SMART MODE:

NORMAL GENOMIC

• Millington GW, Levell NJ
• From genesis to gene sequencing: historical progress in the understanding of skin color.
• Int J Dermatol. 2007; 46: 103-5
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• Since at least biblical times, humans have pondered on why there might be variation in skin color and what might constitute the nature of that difference. In this article, two historical trails are followed, one beginning with the Ancient Greeks, the other with the Ancient Chinese. These two paths converge to provide us with some historical evidence to back recent scientific discoveries in the dynamic regulation of skin pigmentation, focusing on melanocyte-stimulating hormone and its natural antagonist agouti-signaling protein.

• Girardot M, Guibert S, Laforet MP, Gallard Y, Larroque H, Oulmouden A
• The insertion of a full-length Bos taurus LINE element is responsible for a transcriptional deregulation of the Normande Agouti gene.
• Pigment Cell Res. 2006; 19: 346-55
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• Mammalian pigmentation is controlled by the concerted action of Tyr, Tyrp1 and Dct producing eumelanin and/or pheomelanin in melanocytes. The ratio of these two pigments is determined by the agonist alpha-melanocyte stimulating hormone and the antagonist Agouti protein acting on the Mc1r. Here we show that the Agouti gene is over-expressed in Normande breed compared with Prim'Holstein breed. The Normande cattle have a characteristic coat color phenotype with a variable presence of black (eumelanin) hair over a red/brown background. We have found a previously undescribed full-length L1-BT element inserted in the 5'-genomic sequence of the Agouti gene in Normande cattle which promotes the over-expression of alternative transcripts. The variable expression of the alternative transcript directed by the long interspersed nuclear element promoter may be the origin of the brindle coat color pattern of the Normande breed. This new bovine Agouti allele isolated in Normande breed has been named Abr. Finally, as ectopic over-expression of Agouti in Ay mice is responsible for the obesity syndrome, we discuss the possible consequences of Abr for meat and milk production in cattle.

• Voisey J, Gomez-Cabrera Mdel C, Smit DJ, Leonard JH, Sturm RA, van Daal A
• A polymorphism in the agouti signalling protein (ASIP) is associated with decreased levels of mRNA.
• Pigment Cell Res. 2006; 19: 226-31
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• To date, a role for agouti signalling protein (ASIP) in human pigmentation has not been well characterized. It is known that agouti plays a pivotal role in the pigment switch from the dark eumelanin to the light pheomelanin in the mouse. However, because humans do not have an agouti banded hair pattern, its role in human pigmentation has been questioned. We previously identified a single polymorphism in the 3'-untranslated region (UTR) of ASIP that was found at a higher frequency in African-Americans compared with other population groups. To compare allele frequencies between European-Australians and indigenous Australians, the g.8818A --> G polymorphism was genotyped. Significant differences were seen in allele frequencies between these groups (P < 0.0001) with carriage of the G allele highest in Australian Aborigines. In the Caucasian sample set a strong association was observed between the G allele and dark hair colour (P = 0.004) (odds ratio 4.6; 95% CI 1.4-15.27). The functional consequences of this polymorphism are not known but it was postulated that it might result in message instability and premature degradation of the transcript. To test this hypothesis, ASIP mRNA levels were quantified in melanocytes carrying the variant and non-variant alleles. Using quantitative real-time polymerase chain reaction the mean ASIP mRNA ratio of the AA genotype to the AG genotype was 12 (P < 0.05). This study suggests that the 3'-UTR polymorphism results in decreased levels of ASIP and therefore less pheomelanin production.

