The Melanocortin-4 Receptor: Physiology, Pharmacology, and Pathophysiology

Abstract

The melanocortin-4 receptor (MC4R) was cloned in 1993 by degenerate PCR; however, its function was unknown. Subsequent studies suggest that the MC4R might be involved in regulating energy homeostasis. This hypothesis was confirmed in 1997 by a series of seminal studies in mice. In 1998, human genetic studies demonstrated that mutations in the MC4R gene can cause monogenic obesity. We now know that mutations in the MC4R are the most common monogenic form of obesity, with more than 150 distinct mutations reported thus far. This review will summarize the studies on the MC4R, from its cloning and tissue distribution to its physiological roles in regulating energy homeostasis, cachexia, cardiovascular function, glucose and lipid homeostasis, reproduction and sexual function, drug abuse, pain perception, brain inflammation, and anxiety. I will then review the studies on the pharmacology of the receptor, including ligand binding and receptor activation, signaling pathways, as well as its regulation. Finally, the pathophysiology of the MC4R in obesity pathogenesis will be reviewed. Functional studies of the mutant MC4Rs and the therapeutic implications, including small molecules in correcting binding and signaling defect, and their potential as pharmacological chaperones in rescuing intracellularly retained mutants, will be highlighted.

The aim of this review is to summarize the studies on the melanocortin-4 receptor (MC4R), from its cloning and tissue distribution, to its physiological roles in regulating energy homeostasis, cardiovascular function, glucose and lipid homeostasis, reproduction and sexual function, and others. The pharmacology of the receptor, including ligand binding and receptor activation, signaling pathways, as well as its regulation, is also summarized. Mutations in the MC4R are the most common monogenic form of obesity. Functional studies of the mutant MC4Rs and the therapeutic implications, including small molecules acting as pharmacological chaperones in rescuing intracellularly retained mutants, are highlighted.

• I. Introduction

• II. Molecular Cloning and Localization of the Melanocortin-4 Receptor

  • A. Molecular cloning
  • B. Tissue distribution
• III. Physiology of the Melanocortin-4 Receptor

  • A. Energy homeostasis
  • B. Cachexia
  • C. Cardiovascular function
  • D. Glucose and lipid homeostasis
  • E. Reproduction and sexual function
  • F. Miscellaneous functions of the MC4R
• IV. Pharmacology of the Melanocortin-4 Receptor

  • A. Ligand binding and receptor activation
  • B. Signaling pathways
  • C. Internalization, desensitization, and dimerization
• V. Pathophysiology of the Melanocortin-4 Receptor

  • A. Naturally occurring MC4R mutations
  • B. Molecular classification of the MC4R mutants
  • C. Therapeutic implications

• VI. Conclusions and Future Directions

I. Introduction

The melanocortin system consists of several agonists, two antagonists, and five receptors. The agonists, including α-MSH, β-MSH, γ-MSH, and ACTH, as well as some less studied peptides such as γ3-MSH and desacetyl-α-MSH, are all derived from tissue-specific posttranslational processing of a pre-prohormone, pro-opiomelanocortin (POMC) (1,2). In the anterior pituitary gland, POMC is processed into ACTH and other peptides by prohormone convertase (PC) 1. In lower vertebrates such as fish and amphibians, as well as during the fetal and infantile periods in humans, PC2 expressed in the pars intermedia also causes the release of MSH. In skin and hair follicles as well as in the brain (hypothalamus and brainstem), POMC is further processed by PC2 into MSH. POMC is an ancient gene, with expression in sea lamprey, the most ancient vertebrate, suggesting that it existed 700 million years ago (3,4).

The melanocortin system is unique in that two endogenous antagonists, Agouti and Agouti-related peptide (AgRP), exist. So far, no other endogenous antagonists have been identified in other G protein-coupled receptor (GPCR) systems. There are also several other ancillary proteins, such as mahogany and syndecan-3, that modulate receptor function by interacting with Agouti and AgRP (5).

Five melanocortin receptors (MCRs) mediate the diverse actions of these melanocortins. They are numbered MC1R to MC5R according to the sequence of their cloning. After the cloning of the MC1R (6,7) and MC2R (6), three additional MCRs, the MC3R, the MC4R, and the MC5R, were cloned. The MC1R is the classical MSH receptor expressed in skin and hair follicles that regulates pigmentation. The MC2R is the classical ACTH receptor expressed in the adrenal cortex that regulates adrenal steroidogenesis and cell proliferation. The MC3R and the MC4R are expressed primarily in the central nervous system, and are therefore referred to as the neural MCRs. The MC5R is expressed widely, especially in exocrine glands. Knockout experiments showed that the MC5R is involved in regulating exocrine gland secretions (8). For general reviews on the melanocortin system, the reader is referred to several excellent review articles (5,9,10,11,12).

Both MC3R and MC4R are involved in regulating energy homeostasis. The role of the MC3R in regulating food intake is controversial. Data from knockout animals revealed that Mc3r knockout results in increased feed efficiency and adiposity (13,14). Recently, it was shown that the MC3R “is required for entrainment to meal intake” (15). The MC4R regulates both food intake and energy expenditure and is the focus of this article.

II. Molecular Cloning and Localization of the Melanocortin-4 Receptor

A. Molecular cloning

The groups of Gantz and Cone (16,17) independently cloned the human MC4R (hMC4R) through degenerate PCR and homology screening. The human MC4R is an intronless gene with an open reading frame of 999 bp that encodes a protein of 332 amino acids. Alignment of MC4R with other MCRs showed that it has the highest homology with the MC3R, with 58% identity and 76% similarity. By fluorescent in situ hybridization, the MC4R gene was localized to chromosome 18q21.3 (16,18).

Since the cloning of hMC4R, the MC4R has been cloned from mouse, rat, hamster, guinea pig, dog, cat, fox, pig, sheep, cow, and several primates including marmoset, cynomolgus macaque, vervet monkey, and orangutan (see http://www.gpcr.org/7tm/classes/melanocortin-type-4/proteins/). It has also been cloned from several nonmammalian species including fish, chicken, and pigeon. The amino acid sequences between the different species are highly conserved (19,20). For example, there is 93% identity between rat and human MC4Rs and 87% identity between chicken and human MC4Rs (21). Even the zebrafish and spiny dogfish MC4Rs share 71% identity with hMC4R and more than 90% in the transmembrane domains (TMs) (22,23,24). Evolutionary analyses of MC4R sequences from many different species showed that the MC4R has been subject to high levels of continuous purifying selection with codon usage bias, leading to the unusually low levels of silent polymorphisms in humans (20).

The MC4R is a member of family A GPCRs with seven TMs connected by alternating extracellular loops (ELs) and intracellular loops. The N terminus is extracellular, and the C terminus is intracellular. The MC4R, like the other MCRs, has some unique features compared with other family A GPCRs (Fig. 1). For example, the highly conserved disulfide bond linking the top of TM3 and EL2 is missing in the MC4R, although there is an intraloop disulfide bond in EL3 (25). The intracellular loops and ELs are short, especially EL2, making MC4R one of the shortest members in the GPCR superfamily. Highly conserved Pro in TM5 and Asn in TM7 (in the NPxxY motif, N7.49) in family A GPCRs are substituted by a Met and an Asp, respectively, in the MC4R. In Fig. 1, the demarcations of the TMs for hMC4R are based on the crystal structure of rhodopsin (26). [According to the numbering scheme of Ballesteros and Weinstein (27), the most highly conserved residue in each TM is defined as residue 50, preceded by the helix number. Other residues in the same TM are numbered according to their relative position to the most conserved residue. For example, in TM7, Pro in the NPxxY motif is the most highly conserved, therefore numbered as 7.50. Asn is one residue before the Pro, therefore numbered as 7.49. This numbering scheme facilitates the comparison of results obtained from different members of family A GPCRs, and is used herein when appropriate.]

Figure 1.

Sequence alignment of bovine rhodopsin (RHO) with human MC1R, MC3R, and MC4R. The ELs and intracellular loops are boxed (note that the C terminus of rhodopsin is not complete). The divergent N termini are not shown here. The most conserved residues in each TM (50 residue) are in red and are boxed individually. The highly conserved Cys residues at the top of TM3 and EL2 in rhodopsin that form a disulfide bridge are highlighted in gold. The potential palmitoylation sites at the C termini are shaded in light yellow. In MCRs, TM5 is two residues shorter than rhodopsin; otherwise, EL2 would consist of only two residues, which is not likely. Note the following unique characteristics of the MCRs, including the absence of the highly conserved disulfide bridge between the EL2 and the top of TM3 (shaded in gold for rhodopsin). The intracellular loops and ELs of the MCRs are short, especially EL2, making MCRs some of the shortest members in the GPCR superfamily. Highly conserved Pro in TM5 and Asn in TM7 (in the NPxxY motif) in family A GPCRs are substituted by a Met and an Asp, respectively, in all MCRs (shaded in rose). In other family A GPCRs, this Asn is proposed to interact with the highly conserved Asp in TM2. In MCRs, Asp is present at both loci, unlikely to interact with each other. GenBank accession numbers for the genes are: bovine rhodopsin, NM_001014890; human MC1R, NM_002386; human MC3R, NM_019888; and human MC4R, NM_005912. IL, Intracellular loop.

There are four potential N-linked glycosylation sites (three at the N terminus: Asn3, Asn17, and Asn26; and one at EL1: Asn108). Although it is known that the MC4R is glycosylated, there are no reports on the experimental identification of the site(s) that is (are) indeed glycosylated. The exact functions of the glycosylation are also unknown. Two conserved Cys residues at the C terminus, Cys318 and Cys319, might serve as sites for palmitoylation anchoring the C terminus to the plasma membrane, forming a fourth intracellular loop. It is not known whether the MC4R is indeed palmitoylated or where this modification is located.

B. Tissue distribution

In the original cloning article of Gantz et al. (16), through Northern blotting of canine mRNAs, it was shown that the MC4R is primarily expressed in brain. By in situ hybridization in mouse brain sections, it was shown that the MC4R mRNA is localized in regions of the thalamus, hypothalamus, and hippocampus. Extensive labeling of the MC4R was found in CA1 and CA2 but not CA3 and CA4 regions of the hippocampus. The MC4R mRNA is also present in dentate gyrus, cortex, and amygdala (16).

Extensive localization studies on rat brain showed that the MC4R mRNA is widely expressed in adult rat brain, including cortex, thalamus, hypothalamus, brainstem, and spinal cord (17,28,29). In the hypothalamus, it is expressed highly in paraventricular nucleus (PVN), including both parvicellular and magnocellular neurons. A mouse line expressing green fluorescent protein under the control of the MC4R promoter showed similar distribution of green fluorescent protein as observed with in situ hybridization technique (30).

In addition to neurons, astrocytes were also reported to express the MC4R (31,32). Endogenous and synthetic peptide ligands as well as small molecule agonists can increase cAMP accumulation in rat astrocytes (31), consistent with earlier studies demonstrating that α-MSH and ACTH increase cAMP levels in astroglial cells (33,34) and melanocortins stimulate proliferation and induce morphological changes in cultured rat astrocytes (35). ACTH_1–24 was also shown to down-regulate the expression of ciliary neurotrophic factor mRNA in rat astrocytes cultured in vitro (36). Very recently, it was suggested that functional MC4R is also expressed in human epidermal melanocytes that might contribute to melanogenesis (37).

