Discovery and in vivo evaluation of new melanocortin-4 receptor-selective peptides

The melanocortin-4 receptor (MC4R) is involved in several physiological processes, including body weight regulation and grooming behaviour in rats. It has also been suggested that the MC4R mediates the effects of melanocortin ligands on neuropathic pain. Selective compounds are needed to study the exact role of the MC4R in these different processes. We describe here the development and evaluation of new melanocortin compounds that are selective for the MC4R as compared with the other centrally expressed receptors, MC3R and MC5R. First, a library of 18 peptides, in which a melanocortin-based sequence was systematically point-mutated, was screened for binding to and activity on the MC3R, MC4R and MC5R. Compound Ac-Nle-Gly-Lys-D-Phe-Arg-Trp-Gly-NH(2) (JK1) appeared to be the most selective MC4R compound, based on affinity. This compound is 90- and 110-fold selective for the MC4R as compared to the MC3R and MC5R, respectively. Subsequent modification of JK1 yielded compound Ac-Nle-Gly-Lys-D-Nal(2)-Arg-Trp-Gly-NH(2) (JK7)(,) a selective MC4R antagonist with 34-fold MC4R/MC3R and 109-fold MC4R/MC5R selectivity. The compounds were active in vivo as determined in a grooming assay and a model for neuropathic pain in rats. Intravenous (i.v.) injections suggested that they were able to pass the blood-brain barrier.The compounds identified here will be useful in further research on the physiological roles of the MC4R.     

Melanocortin receptor 4 is induced in nerve-injured motor and sensory neurons of mouse

We previously identified melanocortin receptor 4 (MC4R) in a search for genes associated with hypoglossal nerve regeneration. As melanocortins promote nerve regeneration after axonal injury, we investigated whether MC4R functions as a key receptor for peripheral nerve regeneration. In situ hybridization revealed that MC4R mRNA is induced in mouse hypoglossal motor neurons after axonal injury, whereas mRNAs for MC1R, MC2R, MC3R, and MC5R are not expressed either before or after nerve injury. This result was confirmed by RT-PCR. The level of MC4R mRNA expression increased significantly from day 3 after axotomy, reached a peak on day 5, and decreased to the control level on day 14. Similar induction of MC4R was observed in axotomized mouse dorsal root ganglia (DRGs). MC4R mRNA expression was induced exclusively among the MCR family in the L4-6 DRG after sciatic nerve injury. We further examined whether alpha-melanocortin stimulating hormone (alpha-MSH) promotes neurite elongation via MC4R. In mouse DRG neuron culture, alpha-MSH significantly promoted neurite outgrowth at a concentration of 10(-8) mol/L. This neurite-elongation effect was entirely inhibited by the addition of a selective MC4R blocker, JKC-363. Therefore, it is concluded that alpha-MSH could stimulate neurite elongation via MC4R in DRG neurons. The present results suggest that induction of MC4R is crucial for motor and sensory neurons to regenerate after axonal injury.     

Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.     

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.     

Ghrelin-induced food intake and growth hormone secretion are altered in melanocortin 3 and 4 receptor knockout mice

Ghrelin stimulates food intake in part by activating hypothalamic neuropeptide Y (NPY) neurons/agouti related peptide (AGRP) neurons. We investigated the role of AGRP/melanocortin signaling in ghrelin-induced food intake by studying melanocortin 3 and 4 receptor knockout (MC3R KO and MC4R KO) mice. We also determined whether reduced ghrelin levels and/or an altered sensitivity to the GH-stimulating effects of ghrelin accompany the obesity syndromes of MC3R KO and MC4R KO mice. Compared to wild-type (WT) mice, the effects of ghrelin on food intake were reduced in MC3R KO and MC4R KO mice and circulating ghrelin levels were reduced in female MC4R KO mice. Female MC3R KO and MC4R KO mice exhibited a diminished responsiveness to the GH-releasing effects of ghrelin. Thus, deletion of the MC3R or MC4R results in a decreased sensitivity to ghrelin and verifies the involvement in the melanocortin system in ghrelin-induced food intake.     

