RESUMO
The central and peripheral melanocortin system, comprising of five receptors and their endogenous ligands, is responsible for a wide array of physiological functions such as skin pigmentation, sexual function and development, and inflammation. A growing body of both clinical and pre-clinical research is demonstrating the relevance of this system in metabolic health. Disruption of hypothalamic melanocortin signalling is the most common cause of monogenic obesity in humans. Setmelanotide, an FDA-approved analogue of alpha-melanocyte stimulating hormone (α-MSH) that functions by restoring central melanocortin signalling, has proven to be a potent pharmacological tool in the treatment of syndromic obesity. As the first effective therapy targeting the melanocortin system to treat metabolic disorders, its approval has sparked research to further harness the links between these melanocortin receptors and metabolic processes. Here, we outline the structure of the central and peripheral melanocortin system, discuss its critical role in the regulation of food intake, and review promising targets that may hold potential to treat metabolic disorders in humans.
Assuntos
Ingestão de Alimentos , Glucose , Melanocortinas , Receptores de Melanocortina , Animais , Humanos , alfa-MSH/metabolismo , alfa-MSH/análogos & derivados , Ingestão de Alimentos/fisiologia , Glucose/metabolismo , Homeostase , Melanocortinas/metabolismo , Obesidade/metabolismo , Obesidade/tratamento farmacológico , Receptores de Melanocortina/metabolismo , Receptores de Melanocortina/genética , Transdução de SinaisRESUMO
Recent studies from our group on melanocortin 2 receptors (Mc2r) from basal families of actinopterygians have served to resolve that Mrap1 dependence and ACTH selectivity are features of even the most basal ray-finned fishes. However, there have been no studies on Mc2r function of the basal sarcopterygians, the lobe-finned fishes, represented by the extant members coelacanths and lungfishes. Here, we offer the first molecular and functional characterization of an Mc2r from a lobe-finned fish, the West African lungfish (Protopterus annectens). Plasmids containing cDNA constructs of lungfish (lf) Mc2r and Mrap1 were expressed in mammalian and zebrafish cell lines. Cells were then stimulated by human ACTH(1-24) and melanocyte stimulating hormone (α-MSH), as well as alanine-substituted analogs of hACTH(1-24) targeting residues within the H6F7R8W9 and K15K16R17R18P19 motifs. Activation of lfMc2r was assessed using a cAMP-responsive luciferase reporter gene assay. In these assays, lfMc2r required co-expression with lfMrap1, was selective for ACTH over α-MSH at physiological concentrations of the ligands, and was completely inhibited by multiple-alanine substitutions of the HFRW (A6-9) and KKRRP (A15-19) motifs. Single- and partial-alanine substitutions of the HFRW and KKRRP motifs varied in their impacts on receptor-ligand affinity from having no effect to completely inhibiting lfMc2r activation. This characterization of the Mc2r of a lobe-finned fish fulfills the last major extant vertebrate group for which Mc2r function had yet to be characterized. In doing so, we resolve that all basal bony vertebrate groups exhibit Mc2r function that substantially differs from that of the cartilaginous fishes, indicating that rapid and dramatic shift in Mc2r function occurred between the radiation of cartilaginous fishes and the emergence of bony fishes. We support this interpretation with a molecular clock analysis of the melanocortin receptors, which demonstrates the uniquely high rate of sequence divergence in Mc2r. Much remains to be understood regarding the molecular evolution of Mc2r during the early radiation of vertebrates that resulted in the derived functional characteristics of Mrap1 dependence and exclusive selectivity for ACTH.
Assuntos
Receptor Tipo 2 de Melanocortina , alfa-MSH , Animais , Humanos , Hormônio Adrenocorticotrópico/farmacologia , Alanina/genética , Evolução Molecular , Mamíferos/metabolismo , Receptor Tipo 2 de Melanocortina/genética , Receptor Tipo 2 de Melanocortina/metabolismo , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well-known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R-MC5R). The observation of MRAP2 on regulating several non-melanocortin G protein-coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown. METHODS: Here, we performed single-cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs-associated network of two major endocrine organs, the hypothalamus and adrenal gland at single-cell resolution. We also integrated multiple bulk RNA-seq profiles and single-cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co-expression correlation with MRAPs. RESULTS: 36 and 46 metabolic-related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein-protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand-stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno-associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co-injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2-dependent manner. CONCLUSIONS: Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad-spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR-MRAP functional complexes.