Developmentally, it was shown that the MC4R mRNA is first expressed at embryonic day (E) 14 in diencephalon, telencephalon, lamina terminalis, and spinal trigeminal nucleus in the rat. By E19, the MC4R is widely expressed in many regions of the brain (38). The highest level of expression is in the autonomic nervous system. Similar data were obtained when radiolabeled [Nle⁴, D-Phe⁷]-MSH (NDP-MSH) was used as the probe (39). Although NDP-MSH also binds to the MC3R, the binding sites detected with radiolabeled NDP-MSH likely represent MC4R because the MC3R mRNA is only expressed postnatally, not during the fetal period (40). In addition, the MC4R mRNA is also expressed in several peripheral tissues during the fetal period, including the developing heart (E14), lung (E16), muscles involved in respiration such as diaphragm and intercostal muscle (E14), as well as other muscles (41). Although the MC4R mRNA cannot be detected in the adrenal gland, liver, pituitary gland, mesenteric fat, and spleen in the adult rat, it is expressed in heart, lung, kidney, and testis during the fetal period (41). The potential physiological functions of the MC4R in these organs remain to be elucidated. The expression of the MC4R (and its ontogeny) is different from that of the MC3R. The MC3R is expressed primarily within the hypothalamus, with limited expression in the thalamus and brainstem (42).

In nonmammalian species, the MC4R is also abundantly expressed in brain. In the spiny dogfish, it is not expressed in peripheral tissues such as eye, kidney, liver, heart, and muscle (23). However, in some other fish, the MC4R is also expressed in certain peripheral tissues, including eye, ovaries, and gastrointestinal tract in the zebrafish; ovary in the goldfish; and liver, ovary, and testis in the flounder (22,43,44). In the sea bass, in addition to the brain, the MC4R mRNA is expressed in retina and pituitary gland as well as liver, fat, testis, and white muscle at lower levels but not in spleen, gill, intestine, skin, red muscle, heart, and ovary (45). The physiological functions of the MC4Rs in these tissues are not known.

III. Physiology of the Melanocortin-4 Receptor

A. Energy homeostasis

1. Rodent studies

Earlier studies showed that intracerebroventricular (ICV) administration of α-MSH and ACTH decreases food intake in rats (46,47). However, “these studies did not receive the attention they deserved” (10) and the receptor mediating this effect was not known. A renaissance came after the molecular cloning of the MCRs. As mentioned earlier, the MC4R was first cloned by degenerate PCR and homology screening with unknown physiological functions. In 1997, several landmark studies were published that established the critical importance of the MC4R in regulating energy homeostasis.

Based on their studies demonstrating that Agouti, in addition to antagonizing the MC1R, is also an antagonist of the MC4R (48) and the fact that in A^y mice, the well-known obesity mouse model, Agouti is ubiquitously expressed including in the hypothalamus (49), Cone and colleagues (50) hypothesized that the obesity in A^y mice might be due to Agouti antagonizing a central MCR. They showed that ICV administration of MTII (melanotan II) inhibits the hyperphagia in four different mouse models, and this inhibition was blocked by coadministration of SHU9119 (50). [MTII is a superpotent and stable cyclic analog of α-MSH, and SHU9119 is a high-affinity cyclic antagonist of MC3R and MC4R (51).] Unlike leptin, which is not effective in common obesity due to leptin resistance, the effect of MC4R agonism on food intake and body weight is more pronounced in obese than in lean rats (52,53,54). Administration of SHU9119 alone increases feeding (50), even in diet-induced obesity, therefore exacerbating the obesity (55). These experiments supported their hypothesis suggesting that hypothalamic melanocortinergic neurons exert a tonic inhibition on feeding, and the disruption of this mechanism might be responsible for the obesity in A^y mice (50). Injection of these compounds directly into the PVN achieved even more potent alterations in food intake, suggesting that neurons in the PVN, which express very high levels of the MC4R (17), are primary sites of action in melanocortin regulation of feeding behavior (56). The regulation of food intake by these ligands is not due to aversive effects (57), although the MC4R is expressed in brainstem parabrachial neurons (29,58). The suppression of food intake by MTII is due to reduced meal duration and meal size (59,60).

The use of more specific MC4R antagonists such as HS014 and HS024 provided further supporting evidence that MC4R is important in regulating food intake. For example, ICV infusions of these antagonists stimulate feeding in satiated rats, and long-term infusion leads to increased food intake and body weight (61,62,63,64,65,66).

Recently, studies on an enzyme that inactivates α-MSH, prolylcarboxypeptidase, provided further supporting evidence of the importance of maintaining normal levels of active α-MSH in body weight regulation (67). Inhibition of enzyme activity (resulting in increased α-MSH levels) decreases food intake. Mice lacking this enzyme gene are leaner and shorter and are resistant to high-fat diet-induced obesity (67).

Another landmark study published in 1997 is the report of the Mc4r knockout mouse model that provided the definitive evidence that the MC4R is critical for regulating energy homeostasis in mice (68). The homozygous knockout mice have maturity-onset obesity, hyperphagia, increased linear growth, hyperinsulinemia, and hyperglycemia. The heterozygous mice have intermediate body weights compared with the wild-type (WT) littermates and homozygous mice, suggesting that there is a gene dosage effect (68). Mc4r knockout mice have delayed meal termination and reduced sensitivity to cholecystokinin (CCK) (69,70). The obesity in Mc4r knockout mice is exacerbated when the mice are fed a high-fat diet because, unlike the WT mice, which respond to an increase in the fat content of the diet by rapidly increasing diet-induced thermogenesis and by increasing physical activity, the Mc4r knockout mice are impaired in eliciting these responses (71), leading to decreased insulin sensitivity (72). In addition to hyperphagia, the Mc4r knockout mice also have decreased energy expenditure. Change in food intake accounts for 60% of the effect of the MC4R on energy balance. The other 40% is accounted for by changes in energy expenditure (73). In young mice with similar body weights, the Mc4r knockout mice consumed less oxygen than WT littermates, and pair feeding of the Mc4r knockout mice led to more weight gain than the WT mice (74) [however, one study suggested that in young Mc4r knockout mice that are not obese, hyperphagia but not hypometabolism contributes to the early onset in these mice (75)]. MTII has no effect on food intake and energy expenditure in Mc4r knockout mice but is fully active in Mc3r knockout mice (76,77). A recent elegant study of selective reactivation of MC4R expression in specific neurons showed that the MC4R expressed in the PVN and amygdala are involved in the regulation of food intake, whereas MC4R expressed in other neurons are involved in controlling energy expenditure (73). Another study using RNA interference knockdown in the PVN of adult rat also showed that rats with MC4R knockdown exhibited an increase in food intake and excessive body weight gain when exposed to a high-fat diet (78).

The discovery of AgRP in 1997 represents another important breakthrough. Several groups independently cloned the AgRP gene (79,80,81) as an analog of Agouti (Agouti signaling protein in humans). AgRP is an antagonist for the two neural MCRs, MC3R and MC4R. Subsequent studies showed that it is indeed an inverse agonist for human and rodent MC4Rs decreasing basal signaling of WT or constitutively active mutant receptors (82,83,84). AgRP mRNA levels are increased 8- to 10-fold in ob/ob and db/db mice (79,85). Transgenic overexpression of AgRP in mice results in obesity (79,80) but not yellow fur, different from A^y mice. Pharmacological studies showed that AgRP is orexigenic (reviewed in Ref. 86). ICV administration of AgRP increases food intake for at least 24 h and blocks the inhibitory effect of α-MSH (87). Bloom and colleagues (88) further investigated which hypothalamic areas known to express MC4R are involved in the AgRP regulation of feeding by inserting cannulae directly into discrete rat hypothalamic nuclei. They showed that the PVN, the dorsomedial nucleus, and the medial preoptic area were the areas with the greatest response to AgRP, whereas no changes in feeding were seen after the administration of AgRP into the arcuate nucleus (ARC) and lateral hypothalamic area (88). Wirth and Giraudo (89) showed that much lower doses of AgRP were needed to stimulate food intake when directly injected into the PVN, suggesting that the PVN is the primary site for AgRP regulation of food intake. AgRP also reverses leptin-induced inhibition of food intake and body weight in a dose-dependent manner (90). Although it was reported originally that Agrp knockout mice do not have defects in food intake, body weight, or susceptibility to diet-induced obesity (91) likely due to compensation by redundant genes, especially during development, a subsequent knockout study on mice with a different genetic background showed that 6-month-old homozygous Agrp knockout mice have reduced body weights, with increased metabolic rate and motor activity (92). Circulating thyroid hormones and brown adipose tissue (BAT) uncoupling protein (UCP) 1 expression are increased (92). Agrp knockout mice also have longer life span when fed with a high-fat diet (93). Furthermore, targeted postnatal destruction of AgRP/neuropeptide Y neurons results in hypophagia and weight loss, with both appetitive and consummatory behaviors affected, providing strong evidence of these neurons in maintaining energy homeostasis (94,95,96,97,98) (reviewed in Ref. 99). Finally, human genetic studies suggest that single nucleotide polymorphism (SNPs) in AgRP such as A67T might provide protection against obesity, associated with anorexia nervosa and leanness (100,101), although the exact mechanism for this protection remains to be investigated. Functional studies on the AgRP variant did not identify any defect in its interaction with the MC4R (102).

Another line of evidence supporting the melanocortin system in regulating energy homeostasis comes from the studies on mice transgenic for overexpression of syndecan-1. These mice are obese (103), similar to mice overexpressing AgRP. Although syndecan-1 is not normally expressed in the hypothalamus, it was suggested that syndecan-3, expressed in the hypothalamus, was the cause of obesity in transgenic mice with syndecan-1 expression in the hypothalamus. It acts by augmenting AgRP antagonism of α-MSH at the MC4R. Mice lacking syndecan-3 have reduced adipose mass compared with WT mice and are partially resistant to high-fat diet-induced obesity due to reduced food intake in males and increased energy expenditure in females relative to that of WT mice (104). These mice are also more sensitive to exogenously administered MTII, consistent with the hypothesis that in the absence of syndecan-3, MTII binds more efficiently to the MC4R and therefore is more efficacious (105).

MTII also increases energy expenditure as shown by increased oxygen consumption (53,106). The MC4R can directly regulate thermogenesis in BAT (reviewed in Ref. 107). ICV administration of MTII dose-dependently increases sympathetic nerve traffic to thermogenic BAT, and this effect is completely blocked by SHU9119 (108). The MC4R mediates the MTII action because MTII cannot induce UCP1gene expression in Mc4r knockout mice (74), and the renal sympathetic nerve activity is attenuated and abolished, respectively, in heterozygous and homozygous Mc4r knockout mice (109). SHU9119 also abolishes the stimulatory effect of leptin on UCP1 gene expression (110). MTII-induced thermogenic response is maintained in diet-induced obesity (111). MC4R blockade by AgRP or gene targeting leads to defect in high-fat diet-induced up-regulation of UCP1 in interscapular BAT (112). The Mc4r knockout mice, failing to up-regulate UCP1 expression when exposed to cold, are also defective in cold-induced thermogenesis (112). The neurons in the rostral raphe pallidus and its immediate vicinity are important in mediating MC4R action on thermogenesis (113). In summary, the MC4R regulates thermogenesis by activating both the sympathetic nervous system-BAT-UCP1 axis and the hypothalamic-pituitary-thyroid (HPT) axis (107).

