Defect in MAPK Signaling As a Cause for Monogenic Obesity Caused By Inactivating Mutations in the Melanocortin-4 Receptor Gene

The melanocortin-4 receptor (MC4R) is a Family A G protein-coupled receptor that plays an essential role in regulating energy homeostasis, including both energy intake and expenditure. Mutations leading to a reduced MC4R function confer a major gene effect for obesity. More than 170 distinct mutations have been identified in humans. In addition to the conventional Gs-stimulated cAMP pathway, the MC4R also activates MAPKs, especially ERK1/2. We also showed there is biased signaling in the two signaling pathways, with inverse agonists in the Gs-cAMP pathway acting as agonists for the ERK1/2 pathway. In the current study, we sought to determine whether defects in basal or agonist-induced ERK1/2 activation in MC4R mutants might potentially contribute to obesity pathogenesis in patients carrying these mutations. The constitutive and ligand-stimulated ERK1/2 activation were measured in wild type and 73 naturally occurring MC4R mutations. We showed that nineteen mutants had significantly decreased basal pERK1/2 level, and five Class V variants (where no functional defects have been identified previously), C40R, V50M, T112M, A154D and S295P, had impaired ligand-stimulated ERK1/2 activation. Our studies demonstrated for the first time that decreased basal or ligand-stimulated ERK1/2 signaling might contribute to obesity pathogenesis caused by mutations in the MC4R gene. We also observed biased signaling in 25 naturally occurring mutations in the Gs-cAMP and ERK1/2 pathways.     

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

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

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

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

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

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

Pertussis Toxin-sensitive Signaling of Melanocortin-4 Receptors in Hypothalamic GT1-7 Cells Defines Agouti-related Protein as a Biased Agonist

Melanocortin-4 receptor (MC4R)-induced anorexigenic signaling in the hypothalamus controls body weight and energy homeostasis. So far, MC4R-induced signaling has been exclusively attributed to its coupling to G_s proteins. In line with this monogamous G protein coupling profile, most MC4R mutants isolated from obese individuals showed a reduced ability to activate G_s. However, some mutants displayed enhanced G_s coupling, suggesting that signaling pathways independent of G_s may be involved in MC4R-mediated anorexigenic signaling. Here we report that the G_s signaling-deficient MC4R-D90N mutant activates G proteins in a pertussis toxin-sensitive manner, indicating that this mutant is able to selectively interact with G_i/o proteins. Analyzing a hypothalamic cell line (GT1-7 cells), we observed activation of pertussis toxin-sensitive G proteins by the wild-type MC4R as well, reflecting multiple coupling of the MC4R to G_s and G_i/o proteins in an endogenous cell system. Surprisingly, the agouti-related protein, which has been classified as a MC4R antagonist, selectively activates G_i/o signaling in GT1-7 cells. Thus, the agouti-related protein antagonizes melanocortin-dependent G_s activation not only by competitive antagonism but additionally by initiating G_i/o protein-induced signaling as a biased agonist.The melanocortin system has been shown to play a pivotal role in food intake and energy homeostasis. Therefore, dysfunction of the melanocortin system inevitably leads to an obese phenotype in mammals. Accordingly, targeted disruption of the melanocortin-4 receptor (MC4R)² gene in mice causes an obesity-diabetes syndrome characterized by hyperphagia, hyperinsulinemia, and hyperglycemia (1). The importance of MC4R signaling in the regulation of human metabolism has been highlighted by the finding that mutations in the MC4R gene are the most frequent monogenic cause of severe obesity (2–7).Signaling pathways involved in MC4R-mediated regulation of energy homeostasis have been attributed to its coupling to G_s proteins and the resulting activation of the protein kinase A pathway (8, 9). Agouti and agouti-related protein (AGRP) are the only known endogenously occurring neuropeptides that block GPCR activity and are, therefore, classified as MCR antagonists. AGRP has been shown to block melanocortin signaling at MC3R and MC4R subtypes (10). In addition, it has been proposed that AGRP decreases basal as well as forskolin-promoted adenylyl cyclase activity, thus also acting as an inverse agonist on basal MCR activity (11). However, recent studies revealed that the effects of AGRP on appetite control are independent of melanocortin signaling (12, 13). For example, in mice deficient of the melanocortin precursor proopiomelanocortin starvation after AGRP neuron ablation is independent of melanocortin signaling (13). Thus, the orexigenic effects induced by AGRP appear to be mediated in a melanocortin-independent manner by a so far unknown mechanism.Interestingly, the aforementioned MC4R mutants isolated from obese patients exerted inconsistent effects on G_s signaling. For example, the MC4R-G181D or -S94R mutants showed a loss-of-function phenotype, whereas the MC4R-P78L or -R165W variants exhibited reduced function, whereas other mutants (MC4R-G253S, -I317T, -I251L) showed no functional alterations. Even more surprisingly, some mutants (MC4R-S127L, -P230L) constitutively increased G_s-dependent adenylyl cyclase activity (5). Therefore, no clear correlation could be drawn between the cellular phenotype resulting from these mutations and obesity observed in vivo.Melanocortin-independent actions of AGRP and non-uniform effects of obesity-associated mutations on G_s signaling suggest that the MC4R receptor may interact with G proteins other than G_s. The D90N mutation of the MC4R has also been associated with severe early onset obesity (14). This MC4R variant binds melanocortins with unchanged high affinity, but agonist binding does not initiate G_s signaling (14). Thus, the D90N variant represents an excellent tool to analyze putative G_s-independent signaling pathways of the MC4R.Directly measuring incorporation of GTPγ³⁵S, we show herein that the wild-type MC4R and the MC4R-D90N mutant activate pertussis toxin (PTX)-sensitive G_i/o proteins. Multiple coupling of the MC4R to G_s and G_i/o proteins in HEK293 cells is reflected by cAMP accumulation, as treatment of cells with PTX significantly increased melanocortin-induced cAMP accumulation, indicative of simultaneous activation of adenylyl cyclase stimulating and inhibiting pathways. Using a hypothalamic cell line (GT1-7 cells) endogenously expressing MC4R, we demonstrate PTX-sensitive melanocortin-induced GTPγ³⁵S incorporation and an increase in MC4R-mediated cAMP accumulation in response to toxin treatment. In addition, we show that AGRP, assumed to be an antagonist of the MC4R, promotes PTX-sensitive signaling in GT1-7 cells and, thus, exhibits biased agonistic actions on MC4R in hypothalamic cells. These data may explain melanocortin-independent AGRP signaling and define the MC4R as an interface integrating anorexigenic and orexigenic signaling depending on the stimulus received.     