Assuntos
Proteínas de Transporte , Receptores de Melanocortina , Animais , Humanos , Camundongos , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Melanocortinas/metabolismo , Glândulas Suprarrenais/metabolismo , Hipotálamo/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The melanocortin receptors are G-protein-coupled receptors, which are essential components of the hypothalamic-pituitary-adrenal axis, and they mediate the actions of melanocortins (melanocyte-stimulating hormones: α-MSH, ß-MSH, and γ-MSH) as well as the adrenocorticotropin hormone (ACTH) in skin pigmentation, adrenal steroidogenesis, and stress response. Three melanocortin receptor genes (MC1R, MC2R, and MC5R) contribute to the risk of major depressive disorder (MDD), and one melanocortin receptor gene (MC4R) contributes to the risk of type 2 diabetes (T2D). MDD increases T2D risk in drug-naïve patients; thus, MDD and T2D commonly coexist. The five melanocortin receptor genes might confer risk for both disorders. However, they have never been investigated jointly to evaluate their potential contributing roles in the MDD-T2D comorbidity, specifically within families. In 212 Italian families with T2D and MDD, we tested 11 single nucleotide polymorphisms (SNPs) in the MC1R gene, 9 SNPs in MC2R, 3 SNPs in MC3R, 4 SNPs in MC4R, and 2 SNPs in MC5R. The testing used 2-point parametric linkage and linkage disequilibrium (LD) (i.e., association) analysis with four models (dominant with complete penetrance (D1), dominant with incomplete penetrance (D2), recessive with complete penetrance (R1), and recessive with incomplete penetrance (R2)). We detected significant (p ≤ 0.05) linkage and/or LD (i.e., association) to/with MDD for one SNP in MC2R (rs111734014) and one SNP in MC5R (rs2236700), and to/with T2D for three SNPs in MC1R (rs1805007 and rs201192930, and rs2228479), one SNP in MC2R (rs104894660), two SNPs in MC3R (rs3746619 and rs3827103), and one SNP in MC4R genes (Chr18-60372302). The linkage/LD/association was significant across different linkage patterns and different modes of inheritance. All reported variants are novel in MDD and T2D. This is the first study to report risk variants in MC1R, MC2R, and MC3R genes in T2D. MC2R and MC5R genes are replicated in MDD, with one novel variant each. Within our dataset, only the MC2R gene appears to confer risk for both MDD and T2D, albeit with different risk variants. To further clarity the role of the melanocortin receptor genes in MDD-T2D, these findings should be sought among other ethnicities as well.
Assuntos
Transtorno Depressivo Maior , Diabetes Mellitus Tipo 2 , Comorbidade , Depressão , Diabetes Mellitus Tipo 2/genética , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Melanocortinas/genética , Melanocortinas/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismoRESUMO
Melanocortin receptors have emerged as important targets with a very unusual versatility, as their widespread distribution on multiple tissues (e.g. skin, adrenal glands, brain, immune cells, exocrine glands) together with the variety of physiological processes they control (pigmentation, cortisol release, satiety mechanism, inflammation, secretions), place this family of receptors as genuine therapeutic targets for many disorders. This review focuses in the journey of the development of melanocortin receptors as therapeutic targets from the discovery of their existence in the early 1990 s to the approval of the first few drugs of this class. Two major areas of development characterise the current state of melanocortin drug development: their role in obesity, recently culminated with the approval of setmelanotide, and their potential for the treatment of chronic inflammatory and autoimmune diseases like rheumatoid arthritis, multiple sclerosis or fibrosis. The pro-resolving nature of these drugs offers the advantage of acting by mimicking the way our body naturally resolves inflammation, expecting fewer side effects and a more balanced (i.e. non-immunosuppressive) response from them. Here we also review the approaches followed for the design and development of novel compounds, the importance of the GPCR nature of these receptors in the process of drug development, therapeutic value, current challenges and successes, and the potential for the implementation of precision medicine approaches through the incorporation of genetics advances.