Although α-MSH is used in most of the pharmacological experiments, there are several lines of evidence suggesting that β-MSH might also be an important endogenous ligand for the MC4R. β-MSH has higher affinity for the MC4R than α-MSH (114). β-MSH is present in hypothalamic nuclei classically associated with feeding (reviewed in Ref. 115). Pharmacologically, ICV administration of β-MSH or its analog inhibits food intake in rats and mice (116,117,118,119). Mutations in POMC that affect β-MSH cause obesity (120,121). All the evidence supports the hypothesis that β-MSH is also an endogenous agonist for the MC4R involved in regulating energy homeostasis.

In addition to the hypothalamus, the MC4R expressed in the brainstem is also involved in regulating energy homeostasis (122). The nucleus of the solitary tract, apart from the hypothalamus, is the other site of POMC expression in the central nervous system (123,124). The MC4R is expressed abundantly in caudal brainstem structures relevant to energy balance, with the dorsal motor nucleus of the vagus nerve having the highest MC4R expression in the brain (17). Administration of the MCR agonists and antagonists into the fourth ventricle modulates food intake and energy expenditure similar to the administration of these ligands into the third ventricle (125,126,127,128). Administration of MTII into the fourth ventricle also increases UCP1 mRNA in BAT to similar levels of rats administered with MTII into the third ventricle (129).

It is now well accepted that the MC4R in the hypothalamus is critical in mediating the effect of leptin (130) in regulating energy homeostasis. Leptin receptor is expressed in POMC neurons in the ARC (131), and leptin treatment increases POMC mRNA expression (132,133,134). Electrophysiological recordings showed that leptin increases the frequency of action potentials in the POMC neurons (135). Leptin receptor is also expressed in AgRP neurons in the ARC (136), and leptin treatment decreases AgRP mRNA in ob/ob (leptin-deficient) but not db/db (leptin receptor-deficient) mice (90). In ob/ob and db/db mice, hypothalamic AgRP mRNA expression is up-regulated (79,85). Fasting, with plasma leptin concentration decreased, results in up-regulation of AgRP mRNA (90,136,137). Finally, ICV administration of SHU9119 blocked the anorexigenic effect of leptin on food intake, whereas it has no effect on the anorexigenic effect of glucagon-like-peptide-1 (138). It should be pointed out that although the melanocortin system is the predominant mediator of leptin signaling, independent and additive effects of the two systems have also been reported (139,140), with other factors such as age, gender, and diet influencing the interactions between the two systems (141).

Taken together, in an oversimplified way, the response of the leptin-regulated melanocortin circuit to energy status can be summarized in the following way. When the animals are starved, leptin levels decrease, the activity of POMC neurons is decreased, and the activity of AgRP neurons is increased, resulting in decreased MC4R signaling. Increased food intake and decreased energy expenditure ensue. Conversely, when the animals are in the fed state, POMC neurons are activated, AgRP neurons are inhibited, and MC4R signaling is increased. Decreased food intake and increased energy expenditure ensue. Energy homeostasis is maintained in this elegant feedback system. Several outstanding reviews (142,143,144,145,146) have provided more extensive summaries on this topic.

The critical importance of leptin-regulated melanocortin circuit in energy homeostasis in rodents and humans is highlighted by the fact that defects in multiple molecules in this circuit cause obesity. The rodent models include ob/ob mice [deficient in leptin (130)], db/db mice (147) and Zucker fatty rats (148) (deficient in leptin receptor), Pomc knockout mice (149,150), fat/fat mice (deficient in carboxypeptidase E, an enzyme involved in POMC processing) (151), AgRP-overexpressing mice (79,80), and Mc4r knockout mice (68). Mutations in humans include leptin (152,153), leptin receptor (154), POMC (155), PC1 (156), and MC4R (see Section V.A), all resulting in monogenic obesity (reviewed in Refs. 157,158,159,160,161). Finally, loss of the transcription factor single-minded 1 (SIM1) in both mice and humans causes obesity (162,163) because the development of PVN, neurons expressing high levels of MC4R that are critically involved in MC4R regulation of food intake (but not energy expenditure) (73), is disrupted.

In addition to integrating the adipostatic signal of leptin, the MC4R is also involved in responding to acute signals regulating hunger and satiety, such as ghrelin, peptide YY_3-36, and CCK, received primarily in the brainstem (reviewed in Refs. 99,122 and 164). For example, MC4R activation in the PVN is required for CCK-induced suppression of feeding, although MC4R in other brain regions (such as vagal afferents) as well as MC4R-independent mechanisms are also involved (69,70,165).

2. MC4R regulation of energy homeostasis in other mammals

In adult male rhesus monkeys, infusion of NDP-MSH into the lateral cerebral ventricle suppresses food intake dose-dependently, whereas infusion of AgRP stimulates food intake during the scheduled afternoon meal, suggesting that the central melanocortinergic system is a physiological regulator of energy balance in primates (166).

In pigs, Barb et al. (167) showed that ICV administration of NDP-MSH decreases food intake, but treatments with SHU9119 or AgRP fail to stimulate food intake, different from the results obtained in rodents (50,87,138) and sheep (168). In vitro, these ligands did act as antagonists at the WT porcine MC4R (pMC4R) (167). We also showed that pMC4R binds and responds to α-MSH and NDP-MSH similarly as hMC4R; it also binds to AgRP with similar affinity as hMC4R (169). It was suggested that the lack of response to the MC4R antagonists might be due to a mutation in some strains of pigs, D298N (170). Although earlier functional analyses suggested that D298N pMC4R binds to NDP-MSH normally, but is devoid of NDP-MSH-stimulated cAMP production (171), we showed that D298N pMC4R has normal binding and signaling to NDP-MSH; it also binds AgRP normally (169). Therefore, the reason for the lack of response to SHU9119 or AgRP is not known. The early study showed extremely low maximal response to NDP-MSH stimulation (171). Patten et al. (172) also reported that hMC4R D298N has normal response to NDP-MSH stimulation, similar to our data (169). In addition, of the genotype-phenotype association studies published since the original report (170), no consensus could be reached, with some studies supporting (173,174,175,176) and some studies disputing (177,178,179) the original association. Indeed, in a recent study, the opposite trend, i.e., higher average daily food intake for pigs with D298 (WT) than pigs homozygous for N298, was observed (180). Comparison of pigs homozygous for D298 or N298 showed that the MC4R genotype does not significantly affect gene expression, body weight, back fat depth, or any measured serum metabolite concentration (180).

In the sheep, leptin receptors are also expressed in POMC and neuropeptide Y/AgRP neurons (181). Fasting dramatically increases AgRP mRNA and protein levels (168,182,183). AgRP expression is also increased during lactation, when there is a negative energy balance and low leptin levels (184). However, change in POMC expression is more variable in different studies, perhaps suggesting that the POMC gene is less responsive to fasting in sheep, different from rodents (reviewed in Ref. 185). AgRP is a potent stimulator of food intake in both healthy and endotoxin-treated animals (168,185,186). These studies suggest that the MC4R is also a critical component of appetite regulation in sheep, a species that grazes instead of eating intermittently like rodents and other nonruminant species.

3. MC4R regulation of energy homeostasis in lower vertebrates

Recent studies showed that the mechanism of regulation of energy homeostasis by the MC4R is also operational in lower vertebrates. In chickens, administration of both α- and β-MSH suppresses food intake (187,188). Increased c-Fos activity (as a marker of neuronal activation) is observed in periventricular, paraventricular, and infundibular nuclei as well as the ventromedial hypothalamus but not the lateral hypothalamus (188). AgRP attenuates the anorexigenic effect of α-MSH in broiler chicks, and AgRP by itself increases food intake in layer-type chicks fed ad libitum but not in broiler chicks (189). It was suggested that the different orexigenic effect of AgRP and anorexigenic tone exerted by endogenous α-MSH might contribute to the difference in food intake between the two breeds (189). In another study, comparison between two lines of chicks showed that the line with low body weight has a lower threshold for the anorexigenic effect of central α-MSH than the line with high body weight (190). In the chick, the MC3R is not expressed in the brain, whereas the MC4R is (191). Therefore, these effects of α- and β-MSH are likely mediated by the MC4R.

In goldfish, ICV injection of NDP-MSH or MTII inhibits food intake, whereas the MC4R-specific antagonist HS024 increases food intake (43,192). These experiments suggested that the MC4R is exerting a tonic inhibitory effect on food intake. Similar results were obtained in rainbow trout (193). ICV injection of MTII decreases food intake, whereas ICV injection of HS024 and the MC3/4R antagonist SHU9119 increases food intake in rainbow trout (193).

The function of AgRP is also conserved in lower vertebrates. In birds (194) and fish (195,196), AgRP expression is restricted to discrete regions of the brain involved in sensing energy status of the organisms, homologous to the ARC of the mammals. Negative energy balance results in dramatically increased AgRP expression levels (195,196,197). Transgenic zebrafish overexpressing AgRP is obese with increased linear growth and adipocyte hypertrophy (198), similar to transgenic mice overexpressing AgRP.

4. Downstream mediators

Although it is well accepted that the activation of the MC4R results in decreased food intake and increased energy expenditure, the molecules mediating these effects are still unknown. Several candidates, including brain-derived neurotrophic factor (BDNF), CRH, TRH, melanin-concentrating hormone (MCH), and orexins, have been suggested to be involved.

MC4R signaling affects the expression of BDNF in the ventromedial hypothalamus (199). ICV injection of MTII reverses the decrease in BDNF mRNA expression in fasted mice. Impaired MC4R signaling such as in A^y or Mc4r knockout mice results in reduced BDNF mRNA expression in the ventromedial hypothalamus (199). In another study, a selective MC4R agonist, MK1, increases BDNF release from isolated rat hypothalami in vitro, and this effect is blocked by preincubation with SHU9119 (200). In vivo, ICV administration of an anti-BDNF antibody blocks the anorexigenic effect of peripherally administered MK1 (200). These data showed that BDNF release from the hypothalamus induced by MC4R activation is required for the MC4R effect on food intake (200), suggesting that BDNF is a downstream mediator of MC4R signaling in regulating energy balance. Similar observations were also made in the dorsal vagal complex (201).

Central administration of α-MSH results in up-regulation of CRH and TRH mRNA levels and down-regulation of orexin mRNA levels (202,203,204). Double-labeling in situ hybridization showed that a subpopulation of CRH neurons in the PVN also expresses the MC4R (205). Central administration of MTII to conscious and freely moving rats induces a rapid induction of CRH gene transcription in the PVN, accompanied by a rise in plasma corticosterone levels. The increase in plasma corticosterone levels induced by MTII is attenuated by the selective MC4R antagonist HS014 and nonselective CRH receptor antagonist α-helical-CRH_9-41. Pretreatment with α-helical-CRH_9-41 also abolishes half of the inhibitory effect of MTII on food intake (205). Similarly, in chicks, central administration of β-MSH significantly decreases food intake, accompanied by a significant up-regulation of CRH mRNA levels; CRH type 2 receptor antagonist α-helical-CRH blocks the anorexigenic effect of β-MSH (206). In goldfish, CRH is also suggested to mediate the anorexigenic effect of α-MSH (207). These results suggest that CRH may be involved in the anorexigenic action of MSH from fish to mammals.