• Millington GW
• Proopiomelanocortin (POMC): the cutaneous roles of its melanocortin products and receptors.
• Clin Exp Dermatol. 2006; 31: 407-12
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• The precursor protein proopiomelanocortin (POMC) produces many biologically active peptides via a series of enzymatic steps in a tissue-specific manner, yielding the melanocyte-stimulating hormones (MSHs), corticotrophin (ACTH) and beta-endorphin. The gene for alpha-MSH is encoded for by the POMC gene, but alpha-MSH cannot be produced from POMC gene transcription and translation without these specific post-translational proteolytic steps taking place. The MSHs and ACTH bind to the extracellular G-protein-coupled melanocortin receptors (MCR), of which there are five subtypes. Two (MC1R and MC5R) show widespread cutaneous expression. ACTH and alpha-MSH bind to MC1R to influence both pigmentation and the immune system. MC5R regulates the sebaceous glands. Mutations in the MC1R gene lead to fair skin and red hair in humans, which is also seen with inactivating human POMC gene mutations. MC1R mutant receptor expression can also correlate with an increased incidence of the three commonest forms of skin cancer. Other mutations can occur in the POMC system or parallel interacting pathways, such as in prohormone convertase 1 and agouti signalling protein, a human homologue of murine agouti protein. However, they do not necessarily affect skin colour or function in humans, and further studies are needed to clarify these observations.

• Drogemuller C et al.
• The mutation causing the black-and-tan pigmentation phenotype of Mangalitza pigs maps to the porcine ASIP locus but does not affect its coding sequence.
• Mamm Genome. 2006; 17: 58-66
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• The gene for agouti signaling protein (ASIP) is centrally involved in the expression of coat color traits in animals. The Mangalitza pig breed is characterized by a black-and-tan phenotype with black dorsal pigmentation and yellow or white ventral pigmentation. We investigated a Mangalitza x Pietrain cross and observed a coat color segregation pattern in the F(2) generation that can be explained by virtue of two alleles at the MC1R locus and two alleles at the ASIP locus. Complete linkage of the black-and-tan phenotype to microsatellite alleles at the ASIP locus on SSC 17q21 was observed. Corroborated by the knowledge of similar mouse coat color mutants, it seems therefore conceivable that the black-and-tan pigmentation of Mangalitza pigs is caused by an ASIP allele a(t), which is recessive to the wild-type allele A. Toward positional cloning of the a(t) mutation, a 200-kb genomic BAC/PAC contig of this chromosomal region has been constructed and subsequently sequenced. Full-length ASIP cDNAs obtained by RACE differed in their 5' untranslated regions, whereas they shared a common open reading frame. Comparative sequencing of all ASIP exons and ASIP cDNAs between Mangalitza and Pietrain pigs did not reveal any differences associated with the coat color phenotype. Relative qRT-PCR analyses showed different dorsoventral skin expression intensities of the five ASIP transcripts in black-and-tan Mangalitza. The a(t) mutation is therefore probably a regulatory ASIP mutation that alters its dorsoventral expression pattern.

• Mundy NI, Kelly J
• Investigation of the role of the agouti signaling protein gene (ASIP) in coat color evolution in primates.
• Mamm Genome. 2006; 17: 1205-13
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• We investigated variation in the gene encoding the agouti signaling protein (ASIP) in relation to coat color evolution in primates. We found little evidence that mutations in the coding region of ASIP have been involved in color changes among closely related primate species. Among many closely related species with differing coat color, the coding region of ASIP was identical. In two cases (Sulawesi macaque and black lion tamarin) where species with almost completely black coat color had derived point mutations in exon 4 of the ASIP coding sequence, the same mutations did not alter coloration in other mammals and so probably do not affect ASIP function. Evolutionary reconstructions of two key phenotypes that are typically related to ASIP function--transverse phaeomelanin bands on hairs and pale ventral coloration--showed that these usually evolved concurrently, suggesting that loci acting downstream of ASIP may be involved. Analysis of dN/dS ratios revealed a likely change in functional constraint on ASIP following loss of agouti-banded hairs + pale ventral coloration, particularly in catarrhine primates (humans, apes, and Old World monkeys). Together with previous results on a lack of association of coat color with MC1R variation, these results suggest that other loci probably have an important role in primate coat color evolution.