Assuntos
Artrite Reumatoide , Melanocortinas , Humanos , Proteínas de Transporte , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Melanocortinas/metabolismo , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismoRESUMO
The increasing use of marginal lungs for transplantation encourages novel approaches to improve graft quality. Melanocortins and their receptors (MCRs) exert multiple beneficial effects in pulmonary inflammation. We tested the idea that treatment with the synthetic α-melanocyte-stimulating hormone analogue [Nle4,D-Phe7]-α-MSH (NDP-MSH) during ex vivo lung perfusion (EVLP) could exert positive influences in lungs exposed to different injuries. Rats were assigned to one of the following protocols (N = 10 each): 1) ischemia/reperfusion (IR) or 2) cardiac death (CD) followed by ex vivo perfusion. NDP-MSH treatment was performed in five rats of each protocol before lung procurement and during EVLP. Pulmonary function and perfusate concentration of gases, electrolytes, metabolites, nitric-oxide, mediators, and cells were assessed throughout EVLP. ATP content and specific MCR expression were investigated in perfused lungs and in biopsies collected from rats in resting conditions (Native, N = 5). NDP-MSH reduced the release of inflammatory mediators in perfusates of both the IR and the CD groups. Treatment was likewise associated with a lesser amount of leukocytes (IR: p = 0.034; CD: p = 0.002) and reduced lactate production (IR: p = 0.010; CD: p = 0.008). In lungs exposed to IR injury, the NDP-MSH group showed increased ATP content (p = 0.040) compared to controls. In CD lungs, a significant improvement of vascular (p = 0.002) and airway (Ppeak: p < 0.001, compliance: p < 0.050, pO2: p < 0.001) parameters was observed. Finally, the expression of MC1R and MC5R was detected in both native and ex vivo-perfused lungs. The results indicate that NDP-MSH administration preserves lung function through broad positive effects on multiple pathways and suggest that exploitation of the melanocortin system during EVLP could improve reconditioning of marginal lungs before transplantation.
Assuntos
Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Perfusão/métodos , alfa-MSH/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Morte , Ácido Hialurônico/metabolismo , Mediadores da Inflamação/metabolismo , Ácido Láctico/metabolismo , Pulmão/fisiopatologia , Masculino , Técnicas de Cultura de Órgãos , Perfusão/efeitos adversos , Edema Pulmonar/etiologia , Ratos Sprague-Dawley , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismo , Traumatismo por Reperfusão/prevenção & controle , alfa-MSH/farmacologiaRESUMO
The melanocortin-5 receptor (mc5r) plays an important role in exocrine function, lipid metabolism, obesity, and stress response in the vertebrate. However, the functions of the mc5r in mollusks have been rarely investigated. We cloned the full length of Ruditapes philippinarum mc5r like gene (mc5rl) and the sequence structure and phylogenetic relationship of mc5rl were analyzed. Besides, we detected the tissue distribution and the expression pattern of R. philippinarum mc5r like (mc5rl) genes after aerial exposure and low-temperature stress. The full-length cDNA of the mc5rl-1 was 2143 bp, consisting of a 1224 bp open reading frame encoding (ORF) 408 amino acids. Sequence and phylogenetic analyses revealed that the nucleotide and amino acid sequences of Manila clam mc5rl were highly homologous with mc5r of Crassostrea virginica, Crassostrea gigas, Mizuhopecten yessoensis, and Pecten maximus (32%-36%) and low homologous with vertebrates. The results of the distribution of mc5rl genes showed that mc5rl genes were dominant in the mantle, gonad, and hepatopancreas in R. philippinarum. The expression of mc5rl genes was significantly increased after aerial exposure and low-temperature stress in R. philippinarum in hepatopancreas. Aerial exposure and low-temperature stress could induce mc5rl expressed. Mc5rl might serve as a sensor and promote stress response in R. philippinarum. The cloning and expression characteristics of mc5rl will facilitate the investigation of its function in stress response and other physiological processes in R. philippinarum.