POMC and AgRP neurons project to the hypophysiotropic TRH neurons in the PVN and are poised anatomically to regulate the HPT axis (202). It was suggested that these ARC neurons mediate the effects of leptin on the HPT axis. For example, during fasting, decreased release of α-MSH and increased secretion of AgRP result in decreased signaling at the MC4R, thereby inhibiting the HPT axis activity (reviewed in Ref. 208). Centrally administered α-MSH prevents the fasting-induced drop in TRH expression (202) and increases TSH levels (209). In vitro, α-MSH stimulates TRH release in hypothalamic slices that is blocked by AgRP (209). ICV administration of AgRP decreases circulating levels of TSH and thyroid hormone and inhibits proTRH (TRH prohormone) mRNA level in the PVN of WT (210,211) but not Mc4r knockout mice (212). About 70% of TRH neurons are innervated by AgRP but not by POMC (212), suggesting that AgRP acts as an inverse agonist rather than competitive antagonist in these neurons. MC4R activation can directly act on the TRH promoter through cAMP response element binding protein (213). In chicks, however, TRH might not be involved in the anorexigenic effect of β-MSH (206).

Several studies investigated whether MCH or orexin mediates MC4R action in the hypothalamus. Expression of MCH is markedly elevated in Pomc-null mice, suggesting that melanocortins negatively regulate MCH neuronal activity (150). POMC neurons send projections to the orexin neurons (214). One study showed that in several models of disrupted MC4R signaling, including Agouti overexpression in A^y mice and antagonism of the MC4R by AgRP or SHU9119, orexin expression is not changed, although MCH expression is significantly increased (204). However, in another study, it was shown that Pomc-null mice have increased orexin expression in the lateral hypothalamic area and this increase is not reversed by corticosterone (215). Central administration of α-MSH in these mice restores orexin expression back down to that of WT mice, suggesting that α-MSH inhibits orexin expression in Pomc-null mice (215). It was suggested that the discrepant results might be due to residual MC4R signaling in the earlier study vs. the complete absence of melanocortins in the latter study. AgRP was shown to primarily activate orexin (rather than MCH) expression in the lateral hypothalamic area.

Another potential mediator of MC4R action is SIM1. SIM1 is a transcription factor required for the development of the PVN (216). Heterozygous mutation in SIM1 is one cause of monogenic obesity in humans (163). The patient with SIM1 heterozygous mutation had hyperphagia but normal energy expenditure, presenting with early-onset severe obesity (163). Homozygous Sim1 knockout mice die shortly after birth (216). Heterozygous Sim1 knockout mice are viable but have similar phenotypes as the SIM1 haploinsufficient patient, with hyperphagia but normal energy expenditure, resulting in early-onset obesity, together with increased linear growth, hyperinsulinemia, and hyperleptinemia (162). MTII treatment increases hypothalamic Sim1 gene expression (217). Heterozygous Sim1 knockout mice, with similar numbers of PVN neurons as WT mice, have diminished anorectic response to MTII despite a normal increase in their energy expenditure (217). Transgenic mice that overexpress human SIM1 are resistant to diet-induced obesity on a high-fat diet through reduced food intake, although there is no change in energy expenditure (218). The SIM1 transgene also completely corrects the hyperphagia and partially corrects the obesity of A^y mice (the portion due to the hyperphagia; the portion due to energy expenditure could not be corrected) (218). When the MC4R is reactivated in the Sim1 neurons in the PVN in Mc4r knockout mice, hyperphagia is completely corrected (73). These data suggest that Sim1 or its transcriptional target mediates the MC4R action in controlling food intake but not energy expenditure (218).

The molecules listed above are some of the mediators likely involved in mediating the action of the MC4R in regulating energy homeostasis. To gain a better understanding of the complete gene sets mediating MC4R action on energy homeostasis, techniques such as laser capture microdissection and gene expression profiling by microarray are needed (219).

B. Cachexia

Cachexia, wasting of lean body mass due to cancer or infectious disease (such as AIDS), is frequently accompanied by anorexia. Patients with other chronic diseases, such as renal failure, heart failure, and rheumatoid arthritis, are also affected by cachexia. Cachexia is an important prognostic for morbidity and mortality with these chronic diseases. Current therapies for cachexia are not effective, and new therapeutic options are urgently needed.

During the past few years, the melanocortin system has been shown to be involved in the pathogenesis of anorexia and weight loss associated with cachexia (for reviews, see Refs. 220,221,222). Many chronic diseases cause increased levels of proinflammatory cytokines. Marks and colleagues (223) showed that the POMC neurons in the ARC express type I IL-1 receptor and respond to IL-1β stimulation with increased release of α-MSH. An intact MC4R is required for cachexia induced by lipopolysaccharide (LPS) administration (to induce inflammation, systemic administration of LPS is frequently used as a model for anorexia due to acute infection) (224) or tumor (225) or associated with uremia (226). MC4R blockade has been shown to block cytokine-induced anorexia (224,227,228,229). Exogenous α-MSH enhances LPS-induced anorexia (227). Further studies showed that ICV administration of AgRP or SHU9119 also protects against cancer-induced anorexia in both rats and mice (224,230) as well as uremic cachexia (231). The MC4R may also mediate changes in adaptive thermogenesis in cachexia (107).

MC4R antagonism by small molecule MC4R antagonists has been shown to alleviate cachexia in several models. Vos et al. (232) showed that ML00253764, [2-{2-[2-(5-bromo-2-methoxyphenyl)-ethyl]-3-fluorophenyl}-4,5-dihydro-1H-imidazolium hydrochloride], a small molecule MC4R inverse agonist, could reach the central nervous system after sc administration and reduce CT26 (colorectal) tumor-induced weight loss. This was confirmed in another study that grafted mice with Lewis lung carcinoma tumors (233). With another small molecule MC4R antagonist developed by Neurocrine Biosciences, NBI-12i, as well as other compounds, it was shown that cancer and uremic cachexia could be attenuated by ip administration of the compounds (234,235,236). Recently, it was shown that two small molecule MC4R antagonists can penetrate the blood-brain barrier when given orally and almost completely prevent weight loss and fat and muscle wasting induced by the C26 adenocarcinoma tumor (237).

In summary, MC4R antagonists, including inverse agonists, have potential as therapeutics for treating cachexia associated with many chronic illness and inflammatory states.

C. Cardiovascular function

The MC4R is expressed in the nucleus of the solitary tract, a region that is known to be important for regulating cardiovascular function. MC4R activation raises arterial pressure despite decreased food intake, whereas MC4R inhibition causes marked weight gain without raising arterial pressure, partially mediating leptin’s action on these parameters (238,239,240). For example, central blockade of the MC4R by chronic infusion of SHU9119 or AgRP decreases both mean arterial pressure (MAP) and heart rate (HR), although as expected, food intake and weight gain are increased markedly (241,242). Endogenous MC4R activity likely contributes to the elevated arterial pressure in spontaneously hypertensive rats (243). The effects of MC4R on MAP and HR are likely mediated by adrenergic activation (243,244,245), and the MC4R in PVN is likely to be involved in this regulation (246). MC4R agonism in the hindbrain can link sympathetic outflows to cardiac responses (127).

Although the ligands used in some of these studies could not differentiate whether the MC3R or the MC4R or both mediates these pressor effects, studies using knockout mice showed that the MC4R is likely the mediator. ICV administration of α-MSH to WT mice led to increases in both MAP and HR; however, the same treatment does not change these parameters in the Mc4r knockout mice, suggesting that the MC4R mediates the α-MSH action on MAP and HR (247). The Mc4r knockout mice are normotensive with no renal damage despite severe obesity, hyperinsulinemia, and hyperglycemia (247,248).

Clinical studies with MC4R-deficient subjects further extended these findings to humans (249). Although the patients with MC4R haploinsufficiency are obese (see Section V.A), the prevalence of hypertension is significantly lower in these patients than in control subjects with a similar degree of obesity. The blood pressure is also significantly lower than that in control subjects. MC4R agonism led to significant increases in both systolic and diastolic blood pressure (249).

Taken together, these data suggest that although MC4R agonism is a promising therapeutic approach for obesity treatment, there is doubt whether MC4R agonists would offer “a significant therapeutic advantage over currently used appetite suppressants such as sibutramine” (250). Hypertension might be an important adverse side effect for MC4R agonists. Close attention is warranted in any clinical trials.

D. Glucose and lipid homeostasis

In rodents, the MC4R also directly and acutely regulates glucose homeostasis and insulin sensitivity, independent of its effects on food intake and body weight (251,252,253,254,255,256,257,258). In young lean Mc4r knockout mice, plasma insulin level is already increased, and impaired insulin tolerance takes place before the onset of detectable hyperphagia or obesity (251). Central administration of MTII dose-dependently inhibits basal insulin release and improves insulin sensitivity in a number of animal models, including genetic and diet-induced obesity (251,256). MCR expressed in medial hypothalamic nuclei mediates the MTII-induced glucose uptake in peripheral tissues such as skeletal muscle and BAT (259). MC4R signaling in vagal efferents might also be involved (165). ICV administration of NDP-MSH also reduces serum insulin levels, and this effect is blocked by MC4R-specific blocker HS014 (258). In pair-fed animals, ICV administration of α-MSH or MTII markedly enhances the actions of insulin on both glucose uptake and production, whereas SHU9119 exerts opposite effects (252,254). Transgenic overexpression of α-MSH also leads to improved glucose metabolism in both genetic and diet-induced obese models (260,261). A recent study showed that the improvement in hyperinsulinemia after MCR agonist treatment is retained in Mc4r knockout mice, suggesting that other MCRs such as peripheral MC1R and/or MC3R might also be involved in affecting insulin sensitivity (257).

ICV administration of MTII increases skeletal muscle AMP-activated protein kinase activity, even in mice fed with a high-fat diet (262). AMP-activated protein kinase is a critical regulator of skeletal muscle fatty acid β-oxidation; therefore, these data suggest that there is a metabolic link between the hypothalamic melanocortin system and fatty acid mobilization (263). Pharmacological inhibition of MC4R in rats and Mc4r knockout in mice directly and potently promotes lipid uptake, triglyceride synthesis, and fat accumulation in white adipose tissue, whereas increased MCR signaling in the central nervous system triggers lipid mobilization, and these effects are independent of food intake (264). MTII administration increases fat utilization in rats as demonstrated by decreased respiratory quotient (53,265). Similarly, patients with MC4R deficiency also have increased respiratory quotient compared with obese controls (264). By shifting substrate utilization and nutrient partitioning but not elevating circulating triglyceride or free fatty acid levels, the MC4R agonists might be promising therapeutics not just for obesity but also for obesity-associated metabolic disorders such as ectopic lipid deposits and lipotoxicity (263). MC4R activation can also control dietary fat intake, with receptor activation resulting in decreased fat consumption (266) and receptor blockade by Agouti or AgRP or Pomc knockout promoting fat consumption (267,268,269), providing potential further therapeutic benefits.

