduced basal activity with reduced reporter gene activity, whereas the P230L mutant manifested significantly increased basal activity. When the dominant negative G_s mutant was co-expressed, the majority of the mutants, including the P230L mutant, showed reduced basal activity. These results suggest that the i3 loop of MC4R is essential not only for the functional activity but also for the regulation and maintenance of an optimal constitutive activity of MC4R in association with G protein coupling, in the control of energy homeostasis.     

Biased Signaling in Naturally Occurring Mutations in Human Melanocortin-3 Receptor Gene

The melanocortin-3 receptor (MC3R) is primarily expressed in the hypothalamus and plays an important role in the regulation of energy homeostasis. Recently, some studies demonstrated that MC3R also signals through mitogen-activated protein kinases (MAPKs), especially extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 signaling is known to alter gene expression, potentially contributing to the prolonged action of melanocortins on energy homeostasis regulation. In the present study, we performed detailed functional studies on 8 novel naturally occurring MC3R mutations recently reported, and the effects of endogenous MC3R agonist, α-melanocyte stimulating hormone (MSH), on ERK1/2 signaling on all 22 naturally occurring MC3R mutations reported to date. We found that mutants D158Y and L299V were potential pathogenic causes to obesity. Four residues, F82, D158, L249 and L299, played critical roles in different aspects of MC3R function. α-MSH exhibited balanced activity in G_s-cAMP and ERK1/2 signaling pathways in 15 of the 22 mutant MC3Rs. The other 7 mutant MC3Rs were biased to either one of the signaling pathways. In summary, we provided novel data about the structure-function relationship of MC3R, identifying residues important for receptor function. We also demonstrated that some mutations exhibited biased signaling, preferentially activating one intracellular signaling pathway, adding a new layer of complexity to MC3R pharmacology.     

A Novel Melanocortin-4 Receptor Mutation MC4R-P272L Associated with Severe Obesity Has Increased Propensity To Be Ubiquitinated in the ER in the Face of Correct Folding

Heterozygous mutations in the melanocortin-4 receptor (MC4R) gene represent the most frequent cause of monogenic obesity in humans. MC4R mutation analysis in a cohort of 77 children with morbid obesity identified previously unreported heterozygous mutations (P272L, N74I) in two patients inherited from their obese mothers. A rare polymorphism (I251L, allelic frequency: 1/100) reported to protect against obesity was found in another obese patient. When expressed in neuronal cells, the cell surface abundance of wild-type MC4R and of the N74I and I251L variants and the cAMP generated by these receptors in response to exposure to the agonist, α-MSH, were not different. Conversely, MC4R P272L was retained in the endoplasmic reticulum and had reduced cell surface expression and signaling (by ≈3-fold). The chemical chaperone PBA, which promotes protein folding of wild-type MC4R, had minimal effects on the distribution and signaling of the P272L variant. In contrast, incubation with UBE-41, a specific inhibitor of ubiquitin activating enzyme E1, inhibited ubiquitination of MC4R P272L and increased its cell surface expression and signaling to similar levels as wild-type MC4R. UBE41 had much less profound effects on MC4R I316S, another obesity-linked MC4R variant trapped in the ER. These data suggest that P272L is retained in the ER by a propensity to be ubiquitinated in the face of correct folding, which is only minimally shared by MC4R I316S. Thus, studies that combine clinical screening of obese patients and investigation of the functional defects of the obesity-linked MC4R variants can identify specific ways to correct these defects and are the first steps towards personalized medicine.     

Rescue of melanocortin 4 receptor (MC4R) nonsense mutations by aminoglycoside-mediated read-through

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.     

Haplosufficiency of the melanocortin-4 receptor gene in individuals with deletions of 18q

The melanocortin-4 receptor (MC4R) is a seven, transmembrane G-protein-coupled receptor whose ligand, alpha-melanocyte-stimulating hormone (alpha-MSH), is a post-translational derivative of pro-opiomelanocortin (POMC). The regulatory pathway, of which MC4R is a part, has become an area of intense interest because of its potential role in obesity. Three studies have identified individuals with dominantly inherited obesity segregating with mutations in the MC4R gene. It has been hypothesized that the mutation found in these subjects resulted in a loss of gene function resulting in obesity due to haploinsufficiency of the MC4R gene. We have been studying the molecular basis of the phenotype of individuals with large deletions of chromosome 18q. Due to its location at 18q21.3, the MC4R gene is hemizygous in approximately one-third of the individuals in our study. If hemizygosity of the MC4R gene results in haploinsufficiency-induced obesity, then individuals with deletions of 18q whose deletions include the MC4R gene should be obese in comparison with those individuals whose deletion does not include the gene. Our data indicate no difference in obesity among those deleted and not deleted for the gene. This supports the hypothesis that the MC4R gene product is haplosufficient and the involvement of MC4R in obesity may reflect a dominant negative effect.