Assuntos
Bivalves/genética , Perfilação da Expressão Gênica/métodos , Fases de Leitura Aberta/genética , Receptores de Melanocortina/genética , Sequência de Aminoácidos , Animais , Atmosfera , Sequência de Bases , Clonagem Molecular , Temperatura Baixa , DNA Complementar/genética , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína , Receptores de Melanocortina/química , Receptores de Melanocortina/classificação , Homologia de Sequência de AminoácidosRESUMO
Central administration of neuropeptide K (NPK), a 36-amino acid peptide, is associated with anorexigenic effects in rodents and chickens. The mechanisms underlying the potent anorexigenic effects of NPK are still poorly understood. Thus, the aim of the present study was to identify the hypothalamic nuclei and neuropeptides that mediate anorexic effects of NPK in 7â¯day-old Japanese quail (Coturnix japonica). After a 6â¯h fast, intracerebroventricular (ICV) injection of NPK decreased food and water intake for 180â¯min post-injection. Quail injected with NPK had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), lateral hypothalamus, and paraventricular nucleus (PVN) compared to the birds that were injected with the vehicle. In the ARC of NPK-injected quail, there was decreased neuropeptide Y (NPY), NPY receptor sub-type 1, and agouti-related peptide mRNA, and increased CART, POMC, and neurokinin receptor 1 mRNA. NPK-injected quail expressed greater amounts of corticotropin-releasing factor (CRF), CRF receptor sub-type 2, melanocortin receptors 3 and 4, and urocortin 3 mRNA in the PVN. In conclusion, results provide insights into understanding NPK-induced changes in hypothalamic physiology and feeding behavior, and suggest that the anorexigenic effects of NPK involve the ARC and PVN, with increased CRF and melanocortin and reduced NPY signaling.
Assuntos
Anorexia/genética , Coturnix/metabolismo , Hipotálamo/metabolismo , Taquicininas/farmacologia , Animais , Anorexia/induzido quimicamente , Anorexia/metabolismo , Anorexia/patologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Núcleo Arqueado do Hipotálamo/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Coturnix/genética , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipotálamo/efeitos dos fármacos , Infusões Intraventriculares , Proteínas do Tecido Nervoso/genética , Neuropeptídeo Y/genética , Pró-Opiomelanocortina/genética , Proteínas Proto-Oncogênicas c-fos/genética , Receptores de Melanocortina/genética , Taquicininas/metabolismo , Urocortinas/genéticaRESUMO
The melanocortin-5 receptor (MC5R) is a member of the G protein-coupled receptor superfamily that plays a critical role in lipid production, skeletal muscle fatty acid oxidation, and adipocyte lipolysis. Although multiple functions and important value of MC5R in human beings have been fully demonstrated, however, the potential molecular cloning, pharmacological characteristics and key amino acids in poultry and pig were still not fully understood. Herein, we successfully cloned MC5R genes from chicken (Gallus gallus, cMC5R), duck (Anas platyrhynchos, dMC5R), goose (Anser cygnoides domesticus, gMC5R) and pig (Sus scrofa domestica, pMC5R), and compared their genetic and protein difference with hMC5R through phylogenetic analysis and homology models. Besides, we constructed three alanine-substitution mutants for each of MC5Rs through homologous reorganization, including c/d/gMC5R-D119A/F254A/H257A and pMC5R-D204A/F339A/H342A. Subsequently, we focused our investigation on the pharmacological characterization of four wide-type MC5Rs and their mutants in HEK293T cells, including the intracellular cAMP generation and phosphorylation level of ERK1/2. The results showed that these mutants had decreased cAMP levels under the stimulation of ligands, in spite of enhanced basal activity for c/d/gF254A and pH342A, indicating their important roles in the location and activation of receptors. Notably, these MC5Rs and mutants displayed significant species-specific phenotypes in the activation of pERK1/2 with ligands, which was not completely consistent with hMC5R. These findings demonstrated that presence of interspecies differences for MC5Rs, particularly for the pERK1/2 pathway. Taken together, our study expands current knowledge about the molecular and pharmacological characterization of c/d/g/pMC5Rs, providing preliminary data for MC5R-targeted drug screening or genetic breeding of economic animals in the future.