E. Reproduction and sexual function

Several studies suggested that the MC4R is also involved in modulating reproductive function. Although the Mc4r knockout mice are fertile, reproductive aging is advanced in females. In a mouse line expressing a mutant MC4R with reduced function (I194F), decreased numbers of corpus luteum and increased cystic follicles are observed in aged females (270). Male Mc4r knockout mice have erectile dysfunction (271) that can be corrected by exercise (272), suggesting that the reproductive dysfunction might be due to obesity.

It is well known that leptin regulates both energy homeostasis and reproduction. MC4R mediates the effect of leptin on energy homeostasis (63,138). However, whether it also mediates the effect of leptin on reproduction is not well established. The majority of GnRH neurons are closely apposed by fibers expressing immunoreactive β-endorphin (hence α-MSH) (273). AgRP neurons project to the medial preoptic area, an area that also contains GnRH neurons (274). ICV administration of AgRP results in significant increases in plasma LH and FSH in rats and stimulates GnRH release from hypothalamic explants but has no direct effect on LH release from dispersed anterior pituitary cells. The MC4R is expressed in hypothalamic GT1-1 cells, and stimulation of these cells with NDP-MSH increases GnRH secretion (275,276). Ovariectomized female rats primed with estrogen and progesterone display characteristic LH and prolactin (PRL) surges that are completely abolished by starving. MC4R antagonists decrease the magnitude of LH and PRL surges in normally fed rats and significantly block the leptin stimulation of the hormonal surges in starved rats, suggesting that the MC4R might be an important mediator of the leptin stimulation of LH and PRL surges (277). AgRP abolishes LH and PRL surges in these female rats, whereas anti-AgRP antiserum can partially reinstate both LH and PRL surges (278). These results suggest that the melanocortin system is important for the hormonal surges in female rats. MTII but not γ-MSH (selective agonist for MC3R) restores PRL surge in starved rats, suggesting that the MC4R, not the MC3R, mediates the preovulatory surge in PRL (279). In rhesus monkey, central administration of AgRP inhibits pulsatile LH release, suggesting that AgRP may mediate the effect of a negative energy balance on the reproductive system by suppressing the GnRH pulse generator in primates (280). Further studies are needed to investigate what functions the MC4R have in the males.

In the late 1960s, it was shown that ACTH administered into the cerebral ventricles induces penile erections and ejaculation in male rabbits and beagle dogs (reviewed in Ref. 4). Recently, it was shown that the MC4R is expressed in penile tissues. Van der Ploeg et al. (271) showed that the MC4R mRNA is expressed in nerve fibers and mechanoreceptors in the glans of rat and human penis, and rat spinal cord and pelvic ganglion, the major autonomic relay center to the penis. They further showed that a highly selective nonpeptide MC4R agonist, THIQ ([(N-[(3R)-1,2,3,4-tetrahydroisoquinolinium-3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4- (1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl]-2-oxoethylamine (1)]), augments erectile activity initiated by electrical stimulation of the cavernous nerve in WT but not Mc4r knockout mice. Copulatory behavior is enhanced by THIQ administration and is diminished in Mc4r knockout mice. Taken together, these data suggest that the MC4R modulates penile erectile function, likely through neuronal circuitry in spinal cord erectile centers and somatosensory afferent nerve terminals of the penis.

In male rats, MTII given ICV or intrathecally induces penile erection dose-dependently, and SHU9119 completely blocks this response (281). Intrathecal MTII administration induced almost twice as many erections as ICV administration. These results suggest that in addition to activation of MC4R in the brain, MTII also activates the MCRs in distal spinal cord (likely MC4R) to elicit erectile responses (281).

Several clinical studies have also shown that MTII and its derivative PT-141 can induce transient erections in men with erectile dysfunction (282,283) (reviewed in Refs. 284 and 285). PT-141 also selectively stimulates solicitational behaviors in the female rat, suggesting a potential of this compound in treating female sexual desire disorders (286). Increase in penile erection was also observed with another MC4R agonist, LY2112688, a synthetic peptide agonist with 100-fold higher affinity for the MC4R than the MC3R (249). A recent report showed that a MC4R selective agonist developed by Merck, MK-0493, does not induce erectile responses (287). Therefore, the structure of the particular agonist is likely important in determining whether erectile responses are elicited or not.

F. Miscellaneous functions of the MC4R

In addition to the melanocortins, the processing of POMC gives rise to β-endorphin, which activates the μ-opioid receptor. The MC4R and μ-opioid receptor have similar distributions in the spinal cord and the brain, providing the anatomical basis for potential interactions between the two receptors (reviewed in Ref. 288). For example, the MC4R mRNA is expressed in the striatum, nucleus accumbens, and periaqueductal gray, regions implicated in the behavioral effects of opiates (289). α-MSH antagonism of the morphine-induced analgesia, tolerance, and dependence, has been known for more than 25 yr (290). Chronic administration of morphine results in down-regulation of MC4R mRNA expression in the striatum, nucleus accumbens, and periaqueductal gray, but not in other brain regions such as the hypothalamus, frontal cortex, and substantia nigra (289). These data, together with previous studies demonstrating melanocortin antagonism of different functional effects of opiate treatments (reviewed in Ref. 291), led to the suggestion that this MC4R down-regulation might promote the development of opiate tolerance and dependence (289). Repeated (but not acute) administration of cocaine increases MC4R mRNA expression in the striatum, nucleus accumbens, and hippocampus, but not in the cerebral cortex (292,293). This up-regulation in MC4R mRNA comes with functional consequences, accompanied by increased behavioral responses to α-MSH infusion (292). Blockade of the MC4R in the nucleus accumbens blocks the reinforcing, incentive motivational, and locomotor-sensitizing effects of cocaine (293). MTII also potentiates amphetamine reward as measured by the lowering of the threshold for lateral hypothalamic self-stimulation (294).

Because ethanol drinking and food intake are both appetitive and consummatory behaviors, and both ethanol and food have rewarding properties, it was hypothesized that overlapping central pathways are involved with uncontrolled eating and excessive ethanol consumption (295). ICV administration of MTII reduces ethanol self-administration in both WT and Mc3r knockout mice, suggesting that the MC3R is not involved in mediating the effect of MTII; AgRP increases ethanol self-administration (296). The involvement of the MC4R was further confirmed by demonstrating that a highly selective MC4R agonist, cyclo(NH-CH₂-CH₂-CO-His-D-Phe-Arg-Trp-Glu)-NH₂, also reduces ethanol self-administration (296). Administration of MTII also reduces ethanol intake and ethanol preference in alcohol-preferring rats, likely by modulating ethanol-induced changes in opioid peptide levels (297,298). Even peripheral administration of MTII can reduce ethanol intake in mice (299). α-MSH in discrete areas of the brain may also play a role in the antidepressant-like response of ethanol and depression induced by ethanol withdrawal (300).

The MC4R may also be involved in pain perception. It is expressed in dorsal root ganglia (DRG) and spinal cord, suggesting its potential involvement in presynaptic nociception (301). α-MSH or MTII administration results in increased sensitivity to pain (302,303). The intrathecal injection of synthetic MC4R antagonists and AgRP reduces mechanical allodynia in neuropathic rats (304,305). Pharmacological and genetic blockade of the MC4R increases the antinociceptive effects of morphine but does not change its potency for locomotor activity (288,306). Administration of HS014 (MC4R antagonist) with chronic morphine delays the development of tolerance and prevents withdrawal hyperalgesia (307). MC4R mRNA is induced after axonal injury in both hypoglossal motor neurons and DRG (308). In vitro treatment with α-MSH promotes neurite outgrowth in DRG neurons, and a MC4R-specific antagonist blocks this effect (308), similar to findings in Neuro2A cells (309). These results suggest that MC4R antagonists might be used for the treatment of chronic pain by improving the effectiveness of morphine (288,307).

Although the MC1R and the MC3R are the primary MCRs mediating the anti-inflammatory effect of melanocortins (reviewed in Refs. 310 and 311), there is also some evidence that the MC4R is involved in brain inflammation (reviewed in Ref. 312). Melanocortins inhibit the hypothalamic production of proinflammatory molecules such as nitric oxide and prostaglandins induced by IL-1β (313). Small molecule MC4R agonists were also shown to attenuate brain inflammation and promote survival (314,315). The MC4R also has a neuroprotective effect on cerebral ischemia (316,317) (reviewed in Ref. 318). Glial cells that express the MC4R might also be involved in mediating the antiinflammatory and neuroprotective effects of melanocortins (32,312). Treatment of astrocytes with α-MSH decreases the inducible nitric oxide synthase, and cycloxygenase-2 expression induced by LPS and interferon-γ therefore attenuates inflammation. The MC4R antagonist HS024 blocks the effects of α-MSH, suggesting that the MC4R mediates the α-MSH action (32).

The MC4R has also been shown to modulate anxiety behavior, e.g., that induced by IL-1β (319) or ethanol withdrawal (320), by modulating serotonin transmission (reviewed in Ref. 321). Chaki et al. (322) showed that a potent and selective MC4R antagonist they developed has anxiolytic- and antidepressant-like activities in various rodent models, suggesting that MC4R antagonists might be used for treating patients with stress-related disorders such as depression and/or anxiety. The MC4R also mediates the antipyretic actions of α-MSH (323). Although MC4R activation results in increased thermogenesis, it also suppresses fever (324). α-MSH, through the MC4R, can also correct the memory impairment caused by IL-1β treatment (325).

The potential for targeting the MC4R in treating drug addiction, alcohol abuse, anxiety, and depression remains to be further explored.

IV. Pharmacology of the Melanocortin-4 Receptor

A. Ligand binding and receptor activation

Of the four major endogenous melanocortins, the MC4R has the highest affinity for β-MSH, followed by α-MSH and ACTH, and then γ-MSH (β-MSH > α-MSH > ACTH > γ-MSH) (114). Both Agouti and AgRP are antagonists for the MC4R. The dogfish MC4R has similar pharmacological properties as the hMC4R; therefore, the ligand binding sites were established more than 450 million years ago (23).

Peptide agonists bind in a β-turn conformation that organizes the pharmacophore His-Phe-Arg-Trp in an optimal arrangement for binding and activation of the receptor (326). The N terminus and the ELs are not important for peptide agonist binding (327,328,329). Rather, the binding determinants are located in the TMs. Ionic and aromatic residues in the upper regions of the TMs are important for ligand binding and signaling. These residues can form salt bridge and aromatic-aromatic interactions with residues in the HFRW pharmacophore. Mutations of E100 (2.60) in TM2; D122 (3.25) and D126 (3.29) in TM3; and W258 (6.48), F261 (6.51), and H264 (6.54) in TM6 result in decreased binding affinity for NDP-MSH, suggesting that these residues are potential ligand interaction sites, with D122 interacting with Arg in the pharmacophore (330,331,332). Acidic residues were also found to be important for peptide agonist binding in the MC3R (333). Phe284 (7.35) in TM7 has been proposed to interact with Phe in the HFRW pharmacophore through hydrophobic interactions (334).