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

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

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

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

Cyclic adenosine monophosphate-dependent activation of transient receptor potential vanilloid 4 (TRPV4) channels in osteoblast-like MG-63 cells

Parathyroid hormone (PTH) directly interacts with bone remodeling osteoblasts and osteocytes expressing the G-protein coupled receptor PTH receptor 1 (PTH1R), and its osteoanabolic effects mostly involve the cAMP/PKA signaling cascade. Considering that PTH-dependent calcium entry in rat enterocytes is reproduced by the adenylate cyclase agonist forskolin or by cAMP analogues, possible involvement of calcium as a second messenger in PTH-dependent cAMP signaling was investigated in MG-63 cells. First, Ca²⁺ influx was confirmed in Fluo3-loaded MG-63 cells treated with a cell-permeable cAMP analog. Second, PTH (1–34) and forskolin promoted calcium influxes that were completely abrogated by the PKA inhibitor H-89. Ca²⁺ entry was not reproduced when PTH (1–34) was combined with the PKC-activating competitor PTH (3–34). Vanilloid transient potential (TRPV) channel inhibitor Ruthenium Red, but not a voltage-dependent calcium channel (VDCC) inhibitor nifedipine, efficiently stunted Ca²⁺ entry, and comparable abrogation was reproduced in cells treated with TRPV4-selective inhibitor RN-1734 or transfected with TRPV4-specific siRNA. Interestingly, PTH-driven Ca²⁺ through TRPV4 significantly inhibited MG63 cell migration through a mechanism requiring extracellular Ca²⁺. In contrast, the inhibitory effects of forskolin on migration were refractory to TRPV4 silencing or to RN-1734. Altogether, our results indicate that single treatment with PTH (1–34) promotes extracellular calcium entry through TRPV4 channels in MG-63 cells through a cAMP/PKA-dependent mechanism, and that this influx affects cell migration.     

Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R–Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor–Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.Subject terms: Cryoelectron microscopy, Cell signalling     

Human GPR17 missense variants identified in metabolic disease patients have distinct downstream signaling profiles

GPR17 is a G-protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Such evidence is primarily drawn from mouse knockout studies and suggests GPR17 as a potential novel therapeutic target for the treatment of metabolic diseases. However, links between human GPR17 genetic variants, downstream cellular signaling, and metabolic diseases have yet to be reported. Here, we analyzed GPR17 coding sequences from control and disease cohorts consisting of individuals with adverse clinical metabolic deficits including severe insulin resistance, hypercholesterolemia, and obesity. We identified 18 nonsynonymous GPR17 variants, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, membrane localization, and downstream signaling profiles of nine GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). These nine GPR17 variants had similar protein expression and subcellular localization as wild-type GPR17; however, they showed diverse downstream signaling profiles. GPR17-G136S lost the capacity for agonist-mediated cAMP, Ca²⁺, and β-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT, but showed impaired Ca²⁺ and β-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca²⁺ signaling, but unaffected agonist-stimulated β-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with diverse signaling profiles, including differential signaling perturbations that resulted in GPCR signaling bias. Our findings provide a framework for structure–function relationship studies of GPR17 signaling and metabolic disease.     

Molecular Characterisation of Small Molecule Agonists Effect on the Human Glucagon Like Peptide-1 Receptor Internalisation

The glucagon-like peptide receptor (GLP-1R), which is a G-protein coupled receptor (GPCR), signals through both Gαs and Gαq coupled pathways and ERK phosphorylation to stimulate insulin secretion. The aim of this study was to determine molecular details of the effect of small molecule agonists, compounds 2 and B, on GLP-1R mediated cAMP production, intracellular Ca²⁺ accumulation, ERK phosphorylation and its internalisation. In human GLP-1R (hGLP-1R) expressing cells, compounds 2 and B induced cAMP production but caused no intracellular Ca²⁺ accumulation, ERK phosphorylation or hGLP-1R internalisation. GLP-1 antagonists Ex(9–39) and JANT-4 and the orthosteric binding site mutation (V36A) in hGLP-1R failed to inhibit compounds 2 and B induced cAMP production, confirming that their binding site distinct from the GLP-1 binding site on GLP-1R. However, K334A mutation of hGLP-1R, which affects Gα_s coupling, inhibited GLP-1 as well as compounds 2 and B induced cAMP production, indicating that GLP-1, compounds 2 and B binding induce similar conformational changes in the GLP-1R for Gα_s coupling. Additionally, compound 2 or B binding to the hGLP-1R had significantly reduced GLP-1 induced intracellular Ca²⁺ accumulation, ERK phosphorylation and hGLP-1R internalisation. This study illustrates pharmacology of differential activation of GLP-1R by GLP-1 and compounds 2 and B.     

Poor cell surface expression of human melanocortin-4 receptor mutations associated with obesity

The melanocortin-4 receptor (MC4R) plays an important role in the regulation of body weight in rodents. Mutations in the coding region of the MC4R are found more frequently in obese individuals, supporting the hypothesis that also in humans deficient melanocortin signaling may lead to obesity. Family studies that were carried out to demonstrate the relevance of single mutations for obesity were mostly inconclusive, most likely due to small sample size and complexity of the trait. In addition, the existing pharmacological data of the mutant receptors are limited in that for most mutations the effect on receptor expression level and Agouti-related protein (AgRP) pharmacology have not been studied. The aim of the present study was to gain further insight into the impact of the MC4R mutations on receptor function. Eleven missense mutations were tested for cell surface expression, affinity for alpha-melanocyte-stimulating hormone (alpha-MSH) and AgRP-(83-132), and the biological response to alpha-MSH. All mutants were poorly expressed at the cell surface, as measured by 125I-[Nle4-D-Phe7]alpha-MSH binding, and only a few mutants showed altered pharmacology for alpha-MSH and AgRP. Hemagglutinin-tagged mutant receptors were retained in the intracellular environment. These pharmacological data provide a basis to estimate the quantitative effect of MC4R mutations for the development of obesity.     

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

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

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

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

Signaling from the human melanocortin 1 receptor to ERK1 and ERK2 mitogen-activated protein kinases involves transactivation of cKIT

