Chimeras of the agouti-related protein: insights into agonist and antagonist selectivity of melanocortin receptors

The specific melanocortin receptors, MC3R and MC4R, are directly linked to metabolism and body weight control. These receptors are activated by the peptide hormone alpha-MSH and antagonized by the agouti-related protein (AGRP). Whereas alpha-MSH acts broadly on most members of the MCR family (with the exception of MC2R), AGRP is highly specific for only MC3R and MC4R. AGRP is a complex ligand of approximately 100 amino acids. Within AGRP, MCR recognition and antagonism is localized to a 34 residue, cysteine-rich domain that adopts an inhibitor cystine knot (ICK) fold. An oxidatively folded peptide corresponding to this domain, referred to as mini-AGRP, exhibits full antagonist function and selectivity for MC3R and MC4R. Here we investigate a series of chimera proteins based on the mini-AGRP scaffold. Amino acid sequences derived from peptide agonists are grafted into the mini-AGRP active loop, implicated in receptor recognition, with the goal of producing ICK based agonists specific for MC3R and MC4R. Several constructs indeed exhibited potent agonist activity; however, with all chimeras, receptor selectivity is significantly altered. Pharmacologic data indicate that the chimeras do not interact with MC receptors through native AGRP like contacts. A model to explain the data suggest that there is only partial overlap of the agonist versus antagonist binding surfaces within MC receptors. Moreover, accessibility to the binding pocket is highly receptor specific with MC3R being the least tolerant of ligand alterations.     

Structure activity studies of the melanocortin-4 receptor by in vitro mutagenesis: identification of agouti-related protein (AGRP), melanocortin agonist and synthetic peptide antagonist interaction determinants

In vitro mutagenesis of the mouse melanocortin-4 receptor (mMC4R) has been performed, based upon homology molecular modeling and previous melanocortin receptor mutagenesis studies that identified putative ligand-receptor interactions. Twenty-three mMC4 receptor mutants were generated and pharmacologically characterized using several melanocortin-based ligands [alpha-MSH, NDP-MSH, MTII, DNal (1')(7)-MTII, Nal(2')(7)-MTII, SHU9119, and SHU9005]. Selected mutant receptors possessing significant differences in the melanocortin-based peptide agonist and/or antagonist pharmacology were further evaluated using the endogenous antagonist agouti-related protein fragment hAGRP(83-132) and hAGRP(109-118) molecules. These studies of the mouse MC4R provide further experimental data suggesting that the conserved melanocortin receptor residues Glu92 (TM2), Asp114 (TM3), and Asp118 (TM3) (mouse MC4R numbering) are important for melanocortin-based peptide molecular recognition. Additionally, the Glu92 and Asp118 mMC4R residues are important for molecular recognition and binding of AGRP(83-132). We have identified the Phe176 (TM4), Tyr179 (TM4), Phe254 (TM6), and Phe259 (TM6) receptor residues as putatively interacting with the melanocortin-based ligand Phe(7) by differences between alpha-MSH and NDP-MSH agonist potencies. The Glu92, Asp118, and Phe253 mMC4R receptor residues appear to be critical for hAGRP(83-132) molecular recognition and binding while Phe176 appears to be important for functional antagonism of AGRP(83-132) and AGRP(109-118) but not molecular recognition. The Phe253 mMC4R residue appears to be important for AGRP(83-132) molecular recognition and general mMC4 receptor stimulation. The Phe254 and Phe259 mMC4R amino acids may participate in the differentiation of agonist versus antagonist activity of the melanocortin-based peptide antagonists SHU9119 and SHU9005, but not AGRP(83-132) or AGRP(109-118). The Met192 side chain when mutated to a Phe results in a constitutively active mMC4R that does not effect agonist ligand binding or potency. Melanocortin-based peptides modified at the 7 position of MTII with DPhe, DNal(1'), Nal(2'), and DNal(2') have been pharmacologically characterized at these mutant mouse MC4Rs. These data suggest a revised hypothesis for the mechanism of SHU9119 antagonism at the MC4R which may be attributed to the presence of a "bulky" naphthyl moiety at the 7 position (original hypothesis), and additionally that both the stereochemistry and naphthyl ring position (2' versus 1') are important for positioning of the ligand Arg(8) residue with the corresponding mMC4R amino acids.     