It is well known that γ-MSH has selectivity for MC3R over the MC4R (335). Chimera and mutagenesis experiments showed that Y268 (6.58) in TM6 of the MC4R is the primary determinant for the low affinity for a γ-MSH analog, [Nle (4)]Lys-γ₂-MSH (336), whereas residues in TM6, including F267 (6.57), Y268, I269 (6.59), and S270 (6.60), are important in determining the selectivity of [Pro (5), DNal(2′) (8)]-γ-MSH (337).

A recent study used in silico mutagenesis to predict the functional relevance of mutating each residue in hMC4R to all other natural amino acids individually (338). It would be of great interest to experimentally verify the predictions that can potentially contribute significantly to improve our understanding of the MC4R pharmacology.

Another way to study the structure-function relationships of GPCRs is by performing detailed functional characterization of naturally occurring mutants. For example, functional studies of naturally occurring MC4R mutants (see Section V.B) showed that the binding defective mutants are clustered in TM2, EL1, and TM3 (339), implicating this domain as important for ligand binding, consistent with data from previous site-directed mutagenesis experiments (330,331).

Mutation in the most highly conserved Asp in TM2 to Asn, D90N (2.50), causes a loss-of-signaling despite normal ligand binding (Ref. 340 and our unpublished observations), consistent with previous site-directed mutagenesis experiments in other GPCRs (341,342,343). We and others recently showed that mutation of a fully conserved Ser in TM3, S136 (3.39) to Phe, also results in loss of G protein coupling/activation (344,345,346). To gain a better understanding of the role of this codon in receptor function, we generated seven additional mutants, including mutating S136 into hydrophobic amino acids such as Ala and Leu; polar amino acids such as Thr, Tyr, and Cys; and charged amino acids such as Asp and Arg. All seven mutants have normal binding and can signal in response to NDP-MSH stimulation (346). In another study, S136P is retained intracellularly with decreased maximal binding (345); therefore, the phenotype of mutants at S136 depends on the newly introduced residue.

We showed that a naturally occurring mutation in the MC3R, I183N (3.54), at the cytoplasmic end of TM3, causes loss of signaling with normal ligand binding (347). We then generated the corresponding mutation in hMC4R and showed that I151N is also defective in G protein coupling/activation (347).

AgRP has been shown to be not only a competitive antagonist but also an inverse agonist. This was first shown in WT human and constitutively active mouse MC4Rs (82,83). Recently, AgRP was also shown to be an inverse agonist for fish MC4R (45). There is also in vivo evidence supporting the inverse agonist activity of AgRP: AgRP can still regulate energy homeostasis in the absence of endogenous melanocortin agonists (348). Several studies addressed the binding of AgRP to the MC4R. Chimeras generated between the MC1R (which does not bind AgRP) and MC4R (which binds AgRP) (349) showed that the N terminus and EL1 are not involved in AgRP binding; EL2 and EL3 confer binding to AgRP (328). EL3 also confers selectivity for Agouti binding in the MC4R (350). Residues in TM2, TM3, and TM4 are also important for AgRP binding (331,351).

Recently, studies have also been performed on the ligand binding and signaling determinants for small molecule ligands. For example, THIQ was first described by researchers at Merck & Co. (352). Several groups tried to identify how THIQ binds to the MC4R (326,353,354). Using chimeras and site-directed mutagenesis, residues from several TMs, including TM3, TM6, and TM7, were found to be involved in ligand binding. By comparing the MC2R (which does not bind THIQ) and MC4R, both residues conserved between the two receptors (such as E100, D122, D126, F254, and W258), and residues that are not conserved between the two receptors (such as N123, I129, and S131 in TM3) were found to be important for THIQ binding (354). Therefore, THIQ shares some binding determinants with peptide agonists but also has unique binding determinants. Overlapping binding determinants for peptide and nonpeptide ligands were also shown with other analogs (355). Identification of the molecular determinants responsible for receptor binding and signaling of novel peptide and small molecule ligands are important for rational design of drugs targeting the MC4R for treating a variety of diseases.

One widely used method to study GPCR activation is to measure the constitutive activity of the mutant receptors (356). For the MC4Rs, earlier site-directed mutagenesis studies did not report any constitutive activity of the WT or any mutant receptors. The first constitutively active mutant MC4R, in fact, came from characterization of mutant MC4Rs identified from obese patients (357). In this study, L250Q was shown to have very robust constitutive activity. Although it is not compatible with the phenotype (increased activity at the MC4R is expected to result in a lean phenotype or perhaps even anorexia nervosa), it did provide a starting point for further mutagenesis experiments. Multiple mutagenesis together with homology modeling was used to study the functions of L250 in receptor activation (358). Several additional mutants, including S127L, H158R, and P230L, were also reported to cause constitutive activation (346,359,360,361). More detailed studies at these loci have not been reported.

In contrast to other GPCRs, such as LH receptor (362), C5a receptor (363), m1 muscarinic acetylcholine receptor (364), β₂-adrenergic receptor (365), follitropin receptor (365), and TSH receptor (366,367), mutation of a highly conserved Leu in TM3 (3.43) in hMC4R does not cause constitutive activation (356). Rather, L140R has lower basal activity than WT MC4R, suggesting important differences in the role of L3.43 in maintaining the inactive conformation in the MC4R compared with the other GPCRs cited here (356).

We recently showed that H76R and D146N also have high constitutive activities (368). As shown in Fig. 2, ML00253784 can partially decrease the basal signaling of the mutant and WT MC4Rs, proving that these mutants are indeed constitutively active.

Figure 2.

Constitutive activation of two naturally occurring MC4R mutations, H76R and D146N. The basal activities of H76R and D146N are 10- to 15-fold higher than the basal activity of WT MC4R. The small molecule inverse agonist ML00253764 partially decreases the basal activities of the two mutants and the WT MC4R. cAMP levels were measured with RIA (496).

It should be emphasized that for the MC4R, the study of the constitutive activity is not purely of academic interest. It also has potential clinical relevance. Loss of constitutive activity was suggested to be one mechanism for obesity pathogenesis caused by MC4R mutations (369). AgRP serving not just as a competitive antagonist bus also an inverse agonist highlights additionally the physiological relevance of MC4R constitutive activity in regulating energy balance (370,371).

B. Signaling pathways

The classical signaling pathway for the MC4R is by coupling to the heterotrimeric stimulatory G protein (Gs). Receptor activation leads to increased cAMP production, and consequently protein kinase A (PKA) activation. In HEK293 cells stably expressing MC4R, MC4R activation was also shown to increase intracellular calcium (372,373) that was sensitive to cholera toxin (372). In GT1-1 cells, a mouse hypothalamic cell line expressing MC4R endogenously, it was shown that MC4R can increase intracellular calcium through Gq/phospholipase C-dependent signaling pathway (374), although in GT1-7 cells (another mouse hypothalamic cell line closely related to GT1-1 cells expressing MC4R endogenously), no increase in intracellular calcium concentrations can be observed (375). By measuring GTPγS binding, it was shown that the MC4R can also couple to Gi/o proteins (375). In both heterologous cells expressing WT MC4R and GT1-7 cells, MC4R activation stimulates pertussis toxin-sensitive GTPγS binding, implicating coupling to Gi/o proteins (375). Interestingly, AgRP also stimulates GTPγS binding, suggesting that it can decrease cAMP levels by both antagonizing Gs activation and stimulating Gi/o activation (375). The physiological relevance of these signaling pathways has not been investigated in detail.

In addition to activation of the Gs-cAMP-PKA pathway, MC4R also activates the MAPK pathway, and in vivo experiments showed that the activation of ERK1/2 is necessary for MTII-induced suppression of food intake (376). In COS-1 cells expressing rodent MC4R, MTII induce ERK1/2 activation in time- and dose-dependent manners (377). ICV injection of MTII into rat hypothalamus showed that this ERK1/2 activation also happens in vivo (377). Vongs et al. (378) showed that in CHO cells stably expressing hMC4R, NDP-MSH also induces ERK1/2 activation in time- and dose-dependent manners, and this activation is abolished by SHU9119. The GT1-1 cell line that expresses MC4R endogenously also responds to NDP-MSH stimulation with increased ERK1/2 activation (276). In cerebral microvessels, MC4R activation of MAPK leads to potentiation of leptin signaling (379).

Similar to other GPCRs, the signaling pathway leading to ERK1/2 activation is cell-line dependent. In CHO cells expressing hMC4R, phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 block the ERK1/2 activation, whereas PKA inhibitor Rp-cAMPS cannot, suggesting that NDP-MSH-induced ERK1/2 activation is mediated by phosphatidylinositol 3-kinase (378). In GT1-1 cells, increased ERK1/2 activation is mediated by calcium and protein kinase C (276). In vivo, ERK1/2 activation induced by fourth ventricle administration is mediated by the cAMP-PKA pathway (376).

Mulholland and colleagues (380) recently reported that MC4R activation inhibits the c-Jun N-terminal kinase (JNK), therefore inhibiting Ser307 phosphorylation at the insulin receptor substrate-1. These effects were blocked by SHU9119. MC4R agonist augments insulin-stimulated AKT phosphorylation both in vitro and in vivo. Increased insulin-stimulated glucose uptake was observed in GT1-1 cells after NDP-MSH stimulation. These observations suggest that MC4R activation can interact with insulin signaling through modulating JNK activity (380). This is consistent with an earlier observation that central administration of MTII improves insulin tolerance in diet-induced obese rats (256).

In summary, MC4R can couple to all three major classes of G proteins, Gs, Gi/o, and Gq, changing second messengers such as cAMP and calcium and activating MAPK including ERK1/2 and JNK.

C. Internalization, desensitization, and dimerization

Chronic administration of MTII and other agonists has been shown to cause tachyphylaxis (257,381,382), suggesting that there is desensitization. In GT1-7 cells, ligand-induced desensitization was indeed observed (383). In heterologous cells expressing MC4R, it was shown that peptide agonist-activated receptor undergoes GPCR kinase 2-, β-arrestin-, and dynamin-dependent internalization through clathrin-coated pits (383,384). SHU9119 blocks NDP-MSH-stimulated internalization, whereas another MC4R antagonist, HS014, does not induce receptor internalization (384). Nonpeptide agonists induce less internalization (373). AgRP induces MC4R association with the β-arrestins and subsequent internalization (385). Therefore, AgRP not only acts as an inverse agonist antagonizing agonist action; by inducing internalization, AgRP also reduces the amount of MC4R molecules on the cell surface accessible to agonists (385). MC4R expressed heterologously in neuronal cell lines were shown to undergo constitutive endocytosis, also through clathrin-coated pits (386). Prolonged stimulation of the MC4R leads to its translocation from the endosome to the lysosome, likely destined for degradation (384). Very limited recycling of internalized MC4R was observed (384). Detailed studies on the phosphorylation, internalization, and desensitization remain to be performed.