• Nakayama K, Ishida T
• Alu-mediated 100-kb deletion in the primate genome: the loss of the agouti signaling protein gene in the lesser apes.
• Genome Res. 2006; 16: 485-90
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• Agouti signaling protein (ASIP) is an endogenous antagonist of melanocortin receptors that controls a wide range of physiological functions. Its central role in regulation of the melanocortin system implied that ASIP has been relevant to the evolution of various physiological traits in primates. In this study, we have tried to determine DNA sequences of the ASIP gene (ASIP) of various simian species to find molecular evolutionary aspects of ASIP. Unexpectedly, we found that the whole coding region of ASIP was missing only from the gibbon genome; gibbons constitute a large group of hominoid species in Southeast Asia. Our analyses revealed that unequal homologous recombination mediated by two AluSx elements erased a approximately 100-kb region including ASIP from the gibbon genome. The data provide new evidence for the significant roles of Alu elements in the dynamic evolution of the primate genome.

• Jackson PJ et al.
• Structural and molecular evolutionary analysis of Agouti and Agouti-related proteins.
• Chem Biol. 2006; 13: 1297-305
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• Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products.

• Royo LJ, Alvarez I, Fernandez I, Arranz JJ, Gomez E, Goyache F
• The coding sequence of the ASIP gene is identical in nine wild-type coloured cattle breeds.
• J Anim Breed Genet. 2005; 122: 357-60
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• The aim of this study was to ascertain the role of the Agouti signaling peptide (ASIP) gene coding region in the Agouti locus variation within wild-type coat colour in cattle. We determined the Extension genotype in 241 individuals from six Spanish and three French brown cattle breeds representative of wild-type coat variation. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis was carried out in individuals of each Extension genotypes within the same breed in an attempt to identify variants in the three coding exons of the ASIP gene. No SSCP variants were found. Results were confirmed by sequencing the coding exons of the ASIP gene in 20 individuals. Our results suggest that the ASIP coding region does not play a central role in coat colour variation in cattle.

• Landi MT et al.
• MC1R, ASIP, and DNA repair in sporadic and familial melanoma in a Mediterranean population.
• J Natl Cancer Inst. 2005; 97: 998-1007
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• BACKGROUND: Melanoma risk factors include fair pigmentation, multiple nevi, low DNA repair capacity, and CDKN2A or CDK4 mutations. Variants of the melanocortin-1 receptor (MC1R) gene have been associated with fair pigmentation and melanoma risk, and a polymorphism of the Agouti Signaling Protein (ASIP) gene has been associated with dark pigmentation. We examined MC1R and ASIP genotypes in relation to phenotypic characteristics, sporadic and familial melanoma risk, and melanoma thickness as an indicator of disease progression in a Mediterranean population. METHODS: We studied 267 melanoma patients and 382 control subjects from a case-control study and a family study in northeastern Italy. Host factors were assessed by physical examination, questionnaire, spectrophotometer, and minimal erythema dose measurement. MC1R was sequenced, ASIP was genotyped, and DNA repair capacity was measured by the host-cell reactivation assay. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression models. Effect modification of the association between MC1R and melanoma risk by phenotypic characteristics and DNA repair capacity was also assessed. All statistical tests were two-sided. RESULTS: Carrying MC1R variant alleles was associated with a two- to fourfold increase in risk of both sporadic and familial melanoma compared with carrying wild-type MC1R, particularly in individuals carrying multiple variant alleles (OR = 3.9; 95% CI = 3.3 to 4.6). This association was stronger in individuals with fewer additional risk factors (those with dark skin or few nevi). MC1R variant allele carriers were also three to four times more likely than were non-carriers to have thick melanomas. The ASIP polymorphism was not associated with pigmentation, nevi, or melanoma risk. CONCLUSIONS: MC1R was associated with melanoma risk and progression in a Mediterranean population, particularly in the absence of other strong risk factors, such as freckling or many nevi.