The melanocortin system in Fugu: determination of POMC/AGRP/MCR gene repertoire and synteny, as well as pharmacology and anatomical distribution of the MCRs

The G-protein-coupled melanocortin receptors (MCRs) play an important role in a variety of essential functions such as the regulation of pigmentation, energy homeostasis, and steroid production. We performed a comprehensive characterization of the MC system in Fugu (Takifugu rubripes). We show that Fugu has an AGRP gene with high degree of conservation in the C-terminal region in addition to a POMC gene lacking gamma-MSH. The Fugu genome contains single copies of four MCRs, whereas the MC3R is missing. The MC2R and MC5R are found in tandem and remarkably contain one and two introns, respectively. We suggest that these introns were inserted through a reverse splicing mechanism into the DRY motif that is widely conserved through GPCRs. We were able to assemble large blocks around the MCRs in Fugu, showing remarkable synteny with human chromosomes 16 and 18. Detailed pharmacological characterization showed that ACTH had surprisingly high affinity for the Fugu MC1R and MC4R, whereas alpha-MSH had lower affinity. We also showed that the MC2R gene in Fugu codes for an ACTH receptor, which did not respond to alpha-MSH. All the Fugu receptors were able to couple functionally to cAMP production in line with the mammalian orthologs. The anatomical characterization shows that the MC2R is expressed in the brain in addition to the head-kidney, whereas the MC4R and MC5R are found in both brain regions and peripheral tissues. This is the first comprehensive genomic and functional characterization of a GPCR family within the Fugu genome. The study shows that some parts of the MC system are highly conserved through vertebrate evolution, such as regions in POMC coding for ACTH, alpha-MSH, and beta-MSH, the C-terminal region of AGRP, key binding units within the MC1R, MC2R, MC4R, and MC5R, synteny blocks around the MCRs, pharmacological properties of the MC2R, whereas other parts in the system are either missing, such as the MC3R and gamma-MSH, or different as compared to mammals, such as the affinity of ACTH and MSH peptides to MC1R and MC4R and the anatomical expression pattern of the MCRs.     

比格犬MC4R基因多态性与体重相关性的研究

为了分析比格犬黑素皮质素受体-4基因多态性与犬体重的关系, 根据犬MC4R基因DNA外显子序列, 设计MC4R基因特异PCR引物1对, 犬DNA经PCR扩增, 克隆和测序, 寻找和确定犬MC4R基因的多态性位点, 分析多态性与犬体重的关系。结果在比格犬MC4R基因中发现2处单碱基缺失突变, 1个单碱基颠换变异, 存在Psh AⅠ酶切位点, 并基于PshAⅠ酶切位点建立了PCR-RFLP技术。统计分析显示犬MC4R基因型与体重显著相关, 可以考虑将MC4R基因作为犬体重的候选基因。     

Melanocortin subtype-4 receptor agonists containing a piperazine core with substituted aryl sulfonamides

The biological activity for a set of melanocortin-4 receptor (MC4R) agonists containing a piperazine core with an ortho-substituted aryl sulfonamide is described. Compounds from this set had binding and functional activities at MC4R less than 30 nM. The most selective compound in this series was >25,000-fold more potent at MC4R than MC3R, and 490-fold more potent at MC4R than MC5R. This compound also reduced food intake after oral dosing at 25, 50, and 100 mg kg(-1) in fasted mice.     