Similar to other GPCRs, MCRs also form dimers or higher-ordered oligomers. Using bioluminescence resonance energy transfer, MC1R and MC3R expressed in Cos-7 cells were shown to dimerize constitutively (387). Coimmunoprecipitation also showed that MC1R dimerize (388). These dimers form early in the biosynthetic pathway, because intracellularly retained mutants also form dimers. In cotransfection experiments, the intracellularly retained mutants dimerize with WT receptors, thereby exerting dominant negative activity (388). Disulfide bond and noncovalent interactions are likely involved in MC1R dimerization (389).

For the MC4R, Biebermann et al. (340), using sandwich enzyme-linked immunosorbent assays and fluorescence resonance energy transfer techniques, showed that WT MC4R dimerize. Furthermore, they showed that a naturally occurring mutation, D90N, can heterodimerize with WT MC4R and exert dominant negative activity (340). Bioluminescence resonance energy transfer also showed that the MC4R dimerizes constitutively and is not affected by ligand binding (390). Several studies that functionally characterize the naturally occurring MC4R mutations showed that the intracellularly retained mutants do not have dominant negative activity (391,392,393). These studies are in sharp contrast to extensive studies in other GPCRs, where intracellularly retained mutant receptors heterodimerize with WT receptors, resulting in decreased cell surface expression and signaling of cotransfected WT receptors (see Refs. 394,395,396,397 for examples). Although there are a few skeptics (398), GPCR dimerization is now widely accepted and is thought to play an important role in biosynthesis and maturation of these receptors (399,400). The reason for the discordant observations in MC4R compared with other GPCRs remains to be elucidated (reviewed in Ref. 339).

Studies designed to identify the forces involved in dimerization showed that unlike the MC1R, mutations to the extracellular cysteine residues do not affect dimerization, suggesting that the TMs are involved in dimerization (401). The molecular determinants for dimerization and the functions of dimerization await further studies.

V. Pathophysiology of the Melanocortin-4 Receptor

A. Naturally occurring MC4R mutations

In 1998, the groups of Froguel and O’Rahilly (402,403) independently reported the first frameshift mutations in the MC4R gene associated with severe early-onset obesity. Since then, more than 150 distinct mutations have been identified from patient cohorts of different ethnic origins (25,340,344,345,357,359,360,361,392,393,404,405,406,407,408,409,410,411,412,413,414,415,416,417,418,419,420,421,422,423,424,425,426,427,428,429,430,431,432,433,434). These mutations include at least 122 missense mutations, two inframe deletion mutations, and seven nonsense mutations (Fig. 3), as well as dozens of frameshift mutations (not shown in Fig. 3). From Fig. 3, it is very clear that these mutations are scattered throughout the MC4R. A total of at least 105 residues are mutated, representing 32% of the receptor (reviewed in Ref. 435). Several earlier articles provided more detailed summaries on the clinical characteristics of the MC4R-deficient patients and biochemical phenotypes of the mutant proteins and can be consulted (339,413,436,437). I recently published a comprehensive review on this topic (435). The following is an abbreviated account.

Figure 3.

Naturally occurring missense, nonsense, and inframe deletion mutations of the MC4R identified from various patient cohorts. The two polymorphisms that confer protection from obesity are indicated with red filling and letters. The mutations that cause constitutive activation are indicated with blue filling and letters. References describing these mutations are listed in the text. [Reprinted, with minor modification, with permission from Y. X. Tao: Prog Mol Biol Transl Sci 88:173–204, 2009 (435). ©Elsevier 2009.]

Although it is well accepted that the level of MC4R gene expression is important for maintaining energy homeostasis (haploinsufficiency causes obesity), no mutations in the promoter region that regulate the MC4R expression have been identified. The polymorphisms identified so far do not cosegregate with obesity phenotype, and therefore their relevance to the pathogenesis of obesity is questionable (417,438,439). One report suggested that a polymorphism in the MC4R promoter might be related to physical activity phenotype: for the C-2745T variant, the T/T genotype is associated with the highest inactivity score (440). Several recent genome-wide association scans showed that SNPs near MC4R are associated with increased risk for obesity (441,442,443), although the mechanism for these SNPs to change MC4R expression has not been elucidated yet. Overall, it seems that defect in MC4R gene transcription is likely not a major cause of severe early-onset obesity.

The prevalence of MC4R mutations appears to vary depending on the ethnic origin, severity of obesity, and age of obesity onset. Up to 6% of early-onset morbidly obese patients in British Caucasians were found to have MC4R mutations (413). In other studies, lower prevalence was found: 0% in morbidly obese Belgian children and adults (444), 1.9% in obese German children and adolescents (359,432), 1.8% in obese Finnish children (360), 1.7% in obese European Caucasians (430), 4.6% in Pima Indians (417), and 2.3% in North American severely obese adults (445). Lower prevalence is observed in Asian and Mediterranean populations (435,446).

Although there are some subjects homozygous for MC4R mutations (392,408,413,416,447), the vast majority of the subjects are heterozygous. Because not all subjects with MC4R mutations are obese, O’Rahilly et al. (448) suggested that the most appropriate mechanism for the mode of inheritance is codominance with modulation of expression and penetrance of phenotype. Obesity is a multifactorial disease with interaction between genotype and environment. For example, a generational effect was observed in the penetrance of MC4R mutations in causing obesity, with younger generation having higher penetrance, likely due to the obesogenic environment of recent decades compared with decades surrounding World War II (430). Considering these facts, it was suggested that “major gene” or “oligogenic” instead of “monogenic” should be used to describe MC4R mutation and obesity (359,449). MC4R genotype also affects the extent of success of lifestyle intervention in achieving weight reduction. Children with decreased MC4R signaling, despite normal weight loss after lifestyle intervention, have much greater difficulty in maintaining weight loss (450).

The MC4R-deficient patients are hyperphagic. When they are offered a buffet meal, they have increased caloric intake (413). The hyperphagia decreases with increasing age. The degree of hyperphagia correlates with residual receptor activity. Mutations that result in a complete loss of signaling cause more severe obesity (413). Although earlier studies showed that MC4R mutations do not affect energy expenditure (413), different from mouse genetic studies (73,74), Baier and colleagues (431) recently showed that Pima Indians with two pathogenic MC4R mutations (R165Q and a frameshift mutation with A insertion at nucleotide 100) have decreased energy expenditure. Similarly, in Hispanic patients, MC4R variants are also associated with altered energy expenditure (434). In addition to obesity, MC4R mutation carriers were also reported to have increased longitudinal growth (413), similar to the Mc4r knockout mice (68). Patients homozygous for MC4R mutations are more severely obese than patients heterozygous for MC4R mutations (413). The effect of MC4R mutation on body mass index is more pronounced in females than in males: MC4R mutation carriers, compared with MC4R WT relatives, are 9.5 and 4 kg/m² higher in body mass index than middle-aged women and men, respectively (430,449). The exact mechanism for this MC4R genotype-sex interaction is not clear at present. Sex steroids might be contributing factors.

Interestingly, several studies suggested that the MC4R is a double-edged sword that can either cause obesity or protect from obesity. Two MC4R polymorphisms, V103I and I251L, have been shown to confer resistance to obesity (highlighted in red in Fig. 3).

V103I is a common polymorphism with the minor allele appearing at a frequency of 2–4%. Rosmond et al. (451) first reported lower abdominal obesity in heterozygotes for V103I compared with homozygotes for V103. Hebebrand and colleagues (452) then expanded this observation to a larger cohort with meta-analysis, confirming the negative association of the I103 allele with obesity rate. A later meta-analysis consisting of 29,563 individuals (10,975 cases and 18,588 controls) showed that for the V103I polymorphism, the I103 allele had an 18% lower risk of obesity compared with the V103 allele (453). Compared with those homozygous for the WT allele, subjects heterozygous for V103I have significantly lower triglyceride levels, decreased waist circumference, decreased glycosylated hemoglobin, and increased high-density lipoprotein-cholesterol, decreasing the odds of having three or more components of the metabolic syndrome by 50% (454,455). Although earlier functional studies did not find any differences between the V103 and I103 MC4Rs in binding and signaling (constitutive and ligand-induced signaling) (357,391,404,452), recently it was shown that I103 is less sensitive to AgRP inhibition (456) that might result in weaker orexigenic signal conferring lower risk of obesity.

I251L confers even stronger protection from obesity than V103I (457), possibly due to its higher constitutive activity (456). It is negatively associated with childhood and adult severe obesity, with odds ratios ranging from 0.25 to 0.76, averaging 0.52. Together with V103I (average odds ratio, 0.80), these two variants may provide 2% protection of obesity, similar to the roughly 2% cause of obesity by pathogenic loss-of-function mutations in the MC4R (457).

Whether there is an association between MC4R mutations and binge-eating disorder is controversial. Most of the earlier studies did not report the eating behavior of the patients harboring MC4R mutations. Only a few cases of binge-eating patients with functionally relevant MC4R mutations were reported (458,459). In 2003, Branson et al. (460) suggested that all obese patients with MC4R mutations had binge-eating disorder. However, 75% of their subjects had MC4R polymorphisms V103I, T112M, and I251L. These variants have been shown by multiple independent groups to be functioning normally (357,392,404), and V103I and I251L are indeed protective for obesity (see above). We showed that the three novel variants they identified, T11A, F51L, and M200V, as well as T112M, have normal functions (461). These results raise serious doubts about the conclusion reached in the Branson et al. article (460). Subsequent clinical studies demonstrated that indeed, MC4R mutations are not associated with binge-eating disorder (421,462).

In summary, these studies unequivocally demonstrated that the MC4R is a critical regulator of energy homeostasis in humans. Mutations in the MC4R are the most common monogenic form of human obesity.

B. Molecular classification of the MC4R mutants

Identification of a MC4R variant from an obese patient does not necessarily prove that the mutation is the cause of obesity in the patient. Additional data, including cosegregation of the mutation with obesity in the family and absence of the variant in the ethnically matched controls, provide additional support for a causative role of the mutation in causing obesity (463). In addition, functional characterization of the mutant receptor in vitro is indispensable to prove a causative role (463).

The MC4R pharmacology can be studied in several aspects. Ligand binding assays can be done in live cells or membranes. Binding assays done in live cells are preferred because any binding measured represents cell surface binding. Binding assays with crude membrane preparations that include intracellular (such as endoplasmic reticulum or Golgi apparatus) membranes cannot distinguish between binding from cell surface receptors or intracellular receptors. It is not clear whether intracellular MC4Rs can bind ligand. However, other immature GPCRs (such as LH receptor) can bind ligand. The ligand of choice is iodinated NDP-MSH. Iodinated α-MSH can no longer bind the receptor; therefore, it cannot be used in receptor binding assays. The iodinated NDP-MSH needs to be purified by HPLC to isolate the monoiodinated NDP-MSH. Because of the special equipment needed, most laboratories use commercial iodinated NDP-MSH. However, the radioligand is extremely expensive; therefore, displacement experiments rather than saturation binding experiments are performed routinely.

Another property of the mutant receptor that needs to be measured is its signaling ability. Because MC4R activates Gs after ligand binding, resulting in increased intracellular cAMP levels, signaling can be measured by either direct assay of cAMP levels or indirect measurement of increased reporter gene expression driving by increased cAMP levels. These assays will reveal whether mutant receptors have either decreased or absent signaling in response to agonist stimulation. Signaling in the absence of ligand can be measured at the same time to determine the basal activity of the mutant receptor.