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. Upon stimulation by αMSH, MC1R triggers the cAMP and ERK1/ERK2 MAPK pathways. In mouse melanocytes, ERK activation by αMSH binding to Mc1r depends on cAMP, and melanocytes are considered a paradigm for cAMP-dependent ERK activation. However, human MC1R variants associated with red hair, fair skin [red hair color (RHC) phenotype], and increased skin cancer risk display reduced cAMP signaling but activate ERKs as efficiently as wild type in heterologous cells, suggesting independent signaling to ERKs and cAMP in human melanocytes. We show that MC1R signaling activated the ERK pathway in normal human melanocytes and melanoma cells expressing physiological levels of endogenous RHC variants. ERK activation was comparable for wild-type and mutant MC1R and was independent on cAMP because it was neither triggered by stimulation of cAMP synthesis with forskolin nor blocked by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine. Stimulation of MC1R with αMSH did not lead to protein kinase C activation and ERK activation was unaffected by protein kinase C inhibitors. Conversely, pharmacological interference, small interfering RNA studies, expression profiles, and functional reconstitution experiments showed that αMSH-induced ERK activation resulted from Src tyrosine kinase-mediated transactivation of the stem cell factor receptor, a receptor tyrosine kinase essential for proliferation, differentiation, and survival of melanocyte precursors, thus demonstrating a functional link between the stem cell factor receptor and MC1R. Moreover, this transactivation phenomenon is unique because it is unaffected by natural mutations impairing canonical MC1R signaling through the cAMP pathway.     

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

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

An Essential Role for the K+-dependent Na+/Ca2+-exchanger,NCKX4, in Melanocortin-4-receptor-dependent Satiety

K⁺-dependent Na⁺/Ca²⁺-exchangers are broadly expressed in various tissues, and particularly enriched in neurons of the brain. The distinct physiological roles for the different members of this Ca²⁺ transporter family are, however, not well described. Here we show that gene-targeted mice lacking the K⁺-dependent Na⁺/Ca²⁺-exchanger, NCKX4 (gene slc24a4 or Nckx4), display a remarkable anorexia with severe hypophagia and weight loss. Feeding and satiety are coordinated centrally by melanocortin-4 receptors (MC4R) in neurons of the hypothalamic paraventricular nucleus (PVN). The hypophagic response of Nckx4 knock-out mice is accompanied by hyperactivation of neurons in the PVN, evidenced by high levels of c-Fos expression. The activation of PVN neurons in both fasted Nckx4 knock-out and glucose-injected wild-type animals is blocked by Ca²⁺ removal and MC4R antagonists. In cultured hypothalamic neurons, melanocyte stimulating hormone induces an MC4R-dependent and sustained Ca²⁺ signal, which requires phospholipase C activity and plasma membrane Ca²⁺ entry. The Ca²⁺ signal is enhanced in hypothalamic neurons from Nckx4 knock-out animals, and is depressed in cells in which NCKX4 is overexpressed. Finally, MC4R-dependent oxytocin expression in the PVN, a key essential step in satiety, is prevented by blocking phospholipase C activation or Ca²⁺ entry. These findings highlight an essential, and to our knowledge previously unknown, role for Ca²⁺ signaling in the MC4R pathway that leads to satiety, and a novel non-redundant role for NCKX4-mediated Ca²⁺ extrusion in controlling MC4R signaling and feeding behavior. Together, these findings highlight a novel pathway that potentially could be exploited to develop much needed new therapeutics to tackle eating disorders and obesity.     

Structural and signaling requirements of the human melanocortin 4 receptor for MAP kinase activation

In addition to its well known stimulation of cAMP production, the human melanocortin type 4 (hMC4) receptor recently has been shown to mediate p44/42 MAPK activation. This finding opens new questions about the structural and signaling mechanisms that connect the receptor to this alternate cell signaling pathway. Point mutants in the hMC4 receptor that have been associated with obesity were constructed and transfected into HEK 293 cells. Functional analyses then were done to determine if these mutations would similarly impact cAMP formation and p44/42 MAPK signaling. Whereas a D90N mutation in the second transmembrane domain and a D298A mutation in the seventh transmembrane domain impaired both cAMP formation and p44/42 MAPK activation, a more conservative D298N mutation retained cAMP formation but abolished p44/42 MAPK activation. The D298N mutation identified, for the first time, differential structural requirements of the hMC4 receptor for activation of the cAMP and p44/42 MAPK pathways. Furthermore, functional characterizations of a series of chimeric receptors combining the hMC4 receptor and the hMC3 subtype, a receptor that does not couple to p44/42 MAPK activation despite stimulating adenylyl cyclase, indicate that the hMC4 cytoplasmic tail is a necessary structural element for p44/42 MAPK signaling. Subsequent investigation of the signaling requirements for p44/42 MAPK activation demonstrated that the adenylyl cyclase inhibitor 2', 5'-dideoxyadenosine blocked agonist-induced p44/42 MAPK activation, but the PKA inhibitor Rp cAMPS did not. Taken together, these data indicate that cAMP is required, but not sufficient for p44/42 MAPK activation and suggest structural elements required for hMC4 receptor signaling.     

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

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