Assuntos
Mutação/genética , Aves Domésticas/metabolismo , Receptores de Melanocortina/metabolismo , Sus scrofa/genética , Sequência de Aminoácidos , Animais , Galinhas/metabolismo , AMP Cíclico/metabolismo , Patos/metabolismo , Gansos/metabolismo , Células HEK293 , Humanos , Ligantes , Sistema de Sinalização das MAP Quinases , Fosforilação , Filogenia , Receptores de Melanocortina/química , Receptores de Melanocortina/genéticaRESUMO
RT-PCR analysis of gar pituitary and brain indicated that different combinations of gar melanocortin receptor mRNAs are present in the same tissues with mRNAs for gar mrap1 and gar mrap2. Against this background, an objective of this study was to determine whether the ligand sensitivity for either ACTH or α-MSH was affected when gar (g) melanocortin receptors (Mcrs) were co-expressed with either of the accessory proteins gMrap1 or gMrap2 in Chinese Hamster Ovary cells. The results indicated that gMc2r has an obligatory requirement for co-expression with gMrap1 in order for the receptor to be activated by hACTH(1-24). In addition, activation of gMc2r did not occur when the receptor was expressed alone or co-expressed with gMrap2. Furthermore, co-expression of gMc2r with gMrap1 followed by stimulation with NDP-MSH resulted in a low level of activation (only at 10-7â¯M and 10-6â¯M). However, gMc1r, gMc3r, gMc4r, and gMc5r responded to stimulation by NDP-MSH in a more robust manner. Co-expression of gMc1r, gMc3r, gMc4r, and gMc5r with gMRAP1 had no effect on sensitivity to stimulation by NDP-MSH or hACTH(1-24). Co-expression with gMRAP2 had no negative or positive effect on ligand sensitivity for gMc1r, gMc3r, and gMc5r, however this treatment did increase the activation of CHO cells transfected with gMc4r following stimulation with both hACTH(1-24) (pâ¯<â¯0.001), and NDP-MSH (pâ¯<â¯0.001). Co-expression of gMC5R with either gMRAP1 or gMRAP2 increased trafficking of gMC5R to the plasma membrane. These pharmacological observations are compared to the response of melanocortin receptors from other neopterygian fishes, cartilaginous fishes, and tetrapods to stimulation by ACTH(1-24) and forms of α-MSH.
Assuntos
Peixes/metabolismo , Receptores de Melanocortina/metabolismo , Transdução de Sinais , Hormônio Adrenocorticotrópico/farmacologia , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Peixes/genética , Regulação da Expressão Gênica , Genes Reporter , Humanos , Ligantes , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Melanocortina/química , Receptores de Melanocortina/genéticaRESUMO
Tissue-specific immune regulation is an important component of the immune response relevant to many areas of immunology. The focus of this study is on tissue-specific mechanisms that contribute to autoimmune uveitis. Precise gene regulation is necessary for the proper expression of an inflammatory or regulatory response. This precision gene regulation can be accomplished by microRNA at the level of the mRNA transcript. miR-155, in particular, has a complicated role in the immune response with positive and negative inflammatory effects. In this work, we identify a decrease in miR-155 in suppressor macrophages and further examine how tissue-specific production of miR-155 impacts experimental autoimmune uveitis. Importantly, we show that eliminating miR-155 expression by the target tissue before initiation reduces disease severity, but elimination of miR-155 after the onset of inflammation does not alter the course of disease. Additionally, expression of miR-155 by the target tissue before initiation is necessary for the induction of regulatory immunity that protects from further autoimmune disease, but not after the onset of inflammation. In summary, we find a MC5r-dependent decrease in miR-155 in postexperimental autoimmune uveitis APC, miR-155 production by the target tissue is necessary for the initiation of autoimmune uveitis, and may have a role in establishing protective regulatory immunity.