The third property of the mutant receptors is their cell surface expression, especially for mutants that are defective in ligand binding. Immunocytochemistry, flow cytometry, or ELISA can be used for this purpose. These data differentiate whether the defects in binding are due to defective cell surface expression or to defect in ligand binding per se. If no cell surface expression is observed, permeabilization will reveal whether intracellular staining can be observed, demonstrating whether the protein is produced but retained intracellularly or the mutant receptor expression level is impacted. Because transient transfection can overload the cell’s quality control system (see Ref. 397 for an example), resulting in an artificial intracellular retention of the receptors, cells stably transfected with the receptors are preferred for this purpose (435,464).

To catalog the numerous MC4R mutations, we were the first to propose a classification scheme for MC4R mutations (464). This scheme is based on the life cycle of the receptor and modeled after the classification of mutations in low-density lipoprotein receptor and cystic fibrosis transmembrane conductance regulator (465,466) (Fig. 4). [For a detailed listing of the mutants that belong to each class, the reader is referred to my recent review (435).]

Figure 4.

Molecular classification of naturally occurring MC4R mutations. See the text for detailed description. [Reprinted with permission from Y. X. Tao: Mol Cell Endocrinol 239:1–14, 2005 (339). ©Elsevier 2005.]

1. Class I: null mutations

Due to defective protein synthesis and/or accelerated protein degradation, receptor protein levels are decreased. Nonsense mutants might belong to this class, although this assumption needs to be verified experimentally. We recently showed that R7C, C84R, and W174C, with decreased total expression levels, belong to this class (346).

2. Class II: intracellularly retained mutants

The mutant receptors are produced but are retained intracellularly, most likely in the endoplasmic reticulum due to misfolding being detected by the cell’s quality control system. This class comprises the largest set of MC4R mutations reported to date.

3. Class III: binding defective mutants

These mutants are expressed on the cell surface but are defective in ligand binding per se, with decreased binding capacity and/or affinity. Signaling is frequently impaired secondary to the impairment in binding. Because AgRP is the natural antagonist of the MC4R, if the mutants are more sensitive to inhibition by AgRP, the mutants are functionally defective. However, very few studies measured the binding property to AgRP. I316S was shown to have altered relative affinities for α-MSH and AgRP (393), and V103I is less sensitive than WT MC4R to AgRP inhibition (456). Therefore, these mutants can be classified as a subclass within this class.

4. Class IV: signaling-defective mutants

These mutant MC4Rs are transported to the cell surface, bind ligand, but are defective in agonist-stimulated signaling with decreased efficacy and/or potency. Interestingly, although D90N cannot activate Gs, it can activate Gi/o (375). Whether the other signaling-defective mutants can also activate Gi/o remains to be investigated. The ability of naturally occurring mutants to activate MAPK has not been explored in detail either.

Vaisse and colleagues (369) suggested that decreased basal activities of some MC4R mutants might cause obesity. We showed that of nine mutants identified from patients with binge-eating disorder and nonobese or obese subjects, five mutants have decreased constitutive activities, whereas the other four mutants have normal basal activities (461). Further studies, for example vetting in transgenic animals, are needed to demonstrate unequivocally the physiological significance of the constitutive activity of MC4R (435). We suggest that mutants with defective basal signaling are also class IV mutants.

5. Class V: variants with unknown defect

These variants have normal cell surface expression, ligand binding, and signaling (basal and ligand-stimulated). Whether and how these variants cause energy imbalance and therefore obesity is unclear. It should be emphasized that in the in vitro expression system, there are spare receptors (339), whereas in vivo there is likely no spare receptor. In addition, different coupling to G proteins from in vivo situations and amplified potency and efficacy of agonists (31,467,468,469) are also possible with the in vitro expression system. Finally, in vivo, the MC4R is expressed in neurons. The data obtained with heterologous nonneuronal cells might not reveal any neuron-specific aspects of MC4R function (169,435). Therefore, caution is urged in interpretation of the in vitro data, especially when no overt functional defects are observed.

Since our initial proposal of the classification scheme described above, several other groups proposed modified or alternative classification schemes (345,436,437). The pros and cons of the different systems are compared in detail in Ref. 435.

One drawback of our classification scheme is that we did not include mutants that affect processes downstream of receptor activation, such as desensitization, internalization, and resensitization. Defects in these processes can also cause dysfunctional receptor. For example, a constitutively desensitized V2 vasopressin receptor mutant appeared at first as an apparent defect in signaling (470,471). Therefore, further refinement can be introduced.

In summary, it is important to analyze the functional properties when new variant MC4Rs are identified in obese subjects to more accurately determine whether the phenotype of the variant is indeed consistent with the clinical phenotype and to devise strategies for personalized medicine (see Section V.C). A classification system will be useful in cataloging the increasingly large array of MC4R mutations identified (435).

C. Therapeutic implications

Previously, several strategies for correcting the defects in naturally occurring mutations in GPCRs were summarized (472). Class I mutants due to nonsense mutations can potentially be corrected by aminoglycoside antibiotics by decreasing the codon-anticodon fidelity, resulting in read-through of the premature stop codon. Synthetic ligands can be tested for their abilities to correct class III and IV mutants, those mutants that are expressed on the cell surface but could not bind the endogenous ligands or respond to these ligands. Some of these synthetic ligands might bind to residues different from endogenous ligands and therefore activate some of the mutant receptors (339,473). Indeed, some synthetic MC4R ligands can stimulate various MC4R mutants that are impaired in responding to endogenous ligands (473).

Because class II mutants are most numerous, we have been interested in whether small molecule ligands that can pass the plasma and endoplasmic reticulum membranes might act as pharmacological chaperones correcting the folding defect of the mutant receptors. Some of the MC4R mutants have residual signaling activity. Approaches that result in their increased expression on the cell surface could potentially be of therapeutic value. Bouvier and colleagues pioneered this field with the V2 vasopressin receptor (474). Since then, similar results have been achieved in naturally occurring mutations in GnRH receptor (475,476), rhodopsin (477,478), and WT or laboratory-generated mutants in several other GPCRs (479,480,481,482,483,484,485). The clinical utility of pharmacological chaperones was shown by a recent clinical trial (486).

We showed that ML00253764 could rescue two MC4R mutants that are retained intracellularly, C84R and W174C (346). ML00253764, originally described by Vos et al. (232), has a molecular weight of only 377.3. We hypothesized that it might act as a pharmacological chaperone correcting the misfolding of intracellularly retained mutants. To test this hypothesis, cells stably expressing WT, C84R, or W174C MC4Rs, or transfected with the empty vector pcDNA3.1, were treated with 10⁻⁵m ML00253764 for 24 h. We then investigated cell surface expression of the receptors with confocal microscopy qualitatively and flow cytometry quantitatively. The cell surface expression of both mutants is increased to about 35% of that of the untreated WT MC4R (346). Even the cell surface expression of the WT MC4R is increased to approximately 152% after 24-h treatment with ML00253764, suggesting that WT MC4R maturation is not very efficient. To investigate whether the rescued mutant receptors are functional, the cells were stimulated with NDP-MSH. Both mutant receptors have increased cAMP production, up to 80% of the signaling generated by the untreated WT MC4R (346). We have expanded this observation to six additional naturally occurring mutants. As shown in Fig. 5, all six mutants are rescued to the cell surface to different extents. We have also obtained similar data showing that another small molecule MC4R inverse agonist also acts as a pharmacological chaperone (our unpublished observations). These data suggest that small molecule MC4R ligands can indeed act as pharmacological chaperones assisting the trafficking of intracellularly retained mutant receptors, and many of these rescued mutant receptors are capable of eliciting significant signaling. They could potentially be used to treat patients harboring these MC4R mutations. Because they can also increase the cell surface expression of the WT MC4R, they might also be used to treat common obesity for patients that do not have MC4R mutations.

Figure 5.

Pharmacological rescue of six intracellularly retained MC4R mutants by ML00253764. Human embryonic kidney 293 cells stably transfected with WT or mutant MC4Rs or empty vector pcDNA3.1 (as a negative control) were incubated with 10 μm ML00253764 for 24 h at 37 C. Cells were then stained with anti-myc monoclonal antibody to visualize the c-myc epitope at the N terminus of the MC4R by confocal microscopy. Detailed procedures are described in Ref. 346.

VI. Conclusions and Future Directions

Tremendous progress has been made on the MC4R since 1993 when it was cloned. Diverse approaches, including anatomical localization, pharmacological administration, gene targeting, and the continuous development of novel peptide and small molecule agonists and antagonists as new tools, led to the elucidation of numerous functions for the MC4R, including energy homeostasis, cachexia, cardiovascular function, glucose and lipid homeostasis, reproduction and sexual function, drug addiction, pain perception, and mood. Parallel clinical genetic studies demonstrate the pivotal importance of this gene in obesity pathogenesis. Ligands targeting the MC4R can potentially be used to treat obesity (12,99,487), cachexia (221,222), metabolic syndrome, sexual dysfunction, neuropathic pain, drug addiction, and mood disorders such as depression and anxiety.

There are several important questions that remain to be addressed.

1. What are the downstream mediators mediating the MC4R action in regulating the different physiological processes listed above? Do the same molecules mediate the MC4R action in the different processes, or more likely, do different molecules mediate different processes regulated by the MC4R? Proteomic analysis will likely generate novel leads.

2. A very exciting development in GPCR research during the past 2–3 yr is the series of novel crystal structures revealed, including inactive and active structures of GPCRs (488,489,490,491,492,493,494) (reviewed in Ref. 495). These reports represent a major breakthrough since the report of rhodopsin crystal structure in 2000 (26). A crystal structure, either inactive ligand-free form or complexed with ligands, would be warmly welcomed by scientists working on the pharmacology of the MC4R. The MC4R has constitutive activity and is extremely hydrophobic, characteristics likely hindering its purification and crystallization. Inverse agonists can be used to stabilize the receptor during purification and crystallization.

3. Functional studies showed that the majority of the dysfunctional MC4R mutants are retained intracellularly. Therefore, intracellular trafficking is a very important regulatory point for MC4R function. How the MC4R trafficking is regulated, for example, the molecular chaperones involved in MC4R folding, remains largely unknown.

4. Although numerous MC4R mutations were identified in humans, there are no reports of introducing these mutations into mice to develop mouse models. These mouse models will be important in further dissection of the pathophysiology as well as in preclinical testing of ligands that are found to be able to correct the defect in cells such as the pharmacological chaperones we identified.

Numerous laboratories, especially those in pharmaceutical companies, performed extensive studies trying to discover novel small molecule ligands for the MC4R. These studies are not reviewed herein. Although preclinical trials have shown that some of these MC4R compounds are effective, they have not reached clinical use yet. Side effects, including nausea, yawning and stretching, sexual arousal and penile erections, as well as cardiovascular complications such as hypertension, are partly to blame. Additional considerations are selectivity for the MC4R and tachyphylaxis. It has been pointed out that the MC4R can ameliorate other comorbidities such as lipid and glucose dyshomeostasis, in addition to the treatment of obesity, therefore offering great potential in treating metabolic syndrome (12,99). Further research in this rapidly expanding field offers great promise.