Detrimental effects of melanocortin‐1 receptor (MC1R) variants on the clinical outcomes of BRAF V600 metastatic melanoma patients treated with BRAF inhibitors

Melanocortin‐1 receptor (MC1R) plays a key role in skin pigmentation, and its variants are linked with a higher melanoma risk. The influence of MC1R variants on the outcomes of patients with metastatic melanoma (MM) treated with BRAF inhibitors (BRAFi) is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF‐mutated patients with MM treated with BRAFi. We also evaluated the effect of vemurafenib in four ^V600BRAF melanoma cell lines with/without MC1R variants. We found a significant correlation between the presence of MC1R variants and worse outcomes in terms of both overall response rate (ORR; 59% versus 95%, P = 0.011 univariate, P = 0.028 multivariate analysis) and progression‐free survival (PFS) shorter than 6 months (72% versus 33%, P = 0.012 univariate, P = 0.027 multivariate analysis). No difference in overall survival (OS) was reported, probably due to subsequent treatments. Data in vitro showed a significant different phosphorylation of Erk1/2 and p38 MAPK during treatment, associated with a greater increase in vemurafenib IC50 in MC1R variant cell lines.     

Polymorphisms in the melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) genes in Korean vitiligo patients

We have examined the frequency of SNP polymorphisms within the melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) genes in 114 Korean vitiligo patients and 111 normal controls to assess the association of these loci with vitiligo risk. Using direct sequencing techniques, we found the following five MC1R coding region SNPs: Arg67Gln (G200A), Val92Met (G274A), Ile120Thr (T359C), Arg160Arg (C478A), and Gln163Arg (A488G). Of these, the most common were Val92Met at 14% in patients vs. 9% in controls (P = 0.17) and Gln163Arg at 17% in patients vs. 17% in controls (P = 0.84). Presence of the A allele of Val92Met (G274A) was higher in vitiligo patients [P = 0.12, odds ratio (OR) [95% confidence interval (CI)] = 1.68 (0.86-3.25)]. The other three variants showed a frequency <5% of both patients and controls. The ASIP 3'UTR genotype (g.8818A-G) was also assessed in the same subjects. The frequency of the G allele of 3'UTR in ASIP was 17% in vitiligo and 12% in controls [P = 0.14, OR (95% CI) = 1.49 (0.87-2.54)]. Carriage of the G allele was higher in vitiligo patients [P = 0.17, OR (95% CI) = 1.50 (0.83-2.72)], and those who also carried MC1R Val92Met were more prone to vitiligo [eight of 111 patients vs. four of 111 in controls, P = 0.14, OR (95% CI) = 2.75 (0.71-8.69)]. None of these associations, however, reached statistical significance.     

Melanocortin‐1 receptor structure and functional regulation

The melanogenic actions of the melanocortins are mediated by the melanocortin‐1 receptor (MC1R). MC1R is a member of the G‐protein‐coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or α‐melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist‐independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.     

Agonist‐Independent,High Constitutive Activity of the Human Melanocortin 1 Receptor

The melanocortins (α‐melanocyte‐stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain‐of‐function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss‐of‐function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss‐of‐function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist‐independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist‐independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over‐expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E^so‐3J allele. Stable or transient expression of wild‐type MC1R, but not of loss‐of‐function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs‐coupled receptors. Therefore, human MC1R displays a strong agonist‐independent constitutive activity.     

Keratinocytes Suppress TRP‐1 Expression and Reduce Cell Number of Co‐cultured Melanocytes – Implications For Grafting of Patients with Vitiligo