Assuntos
Doenças Autoimunes/genética , Macrófagos/imunologia , MicroRNAs/genética , Receptores de Melanocortina/genética , Epitélio Pigmentado da Retina/imunologia , Uveíte/genética , Animais , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Adjuvante de Freund/administração & dosagem , Regulação da Expressão Gênica/imunologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/imunologia , Especificidade de Órgãos , Receptores de Melanocortina/imunologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Uveíte/induzido quimicamente , Uveíte/imunologia , Uveíte/patologiaRESUMO
The five melanocortin receptors (MC1R-MC5R) are involved in numerous biological pathways, including steroidogenesis, pigmentation, and food intake. In particular, MC3R and MC4R knockout mice suggest that the MC3R and MC4R regulate energy homeostasis in a non-redundant manner. While MC4R-selective agonists have been utilized as appetite modulating agents, the lack of MC3R-selective agonists has impeded progress in modulating this receptor in vivo. In this study, the (pI)DPhe position of the tetrapeptide Ac-His-Arg-(pI)DPhe-Tic-NH2 (an MC3R agonist/MC4R antagonist ligand) was investigated with a library of 12 compounds. The compounds in this library were found to have higher agonist efficacy and potency at the mouse (m) MC3R compared to the MC4R, indicating that the Arg-DPhe motif preferentially activates the mMC3R over the mMC4R. This observation may be used in the design of new MC3R-selective ligands, leading to novel probe and therapeutic lead compounds that will be useful for treating metabolic disorders.
Assuntos
Oligopeptídeos , Receptores de Melanocortina/agonistas , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismo , Relação Estrutura-AtividadeAssuntos
Hormônio Liberador da Corticotropina/genética , Polimorfismo de Nucleotídeo Único , Pró-Opiomelanocortina/genética , Psoríase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína Agouti Sinalizadora/genética , Proteína Relacionada com Agouti/genética , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Oxirredutases Intramoleculares/genética , Masculino , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/genética , Fenótipo , Psoríase/diagnóstico , Receptores de Melanocortina/genética , Fatores de Risco , Adulto JovemRESUMO
The presence of Mrap1 and Mrap2 orthologs in the genome of the elephant shark (es), a cartilaginous fish, presented an opportunity to evaluate the potential interactions between these accessory proteins and melanocortin receptors of a cartilaginous fish. RT-PCR analysis indicated that Mrap1 mRNA was present in interrenal, brain, and pituitary tissue with mRNA for Mc2R, Mc3R, Mc4R, and Mc5r. Co-expression of esMrap1 cDNA with esMc2r cDNA or esMc5r cDNA in CHO cells increased sensitivity to stimulation with ACTH(1-24) 10 fold and 100 fold, respectfully, but had no effect on sensitivity to stimulation with DesAc-αMSH [i.e., ACTH(1-13)NH2] for either receptor, and had no effect on the ligand sensitivity of either esMc3r or esMc4r. Fluorescence image analysis indicated co-localization of esMrap1/esMc2r, and esMrap1/esMc5r on the plasma membrane; however, cell surface ELISA analysis indicated that co-expression with esMrap1 had no effect, positive or negative, on the trafficking of either esMc2r or esMc5r to the plasma membrane. RT-PCR analysis also indicated that Mrap2 mRNA, as well as, mRNAs for Mc2r, Mc3r, Mc4r, and Mc5r could be detected in brain tissue, however no Mrap2 mRNA was detected in interrenal tissue. Co-expression of esMrap2 in CHO cells with, respectively, esMc2r, esMc4r, or esMc5r had no effect on ligand sensitivity. However, co-expression of esMrap2 with esMc3r did lower sensitivity to stimulation by DesAc-αMSH 10 fold. These observations are discussed in the context of the parallel evolution of melanocortin receptors and their accessory proteins, and the hypothalamus/pituitary/adrenal axis and the hypothalamus/pituitary/interrenal axis in bony vertebrates and cartilaginous fishes.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Receptor Tipo 1 de Melanocortina/metabolismo , Receptor Tipo 2 de Melanocortina/metabolismo , Receptores de Melanocortina/genética , Animais , Peixes , TubarõesRESUMO