Sustained orexigenic effect of Agouti related protein may be not mediated by the melanocortin 4 receptor

Intracerebroventricular (ICV) injection of Agouti related protein (AgRP), an endogenous melanocortin 3 and 4 receptor (MC3/4-R) antagonist, produces a prolonged increase in food intake. To clarify the roles of the MC3-R and MC4-R in AgRP-induced hyperphagia, the feeding effect of AgRP (83-132) was compared with that of the selective MC4-R antagonist, JKC-363 (cyclic [Mpr11, D-Nal14, Cys18, Asp22-NH2]-beta-MSH11-22). Single ICV administration of AgRP (83-132) increased food intake for 48 h whilst ICV JKC-363 increased food intake for 8h. An increase in body weight at 24 and 48 h was observed following AgRP (83-132) but not JKC-363 treatment. These data suggest that the sustained orexigenic action of AgRP (83-132) may not be through MC4-R antagonism.     

Dynamic regulation of hypothalamic neuropeptide gene expression and food intake by melanocortin analogues and reversal with melanocortin-4 receptor antagonist

Melanocortins are known to be involved in the inhibition of food intake and energy metabolism. Acute and chronic intracerebroventricular administration of several different analogues of alpha-MSH, such as alpha-MSH, NDP-MSH, alpha-MSH-ND, [Gln(6)]alpha-MSH-ND, and [Lys(6)]alpha-MSH-ND, which were substituted in the position of His(6) with Gln and Lys, and cyclic16k-MSH to C57J/BL6 mice resulted in a significant inhibition of both time course food intake and body weight gain, compared to the saline-administered control. However, [Gln(6)]alpha-MSH-ND(6-10), the truncated form of [Gln(6)]alpha-MSH-ND, had no inhibitory effects on food intake. In situ hybridization analysis revealed that the expression levels of AGRP and NPY in the hypothalamus were significantly and rapidly diminished while POMC expression was strongly induced by [Gln(6)]alpha-MSH-ND. Administration of JKC-363, a selective MC4R-specific antagonist, coupled with [Gln(6)]alpha-MSH-ND, specifically reversed the [Gln(6)]alpha-MSH-ND-induced inhibition of food intake, but also reversed the hypothalamic expression levels of neuropeptides such as AGRP, NPY, MCH, and POMC, which suggests [Gln(6)]alpha-MSH-ND can function as a selective MC4R agonist.     

Isolation and characterization of antagonist and agonist peptides to the human melanocortin 1 receptor

We identified a large number of peptide mimotopes of the adrenocorticotropic hormone (ACTH) and the alpha-melanocyte stimulating hormone (alpha-MSH) to analyze better the structure-function relationships of these hormones with the human MC1 receptor (hMC1R). We have investigated the use of phage-display technology to isolate specific peptides of this receptor by using three monoclonal anti-ACTH antibodies (mAbs). A library of 10(8) phage-peptides displaying randomized decapeptides was constructed and used to select phage-peptides that bind to mAbs. Forty-five phage-peptides have been isolated and from their amino acid sequences, we have identified two consensus sequences, EXFRWGKPA and WGXPVGKP, corresponding to the regions 5-13 and 9-16 of ACTH, respectively. A biological assay on cells expressing the hMC1-R was developed to determine the capacity of phage-peptides to stimulate the receptor. Only two phage-peptides showed detectable activity. Thirty-one peptides were synthesized to analyze their biological effect. We identified two weak agonists, EC50=16 and 11 microM, two strong agonists, EC50=25 and 14 nM and a partial antagonist, IC50=36 microM. This work confirmed the modulator agonist role of the regions 11-12 of alpha-MSH and ACTH, and the importance of the methionine residue at position 4 for the stimulation of the hMC1-R. We also identified analogues of the regions 8-17 of ACTH that exhibited a weak activator effect, and of one analogue of the N-terminal regions 1-9 of ACTH and alpha-MSH having a partial antagonist effect. These results may be useful in the development of potential agonists or antagonists of the hMC1R.     

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.     

Molecular determinants of human melanocortin-4 receptor responsible for antagonist SHU9119 selective activity

The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met(128) in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu(133) of hMC4R plays a key role in melanocortin receptor subtype specificity.     

Agouti signalling protein is an inverse agonist to the wildtype and agonist to the melanic variant of the melanocortin-1 receptor in the grey squirrel (Sciurus carolinensis)

The melanocortin-1 receptor (MC1R) is a key regulator of mammalian pigmentation. Melanism in the grey squirrel is associated with an eight amino acid deletion in the mutant melanocortin-1 receptor with 24 base pair deletion (MC1RΔ24) variant. We demonstrate that the MC1RΔ24 exhibits a higher basal activity than the wildtype MC1R (MC1R-wt). We demonstrate that agouti signalling protein (ASIP) is an inverse agonist to the MC1R-wt but is an agonist to the MC1RΔ24. We conclude that the deletion in the MC1RΔ24 leads to a receptor with a high basal activity which is further activated by ASIP. This is the first report of ASIP acting as an agonist to MC1R.     