A pilot study for grafting of patients with vitiligo using cultured epithelial autografts containing melanocytes gave disappointing clinical results, with pigmentation achieved in only one out of five patients. Irrespective of the fate of melanocytes grafted back onto the patients, we experienced problems in identifying melanocytes within these well‐integrated keratinocyte sheets. This led us to explore the fate of these cells within these sheets in vitro and to seek to improve their number and function within the sheets. We report that the introduction of a fibroblast feeder layer can improve melanocyte number within melanocyte/keratinocyte co‐cultures initially, but at very high keratinocyte density, there is a marked loss of melanocytes (as detected by staining for S100). Additionally, we found that keratinocytes not only down‐regulate melanocyte number, but also pigmentary function; thus, it was possible to identify melanocytes that were S100 positive but tyrosinase‐related protein‐1 (TRP‐1) negative in confluent well‐integrated keratinocyte sheets. In summary, our data suggest that keratinocytes at high density initially suppress melanocyte pigmentation (as evidenced by a lack of TRP‐1 expression) and then cause a physical loss of melanocytes. The introduction of a fibroblast feeder layer can help maintain melanocyte number while keratinocytes are subconfluent, but fails to oppose the inhibitory influence of the keratinocytes on melanocyte TRP‐1 expression.     

Melanocortin‐1 receptor (MC1R) genotypes do not correlate with size in two cohorts of medium‐to‐giant congenital melanocytic nevi

Congenital melanocytic nevi (CMN) are cutaneous malformations whose prevalence is inversely correlated with projected adult size. CMN are caused by somatic mutations, but epidemiological studies suggest that germline genetic factors may influence CMN development. In CMN patients from the U.K., genetic variants in MC1R, such as p.V92M and loss‐of‐function variants, have been previously associated with larger CMN. We analyzed the association of MC1R variants with CMN characteristics in two distinct cohorts of medium‐to‐giant CMN patients from Spain (N = 113) and from France, Norway, Canada, and the United States (N = 53), similar at the clinical and phenotypical level except for the number of nevi per patient. We found that the p.V92M or loss‐of‐function MC1R variants either alone or in combination did not correlate with CMN size, in contrast to the U.K. CMN patients. An additional case–control analysis with 259 unaffected Spanish individuals showed a higher frequency of MC1R compound heterozygous or homozygous variant genotypes in Spanish CMN patients compared to the control population (15.9% vs. 9.3%; p = .075). Altogether, this study suggests that MC1R variants are not associated with CMN size in these non‐UK cohorts. Additional studies are required to define the potential role of MC1R as a risk factor in CMN development.     

Agouti Protein Inhibits the Production of Eumelanin and Phaeomelanin in the Presence and Absence of α-Melanocyte Stimulating Hormone

Melanocytes synthesise two types of melanin: the brown-black eumelanin and the red-yellow phaeomelanin. In mice, the relative proportions of these two melanins are regulated by α-MSH, which preferentially increases the synthesis of eumelanin and by the Agouti protein (AP), the expression of which correlates with the growth of yellow phaeomelanin-containing hair. It has been proposed that AP acts by antagonizing the action of α-MSH at the MCI receptor, although it has been suggested that it may also act independently of α-MSH. In the present study we show that AP inhibits melanogenesis in B16F1 melanoma cells in the presence and absence of α-MSH and also causes dose-related decreases in the synthesis of both eumelanin and phaeomelanin. In the presence of α-MSH AP had a greater effect on eumelanin production and this is consistent with an antagonistic action at the MCI receptor. In the absence of α-MSH however, AP produced similar reductions in the synthesis of both melanins. These changes were not seen in B16G4F cells which lack the MCI receptor, suggesting that even in the absence of α-MSH AP acts at the MCI receptor. How this action is mediated at the intracellular level is not yet clear, although it appears to be associated with a decrease in tyrosinase activity.     

MC1R,the cAMP pathway,and the response to solar UV: extending the horizon beyond pigmentation

The melanocortin 1 receptor (MC1R) is a G protein‐coupled receptor crucial for the regulation of melanocyte proliferation and function. Upon binding melanocortins, MC1R activates several signaling cascades, notably the cAMP pathway leading to synthesis of photoprotective eumelanin. Polymorphisms in the MC1R gene are a major source of normal variation of human hair color and skin pigmentation, response to ultraviolet radiation (UVR), and skin cancer susceptibility. The identification of a surprisingly high number of MC1R natural variants strongly associated with pigmentary phenotypes and increased skin cancer risk has prompted research on the functional properties of the wild‐type receptor and frequent mutant alleles. We summarize current knowledge on MC1R structural and functional properties, as well as on its intracellular trafficking and signaling. We also review the current knowledge about the function of MC1R as a skin cancer, particularly melanoma, susceptibility gene and how it modulates the response of melanocytes to UVR.     