• Cerda-Reverter JM, Haitina T, Schioth HB, Peter RE
• Gene structure of the goldfish agouti-signaling protein: a putative role in the dorsal-ventral pigment pattern of fish.
• Endocrinology. 2005; 146: 1597-610
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• One of the most successful chromatic adaptations in vertebrates is the dorsal-ventral pigment pattern in which the dorsal skin is darkly colored, whereas the ventrum is light. In fish, the latter pattern is achieved because a melanization inhibition factor inhibits melanoblast differentiation and supports iridophore proliferation in the ventrum. In rodents, the patterned pigmentation results from regional production of the agouti-signaling protein (ASP). This peptide controls the switch between production of eumelanin and pheomelanin by antagonizing alphaMSH effects on melanocortin receptor (MCR) 1 in the melanocytes. In addition, ASP inhibits the differentiation and proliferation of melanoblast. Thus, the mammalian ASP may be homologous to the poikilotherm melanization inhibition factor. By screening of a genomic library, we deduced the amino acid sequence of goldfish ASP. The ASP gene is a four-exon gene spanning 3097 bp that encodes a 125-amino acid precursor. Northern blot analysis identified two different ASP mRNAs in ventral skin of red- and black-pigmented and albino fish, but no expression levels were observed in the dorsal skin of the same fish. The dorsal-ventral expression polarity was also detected in both black dorsally pigmented fish and albino fish. Pharmacological studies demonstrate that goldfish ASP acts as a melanocortin antagonist at Fugu MC1R and goldfish MC4R. In addition, goldfish ASP inhibited Nle4, D-Phe7-MSH-stimulated pigment dispersion in medaka melanophores. Our studies support agouti signaling protein as the melanization inhibition factor, a key factor in the development of the dorsal-ventral pigment pattern in fish.

• Sharov AA et al.
• Bone morphogenetic protein (BMP) signaling controls hair pigmentation by means of cross-talk with the melanocortin receptor-1 pathway.
• Proc Natl Acad Sci U S A. 2005; 102: 93-8
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• Hair pigmentation is controlled by tightly coordinated programs of melanin synthesis and involves signaling through the melanocortin type 1 receptor (MC-1R) that regulates the switch between pheomelanogenesis and eumelanogenesis. However, the involvement of other signaling systems, including the bone morphogenetic protein (BMP) pathway, in the control of hair pigmentation remains to be elucidated. To assess the effects of BMP signaling on hair pigmentation, transgenic mice overexpressing the BMP antagonist noggin (promoter: keratin 5) were generated. Whereas wild-type C3H/HeJ mice have a subapical yellow band on otherwise black dorsal hairs, K5-Noggin mice are characterized by the absence of a yellow band and near-black pigment in dorsal coat. Noggin overexpression is accompanied by strongly reduced levels of Agouti signal protein and enhanced expression of microphthalmia transcription factor in the midphase of the hair-growth cycle. Wild-type color in K5-Noggin mice is restored by administration of a synthetic MC-1R antagonist resulting in the reappearance of a subapical yellow band. BMP-4 stimulates the expression of Agouti transcripts and protein in primary epidermal keratinocytes, and BMP signaling positively regulates dermal papilla-specific enhancer of the Agouti gene in primary dermal fibroblasts. Taken together, these data suggests that BMP signaling controls the expression of Agouti protein in the hair follicle and provide evidence for interaction between BMP and MC-1R signaling pathways to modulate the balance between pheomelanogenesis and eumelanogenesis during hair growth.