鸽黑素皮质素受体3、4(MC3R、MC4R)基因多态性与生长性状的相关分析

利用PCR-SSCP技术和DNA测序方法检测广东石岐肉鸽和哈尔滨地区灰色家鸽MC3R和MC4R基因部分编码区序列的单核苷酸多态性, 分析了MC3R基因T91G突变位点和MC4R基因A903G突变位点导致的基因型与两群体鸽生长和体组成性状的关系。结果表明, 这两个多态位点所导致的基因型对石岐肉鸽活重、屠体重、全净膛重均有显著影响(P<0.05); 另外, 利用这两个突变位点所产生的合并基因型在鸽群体中与生长和体组成性状作最小二乘分析, 结果表明, 两位点合并后的基因型对全净膛重影响显著(P<0.05)。多重比较结果表明, BBAA型全净膛重显著大于AABB型, BBAA型对于体重增长是有利基因型。     

黑皮质素受体-4调节通路的研究进展

黑皮质素系统来自阿片-促黑素细胞皮质素原,在中枢摄食行为和能量平衡代谢中起到重要作用,此系统生理功能的发挥主要通过与下丘脑神经元细胞上特定膜受体（黑皮质素受体）结合完成。黑皮质素受体（MCR）有五种亚型（MC1R-MC5R）,其中参与体重调节的受体主要是黑皮质素受体3（MC3R）和黑皮质素受体4（MC4R）。MC4R属于G蛋白耦联受体,具有七次跨膜结构。作为一种膜受体,MC4R发挥体重调节作用,一方面受外界激动剂或拮抗剂的调节;另一方面,此受体活化后会影响到细胞内的信号调节通路。研究MC4R的功能首先要了解受体的结构,本文对G蛋白耦联受体的结构进行了较详细的叙述,MC4R经信号调节通路,激活腺苷酸环化酶,增加cAMP的浓度,最终通过影响细胞内基因的转录和翻译,来调节体重和能量的消耗。     

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.     

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.     

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

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

Design,Synthesis, Structural and Functional Characterization of Novel Melanocortin Agonists Based on the Cyclotide Kalata B1

Obesity is an increasingly important global health problem that lacks current treatment options. The melanocortin receptor 4 (MC4R) is a target for obesity therapies because its activation triggers appetite suppression and increases energy expenditure. Cyclotides have been suggested as scaffolds for the insertion and stabilization of pharmaceutically active peptides. In this study, we explored the development of appetite-reducing peptides by synthesizing MC4R agonists based on the insertion of the His-Phe-Arg-Trp sequence into the cyclotide kalata B1. The ability of the analogues to fold similarly to kalata B1 but display MC4R activity were investigated. Four peptides were synthesized using t-butoxycarbonyl peptide chemistry with a C-terminal thioester to facilitate backbone cyclization. The structures of the peptides were found to be similar to kalata B1, evaluated by Hα NMR chemical shifts. KB1(GHFRWG;23–28) had a K_i of 29 nm at the MC4R and was 107 or 314 times more selective over this receptor than MC1R or MC5R, respectively, and had no detectable binding to MC3R. The peptide had higher affinity for the MC4R than the endogenous agonist, α-melanocyte stimulation hormone, but it was less potent at the MC4R, with an EC₅₀ of 580 nm for activation of the MC4R. In conclusion, we synthesized melanocortin analogues of kalata B1 that preserve the structural scaffold and display receptor binding and functional activity. KB1(GHFRWG;23–28) is potent and selective for the MC4R. This compound validates the use of cyclotides as scaffolds and has the potential to be a new lead for the treatment of obesity.     

用放射杂交板定位鸡的MC4R基因以及其在鸡和人染色体上同源区的比较分析

黑素皮质素受体(melanocortin-4 receptor,MC4R)基因的突变与猪、鼠和人等的食欲、肥胖和生长有关联性,然而对鸡的MC4R基因的功能却知之甚少。为了确定鸡的MC4R基因在染色体上的位置,使用鸡-仓鼠杂交板(ChickRH6)做了该基因的定位工作。通过扩增ChickRH6杂交板上的93个样品,然后经整合分析将mC4R基因定位在2号染色体上的标记MCW0062、BCL2和OVY附近,即2q12。这个连锁图上的5个标记基于两点分析与MC4R的LOD值都大于5。同时,以MC4R基因为标记做了鸡和人的染色体比较分析。结果显示鸡的2号染色体(GGA2)和人的18号染色体(HSA18)存在同源区,且基因BCL2和肥胖基因(obesity)位于MC4R基因附近。推测鸡的MC4R基因与人的MC4R基因可能具有相似的功能。该研究揭示了鸡和人MC4R基因的染色体分布,并用杂交放射板将鸡的MC4R基因定位在2号染色体的12区带。     

Linkage and association studies between the melanocortin receptors 4 and 5 genes and obesity-related phenotypes in the Québec Family Study.