Identification and Characterization of Single‐Nucleotide Polymorphisms in MCH‐R1 and MCH‐R2*

Objective: To identify and functionally characterize single‐nucleotide polymorphisms (SNPs) in melanin‐concentrating hormone (MCH)‐R1 and ‐R2. Research Methods and Procedures: The entire coding regions and intron/exon splice junction regions of MCH‐R1 and MCH‐R2 were sequenced from anonymous white (n = 45) and African‐American (n = 46) individuals. DNA was analyzed, and SNPs were identified using Phred, Phrap, and Consed software. DNA constructs containing MCH‐R1 and MCH‐R2 SNPs were generated and expressed in CHO cells. The effect of the SNPs in MCH‐R1 and MCH‐R2 were assessed in receptor binding assays and functional assays measuring changes in intracellular cAMP and Ca²⁺ levels. Results: We identified 12 SNPs in the MCH‐R1 gene. Two of these SNPs are in coding regions, and one produces an arginine‐for‐glycine substitution at residue 34 in the MCH‐R1 sequence. This SNP is present at a minor allele frequency of 15% in the African‐American population tested in this study. We identified eight SNPs in the MCH‐R2 gene. Four of these SNPs are in coding regions, and two produce amino acid substitutions. Lysine substitutes for arginine at residue 63 of the African‐American population, and glutamine substitutes for arginine at residue 152 in whites (minor allele frequency of 2% for both SNPs). No changes in receptor binding or functional signaling were observed with the SNP mutations in MCH‐R1 or MCH‐R2. Discussion: These data indicate that potential therapeutics designed to act at the MCH receptor are unlikely to have altered effects in subpopulations that express variant forms of MCH‐R1 or MCH‐R2.     

Sex‐specific allelic transmission bias suggests sexual conflict at MC1R

Sexual conflict arises when selection in one sex causes the displacement of the other sex from its phenotypic optimum, leading to an inevitable tension within the genome – called intralocus sexual conflict. Although the autosomal melanocortin‐1‐receptor gene (MC1R) can generate colour variation in sexually dichromatic species, most previous studies have not considered the possibility that MC1R may be subject to sexual conflict. In the barn owl (Tyto alba), the allele MC1R_WHITE is associated with whitish plumage coloration, typical of males, and the allele MC1R_RUFOUS is associated with dark rufous coloration, typical of females, although each sex can express any phenotype. Because each colour variant is adapted to specific environmental conditions, the allele MC1R_WHITE may be more strongly selected in males and the allele MC1R_RUFOUS in females. We therefore investigated whether MC1R genotypes are in excess or deficit in male and female fledglings compared with the expected Hardy–Weinberg proportions. Our results show an overall deficit of 7.5% in the proportion of heterozygotes in males and of 12.9% in females. In males, interannual variation in assortative pairing with respect to MC1R explained the year‐specific deviations from Hardy–Weinberg proportions, whereas in females, the deficit was better explained by the interannual variation in the probability of inheriting the MC1R_WHITE or MC1R_RUFOUS allele. Additionally, we observed that sons inherit the MC1R_RUFOUS allele from their fathers on average slightly less often than expected under the first Mendelian law. Transmission ratio distortion may be adaptive in this sexually dichromatic species if males and females are, respectively, selected to display white and rufous plumages.     

SREBP‐1c,Pdx‐1, and GLP‐1R involved in palmitate–EPA regulated glucose‐stimulated insulin secretion in INS‐1 cells

Impairment of glucose‐stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up‐regulate the expression level of sterol regulatory element‐binding protein (SREBP)‐1c and down‐regulate the levels of pancreatic and duodenal homeobox (Pdx)‐1 and glucagon‐like peptide (GLP)‐1 receptor (GLP‐1R) in INS‐1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock‐down or over‐express SREBP‐1c and Pdx‐1, respectively. It was found that palmitate failed to suppress the expression of Pdx‐1 and GLP‐1R in SREBP‐1c‐deficient INS‐1 cells. Moreover, down‐regulation of Pdx‐1 could cause the low expression of GLP‐1R with/without palmitate treatment. Additionally, either SREBP‐1c down‐regulation or Pdx‐1 over‐expression could partially alleviate palmitate‐induced GSIS impairment. These results suggested that sequent SREBP‐1c‐Pdx‐1‐GLP‐1R signal pathway was involved in the palmitate‐caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634–642, 2010. © 2010 Wiley‐Liss, Inc.     