The melanocortin-5 receptor (MC5R) plays an important role in the regulation of exocrine secretion in mammals. Its function in fish is not well established. In this study, we reported the molecular cloning, tissue expression, and pharmacological characterization of Megalobrama amblycephala MC5R (MamMC5R), as well as the effect of catching stress on its expression. The full-length cDNA of Mammc5r gene was 1237 bp, consisted of a 990-bp open reading frame encoding 329 amino acids. Sequence analyses revealed that the nucleotide and amino acid sequences of Mammc5r were highly homologous (> 90%) with MC5Rs of zebrafish, common carp, and goldfish. Tissue expression profile analysis showed that Mammc5r was widely expressed in both central and peripheral tissues, with the highest expression in the brain. Catching stress significantly changed the expression of Mammc5r in the skin, brain, and eye. In the skin, the expression level of Mammc5r was significantly reduced at 1 h and 4 h and increased at 24 h after catching stress. The Mammc5r expression level was rapidly upregulated in the brain and eye at 1 h and then decreased to the level before stress at 4 h and 24 h. With human MC5R (HsaMC5R) as a control, several agonists including α-melanocyte-stimulating hormone (α-MSH), and ß-MSH in addition to an analogue [Nle4, D-Phe7]-α-MSH (NDP-MSH), were used to investigate the binding and signaling properties of MamMC5R. The results revealed that MamMC5R had the highest affinity for NDP-MSH, followed by α- and ß-MSH. Taken together, the data suggested that MamMC5R might play a role in stress response in M. amblycephala.
Assuntos
Clonagem Molecular , Cyprinidae/metabolismo , Proteínas de Peixes/metabolismo , Receptores de Melanocortina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cyprinidae/genética , DNA Complementar , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Masculino , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Melanocortina/química , Receptores de Melanocortina/genética , Transdução de Sinais , Distribuição Tecidual , TranscriptomaRESUMO
Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1)-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiology, TRPV1 knock-out (KO), and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5) carrying a Cre-dependent channelrhodopsin-2 (ChR2)-enhanced yellow fluorescent protein (eYFP) expression cassette under the control of the two neuronal POMC enhancers (nPEs). Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Ingestão de Alimentos/genética , Neurônios/metabolismo , Pró-Opiomelanocortina/genética , Canais de Cátion TRPV/genética , Animais , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capsaicina/farmacologia , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Optogenética , Condicionamento Físico Animal , Pró-Opiomelanocortina/metabolismo , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismo , Transdução de Sinais , Análise de Célula Única , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/deficiência , TemperaturaRESUMO
The melanocortin receptors (MCRs) and their accessory proteins (MRAPs) are involved in regulation of a diverse range of endocrine pathways. Genetic variants of these components result in phenotypic variation and disease. The MC1R is expressed in skin and variants in the MC1R gene are associated with ginger hair color. The MC2R mediates the action of ACTH in the adrenal gland to stimulate glucocorticoid production and MC2R mutations result in familial glucocorticoid deficiency (FGD). MC3R and MC4R are involved in metabolic regulation and their gene variants are associated with severe pediatric obesity, whereas the function of MC5R remains to be fully elucidated. MRAPs have been shown to modulate the function of MCRs and genetic variants in MRAPs are associated with diseases including FGD type 2 and potentially early onset obesity. This review provides an insight into recent advances in MCRs and MRAPs physiology, focusing on the disorders associated with their dysfunction.