Down-regulation of melanocortin receptor signaling mediated by the amino terminus of Agouti protein in Xenopus melanophores.

Agouti protein and Agouti-related protein (Agrp) regulate pigmentation and body weight, respectively, by antagonizing melanocortin receptor signaling. A carboxyl-terminal fragment of Agouti protein, Ser73-Cys131, is sufficient for melanocortin receptor antagonism, but Western blot analysis of skin extracts reveals that the electrophoretic mobility of native Agouti protein corresponds to the mature full-length form, His23-Cys131. To investigate the potential role of the amino-terminal residues, we compared the function of full-length and carboxyl-terminal fragments of Agrp and Agouti protein in a sensitive bioassay based on pigment dispersion in Xenopus melanophores. We find that carboxyl-terminal Agouti protein, and all forms of Agrp tested, act solely by competitive antagonism of melanocortin action. However, full-length Agouti protein acts by an additional mechanism that is time- and temperature-dependent, depresses maximal levels of pigment dispersion, and is therefore likely to be mediated by receptor down-regulation. Apparent down-regulation is not observed for a mixture of amino-terminal and carboxyl-terminal fragments. We propose that the phenotypic effects of Agouti in vivo represent a bipartite mechanism: competitive antagonism of agonist binding by the carboxyl-terminal portion of Agouti protein and down-regulation of melanocortin receptor signaling by an unknown mechanism that requires residues in the amino terminus of the Agouti protein.     

Study of a structurally similar kappa opioid receptor agonist and antagonist pair by molecular dynamics simulations

Among the structurally similar guanidinonaltrindole (GNTI) compounds, 5′-GNTI is an antagonist while 6′-GNTI is an agonist of the κOR opioid receptor. To explore how a subtle alteration of the ligand structure influences the receptor activity, we investigated two concurrent processes: the final steps of ligand binding at the receptor binding site and the initial steps of receptor activation. To trace these early activation steps, the membranous part of the receptor was built on an inactive receptor template while the extracellular loops were built using the ab initio CABS method. We used the simulated annealing procedure for ligand docking and all-atom molecular dynamics simulations to determine the immediate changes in the structure of the ligand–receptor complex. The binding of an agonist, in contrast to an antagonist, induced the breakage of the “3–7 lock” between helices TM3 and TM7. We also observed an action of the extended rotamer toggle switch which suggests that those two switches are interdependent.     

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. -Melanocyte stimulating hormone (-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111–113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7–9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3–5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH₂ peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.     

Common mechanism for the estrogen agonist and antagonist activities of droloxifene

The incidence of postmenopausal osteoporosis is increasing as the population ages. Even though estrogen replacement therapy has proven beneficial in reducing the number of skeletal fractures, the known risks and associated side-effects of estrogen replacement therapy make compliance poor. Recent research has focused on the development of tissue specific estrogen agonist/anatagonists such as droloxifene which can prevent estrogen deficiency-induced bone loss without causing uterine hypertrophy. Furthermore, droloxifene acts as a full estrogen antagonist on breast tissue and is being evaluated for treatment of advanced breast cancer. In this report we propose a common mechanism of action for droloxifene that underlies its estrogen agonist and antagonist effects in different tissues. Droloxifene and estrogen, which have identical effects on bone in vivo, both induced p53 expression and apoptosis in cells of in vitro rat bone marrow cultures resulting in a decrease in the number of bone-resorbing osteoclasts. Droloxifene is growth inhibitory in MCF-7 human breast cancer cells and therefore acts as an antagonist, whereas estrogen is mitogenic to these cells and acts as an agonist. Droloxifene, but not estrogen, induced p53 expression and apoptosis in MCF-7 cells. These results indicate that the induction of apoptosis by droloxifene may be the common mechanism for both its estrogen agonist effects in bone and its antagonist effects in breast tissue. J. Cell. Biochem. 65:159–171. © 1997 Wiley-Liss, Inc.     