BACKGROUND: The agouti yellow mouse shows adult onset of moderate obesity and diabetes. A depressed basal lipolytic rate in adipocytes or a decreased adrenergic tone arising from antagonizing alpha-melanocyte-stimulating hormone (MSH) activation of melanocortin receptors (MCR) could be at the origin of the obesity phenotype. MATERIAL AND METHODS: MCR 4 and 5 (MC4R, MC5R) genes were studied in the Québec Family Study. Sequence variations were detected by Southern blot probing of restricted genomic DNA, and mRNA tissue expression was detected by RT-PCR. Subjects with a wide range of weight were used for single-point sib-pair linkage studies (maximum of 289 sibships from 124 nuclear families). Analysis of variance across genotypes in unrelated males (n = 143) and females (n = 156) was also undertaken. Body mass index (BMI), sum of six skin-folds (SF6), fat mass (FM), percent body fat (%FAT), respiratory quotient (RQ), resting metabolic rate (RMR), fasting glucose and insulin, and glucose and insulin area during an oral glucose tolerance test were analyzed. RESULTS: MC4R showed polymorphism with NcoI, and MC5R, with PstI and PvuII, with a heterozygosity of 0.38, 0.10, and 0.20, respectively. Linkages were observed between MC5R and BMI (p = 0.001), SF6 (p = 0.005), FM (p = 0.001), and RMR (p = 0.002), whereas associations were observed in females between MC5R and BMI (p = 0.003), and between MC4R and FM (p = 0.002) and %FAT (p = 0.004). After correction for multiple tests, these p values are lowered by one tenth. MC4R and MC5R mRNAs have been detected in brain, adipose tissue, and skeletal muscle. CONCLUSIONS: MC4R and MC5R exhibit evidence of linkage or association with obesity phenotypes, but this evidence is strongest for MC5R.     

Mahoganoid and mahogany mutations rectify the obesity of the yellow mouse by effects on endosomal traffic of MC4R protein

The ubiquitous overexpression of agouti-signaling protein (ASP), a paracrine-signaling molecule that regulates pigment-type switching in the hair follicle of the mouse, is responsible for the obesity and yellow pelage of the Yellow mouse (A(y)). Mahogany (Attractin, Atrn/mg) and mahoganoid (Mahogunin Ring Finger-1, Mgrn1/md) are mutations epistatic to A(y). These mutations have been described as suppressors of ASP action, blocking its antagonizing effects on the melanocortin 1 and 4 receptors (MC1R and MC4R) in the skin and the brain, respectively, via unknown mechanisms. Here, we describe the molecular bases for the md- and mg-dependent rescue of the A(y) phenotype at the MC4R. We show that overexpression of ASP inhibits the rise in cAMP levels in response to α-melanocyte-stimulating hormone, an MC4R agonist, by blocking ligand binding and by directing MC4R trafficking to the lysosome. Loss-of-function of either attractin or MGRN1 blocks ASP-dependent MC4R degradation and promotes increased trafficking of internalized MC4R to the cell surface, but it does not restore α-melanocyte-stimulating hormone-dependent cAMP signaling. We propose that MGRN1 and attractin are components of an evolutionarily conserved receptor trafficking pathway and that the md and mg mutations rescue the A(y) phenotypes by a primarily cAMP-independent mechanism promoting trafficking of MC4R and likely MC1R away from the lysosome toward the cell surface.