Assuntos
Proteínas de Transporte/fisiologia , Doença/genética , Receptores de Melanocortina/fisiologia , Glândulas Suprarrenais/fisiologia , Animais , Proteínas de Transporte/metabolismo , Criança , Humanos , Mutação/fisiologia , Ligação Proteica , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismoRESUMO
Previous studies on bony vertebrate MC2R orthologs (i.e., ray finned fishes, amphibians, reptiles, birds, and mammals) have shown that these MC2R orthologs have an obligatory requirement for interaction with bony vertebrate MRAP1 orthologs to a) allow for the trafficking of the MC2R ortholog to the plasma membrane; and b) to allow activation by ACTH, but not by any MSH-sized ligand. In addition, previous studies have found that co-expression of teleost and mammalian MC4R orthologs with corresponding MRAP2 has positive effects on sensitivity to stimulation by αMSH or ACTH. MRAP1 and MRAP2 paralogs have been detected in the genome of a cartilaginous fish (elephant shark), yet two cartilaginous fish MC2R orthologs (elephant shark and red stingray) do not apparently require MRAP1 for trafficking to the plasma membrane when expressed in Chinese Hamster Ovary (CHO) cells, and both orthologs can be activated by either ACTH or MSH-sized ligands. This study was done to determine whether sensitivity to stimulation by ACTH(1-24) or Des-Acetyl-αMSH is affected when stingray (sr) MC1R, MC2R, MC3R, MC4R or MC5R were co-expressed in CHO cells with either elephant shark (es) MRAP1 or esMRAP2. The results indicated that co-expression with heterologous MRAP1 increased the sensitivity of all five stingray melanocortin receptors for srACTH(1-24), but had not statistically significant effect on stimulation by srDes-Acetyl-αMSH for any of the stingray melanocortin receptors. Conversely, co-expression with esMRAP2 only enhanced sensitivity for srDes-Acetyl-αMSH for srMC4R, but had no effect on the other stingray orthologs, and there was no increase in sensitivity for srACTH(1-24) for any of the stingray melanocortin receptors. It appears then that some stingray melanocortin receptors have retained the ability to interact with a cartilaginous MRAP1 paralog. These results are discussed with reference to radiation of MRAP-related accessory proteins in cartilaginous fishes.
Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Proteínas de Transporte/metabolismo , Receptores de Melanocortina/metabolismo , Tubarões/metabolismo , alfa-MSH/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Ligantes , Receptores de Melanocortina/genéticaRESUMO
Body coloration in flatfish is one of the most distinctive asymmetries in the animal kingdom, although the fundamental molecular mechanism of the pigmentation is unclear. In the dorso-ventral coloration (countershading) of other teleost fishes, ventral-specific expression of agouti signaling protein 1 (ASIP1), an endogenous antagonist of melanocortin 1 receptor (MC1R), has been reported to play a pivotal role. Contribution of ASIP1 is also suggested in the asymmetrical pigmentation of flatfish. In order to confirm the contribution of ASIP1 and further examine receptor function in the body coloration of Japanese flounder, expression levels of asip1, mc1r, melanocortin 5 receptor (mc5r), and melanin-concentrating hormone receptor 2 (mchr2) were measured in the normally pigmented area of the left side, the normally non-pigmented area of the right side, and the abnormally pigmented (exhibiting hypermelanosis) area of the right side. Measurement was also carried out under conditions of hypermelanosis stimulated by cortisol and during the transition from non-pigmentation to pigmentation in areas of hypermelanosis. Contrary to our expectations, no difference was detected in asip1 expression between pigmented and non-pigmented areas. There was also no difference between normal and hormonally stimulated pigmented conditions in areas of hypermelanosis or during the transition process. Instead, the expression levels of mc1r, mc5r, and mchr2 were consistently higher in pigmented areas, and were especially increased under hormonally stimulated conditions. In addition, expressions of these receptor genes increased prior to pigmentation in areas of future hypermelanosis. Our results suggest that MC1Rand MC5R, but not necessarily ASIP1, contribute to pigmentation and hypermelanosis in Japanese flounder. We propose a yet unknown molecular mechanism for asymmetrical pigmentation in flatfish that is distinct from that of countershading in other vertebrates.