The natural inverse agonist agouti-related protein induces arrestin-mediated endocytosis of melanocortin-3 and -4 receptors

Agouti-related protein (Agrp), one of the two naturally occurring inverse agonists known to inhibit G protein-coupled receptor activity, regulates energy expenditure by decreasing basal and blocking agonist-promoted melanocortin receptor (MCR) signaling. Here we report that, in addition to its inverse agonistic activities, Agrp exhibits agonistic properties on the endocytosis pathway of melanocortin receptors. Sustained exposure of human embryonic kidney 293 cells to Agrp induced endocytosis of the MC3R or the MC4R. The extent and kinetics of Agrp-promoted MCR endocytosis were similar to the endocytosis induced by melanocortins. Using the bioluminescence resonance energy transfer technique, we further showed that after binding of Agrp both MCRs interacted with beta-arrestins. In line with this observation, in COS-7 cells co-expression of beta-arrestins enhanced Agrp-induced MCR endocytosis, whereas in human embryonic kidney 293 cells co-transfection of beta-arrestin-specific small interference RNAs diminished Agrp-promoted endocytosis. This new regulatory mechanism was likewise detectable in a cell line derived from murine hypothalamic neurons endogenously expressing MC4R, pointing to the physiological relevance of Agrp-promoted receptor endocytosis. In conclusion, we demonstrated that Agrp does not solely act by directly blocking MCR signaling but also by reducing the amount of MCR molecules accessible to melanocortins at the cell surface. This beta-arrestin-dependent mechanism reveals a new aspect of MCR signaling in particular and refines the concept of G protein-coupled receptor antagonism in general.     

Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.     

Discovery of a spiroindane based compound as a potent,selective, orally bioavailable melanocortin subtype-4 receptor agonist

We report the design, synthesis and properties of spiroindane based compound 1, a potent, selective, orally bioavailable, non-peptide melanocortin subtype-4 receptor agonist. Compound 1 shows excellent erectogenic activity in the rodent models.     

A potent and selective nonpeptide antagonist of the melanocortin-4 receptor induces food intake in satiated mice

Optimization on a series of piperazinebenzylamines resulted in analogues with low nanomolar binding at the human MC4 receptor but weak affinity (Ki > 500 nM) at the MC3 receptor. Compound 14c was identified to be a potent MC4R antagonist (Ki = 3.2 nM) with a selectivity of 240-fold over MC3R. It proved to be an insurmountable antagonist in a cAMP assay. Compound 14c potently stimulated food intake in satiated mice when given by intracerebroventricular administration.     

Peptide and small molecules rescue the functional activity and agonist potency of dysfunctional human melanocortin-4 receptor polymorphisms

The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal weight control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms observed as both heterozygous and homozygous forms. A number of laboratories have been studying these hMC4R polymorphisms attempting to understand the molecular mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small molecules attempting to identify ligands that can pharmacologically rescue the hMC4R polymorphic agonist response. The ligands examined in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg-Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH2, AMW3-106), and the small molecules JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examined in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the molecular mechanism(s) involving these receptor modifications.     

Molecular basis of melanocortin-4 receptor for AGRP inverse agonism

We have investigated receptor structural components of the melanocortin-4 receptor (MC4R) responsible for ligand-dependent inverse agonism. We utilized agouti-related protein (AGRP), an inverse agonist which reduces MC4R basal cAMP production, as a tool to determine the molecular mechanism. We tested a series of chimeric receptors and utilized MC4R and MC1R as templates, in which AGRP is an inverse agonist for MC4R but not for MC1R. Our results indicate that replacements of the extracellular loops 1, 2 and 3 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity. However, replacement of the N terminus of MC4R with the same region of MC1R decreases AGRP inverse agonism. Replacement of transmembrane domains 3, 4, 5 and 6 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity but mutation of D90A in transmembrane 2 (TM2) and D298A in TM7 abolished AGRP inverse activity. Deletion of the distal MC4R C terminus fails to maintain AGRP mediated reduction in basal cAMP production although it maintains NDP-MSH mediated cAMP production. In conclusion, our results indicate that the N terminus and the distal C terminus of MC4R do appear to play important roles in AGRP inverse agonism but not NDP-MSH mediated receptor activation. Our results also indicate that the residues D90 in TM2 and D298 in TM7 of hMC4R are involved in not only NDP-MSH mediated receptor activation but also AGRP mediated inverse agonism.     

Involvement of melanocortin-4 receptor in anxiety and depression

The melanocortins, which are derived from proopiomelanocortin, have a variety of physiological functions mediated membrane surface receptors. To date, five subtypes have been cloned. With the cloning of melanocortin receptors, studies with genetic models, and development of selective compounds, the physiological roles of the five melanocortin receptors have begun to be understood. The melanocortin-4 receptor (MC4R), which is predominantly expressed in the central nervous system, has in particular become the focus of much attention in recent years because of the critical roles it plays in a wide range of functions, including feeding, sexual behavior, and stress. Recent development of selective antagonists for the MC4R has provided pharmacological evidence that blockade of MC4R could be a useful way of alleviating numerous conditions such as anxiety/depression, pain, and addiction to drugs of abuse.