Melanocortin‐1‐receptor (MC1R) variation is not associated with parasite burden in a neotropical bird,the bananaquit (Coereba flaveola)

It has been suggested that selection on melanocortin‐1‐receptor (MC1R) polymorphism, a common cause of melanic colour variation in vertebrates, results from pleiotropic effects of the gene in the immune system. Here we present the first test of whether MC1R variation is associated with differences in parasite abundance in a natural population. Bananaquits (Coereba flaveola) (Linnaeus, 1758) living on Grenada in the Caribbean exhibit a melanic plumage dimorphism as a result of a mutation in MC1R. The proportion of black individuals increases clinally towards the central, wetter parts of the island. We captured bananaquits through the cline and quantified parasite abundances. Avian malaria, feather mites, and mallophaga lice varied significantly in abundance across the cline; however, neither these infections, nor coccidia, nor arboviruses showed overall differences between the morphs. Feather mites tended to be more abundant on black individuals, in areas where the black morph was more common. This may result from differences in microhabitat use by the two morphs. These patterns do not support the idea that MC1R variation in itself results in differing susceptibility to parasites. © 2013 The Linnean Society of London     

3个猪品种黑素皮质素受体1(MC1R)基因变异研究

利用测序、PCR-RFLP和PCR-SSCP等技术对杜洛克、长白、大白猪MC1R基因进行研究发现了5个多态位点。其中,668位点G→C突变发生在5′UTR,其余4个多态位点nt894insCC(894位点CC插入),1318C→T,1554G→A和1197G→A发生在编码区。nt894insCC导致编码蛋白过早终止。1318C→T,1554G→A和1197G→A突变分别导致a164Val,Ala243Thr和Asp124Asn氨基酸的改变。所有长白、大白猪个体在894位点均存在CC插入,其余多态位点基因型分别为668GG,1197AA,1318CC,1554GG。所有杜洛克个体在894位点均不存在CC插入,其余多态位点基因型分别为668CC,1197GG,1318TT,1554AA。所有突变位点无杂合子出现。由此可以推测,668G→C,1318C→T和1554G→A可能与杜洛克的红毛色存在相关,导致1197G→A突变无意义的894位点CC插入可能与长白、大白猪白毛色存在相关。     

黑素皮质激素受体1（MC1R）基因系统发育树与犬的毛色

犬的驯养迄今约有1万多年,由于不同环境和不同目的人工选择形成了犬品种间或品种内极丰富的毛色多样性,经证实,这些犬的很多毛色类型与MC1R相关 ,MC1R在一些物种中有同源基因 本文阐述了犬MC1R多态性研究进展,并选择其它9个有代表性的哺乳动物物种与犬MC1R同源基因进行了比较,以此建立系统发育树。结果显示,10个物种的MC1R基因的分子进化关系与物种的经典分类学地位基本相符。      

The Synaptic and Neuronal Functions of the X‐Linked Intellectual Disability Protein Interleukin‐1 Receptor Accessory Protein Like 1 (IL1RAPL1)

Since the first observation that described a patient with a mutation in IL1RAPL1 gene associated with intellectual disability in 1999, the function of IL1RAPL1 has been extensively studied by a number of laboratories. In this review, we summarize all the major data describing the synaptic and neuronal functions of IL1RAPL1 and recapitulate most of the genetic deletion identified in humans and associated to intellectual disability (ID) and autism spectrum disorders (ASD). All the data clearly demonstrate that IL1RAPL1 is a synaptic adhesion molecule localized at the postsynaptic membrane. Mutations in IL1RAPL1 gene cause either the absence of the protein or the production of a dysfunctional protein. More recently it has been demonstrated that IL1RAPL1 regulated dendrite formation and mediates the activity of IL‐1β on dendrite morphology. All these data will possibly contribute to identifying therapies for patients carrying mutations in IL1RAPL1 gene.