• Beaumont KA, Newton RA, Smit DJ, Leonard JH, Stow JL, Sturm RA
• Altered cell surface expression of human MC1R variant receptor alleles associated with red hair and skin cancer risk.
• Hum Mol Genet. 2005; 14: 2145-54
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• The human melanocortin-1 receptor gene (MC1R) encodes a G-protein coupled receptor that is primarily expressed on melanocytes, where it plays a key role in pigmentation regulation. Variant alleles are associated with red hair colour and fair skin, known as the RHC phenotype, as well as skin cancer risk. The R151C, R160W and D294H alleles, designated 'R', are strongly associated with the RHC phenotype and have been proposed to result in loss of function receptors due to impaired G-protein coupling. We recently provided evidence that the R151C and R160W variants can efficiently couple to G-proteins in response to alpha-melanocyte stimulating hormone. The possibility that altered cellular localization of the R151C and R160W variant receptors could underlie their association with RHC was therefore considered. Using immunofluorescence and ligand binding studies, we found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localization of the wild-type and D294H alleles but markedly reduced cell surface expression of the R151C and R160W receptors. In additional exogenous expression studies, the R variant D84E and the rare I155T variant, also demonstrated a significant reduction in plasma membrane receptor numbers. The V60L, V92M and R163Q weakly associated RHC alleles, designated 'r', were expressed with normal or intermediate cell surface receptor levels. These results indicate that reduced receptor coupling activity may not be the only contributing factor to the genetic association between the MC1R variants and the RHC phenotype, with MC1R polymorphisms now linked to a change in receptor localization.

• Rouzaud F, Kadekaro AL, Abdel-Malek ZA, Hearing VJ
• MC1R and the response of melanocytes to ultraviolet radiation.
• Mutat Res. 2005; 571: 133-52
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• The constitutive color of our skin plays a dramatic role in our photoprotection from solar ultraviolet radiation (UVR) that reaches the Earth and in minimizing DNA damage that gives rise to skin cancer. More than 120 genes have been identified and shown to regulate pigmentation, one of the key genes being melanocortin 1 receptor (MC1R) that encodes the melanocortin 1 receptor (MC1R), a seven-transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Modulation of MC1R function regulates melanin synthesis by melanocytes qualitatively and quantitatively. The MC1R is regulated by the physiological agonists alpha-melanocyte-stimulating hormone (alphaMSH) and adrenocorticotropic hormone (ACTH), and antagonist agouti signaling protein (ASP). Activation of the MC1R by binding of an agonist stimulates the synthesis of eumelanin primarily via activation of adenylate cyclase. The significance of cutaneous pigmentation lies in the photoprotective effect of melanin, particularly eumelanin, against sun-induced carcinogenesis. Epidermal melanocytes and keratinocytes respond to UVR by increasing their expression of alphaMSH and ACTH, which up-regulate the expression of MC1R, and consequently enhance the response of melanocytes to melanocortins. Constitutive skin pigmentation dramatically affects the incidence of skin cancer. The pigmentary phenotype characterized by red hair, fair complexion, inability to tan and tendency to freckle is an independent risk factor for all skin cancers, including melanoma. The MC1R gene is highly polymorphic in human populations, and allelic variation at this locus accounts, to a large extent, for the variation in pigmentary phenotypes and skin phototypes (SPT) in humans. Several allelic variants of the MC1R gene are associated with the red hair and fair skin (RHC) phenotype, and carrying one of these variants is thought to diminish the ability of the epidermis to respond to DNA damage elicited by UVR. The MC1R gene is considered a melanoma susceptibility gene, and its significance in determining the risk for skin cancer is of tremendous interest.

• Bonilla C et al.
• The 8818G allele of the agouti signaling protein (ASIP) gene is ancestral and is associated with darker skin color in African Americans.
• Hum Genet. 2005; 116: 402-6
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• Skin color, a predictor of social interactions and risk factor for several types of cancer, is due to two contrasting forms of melanin, the darker eumelanin and lighter phaeomelanin. The lighter pigment phaeomelanin is the product of the antagonistic function of the agouti signaling protein (ASIP) on the alpha-melanocyte stimulating hormone receptor (MC1R). Studies have shown that a single-nucleotide polymorphism (SNP) in the 3'UTR of the ASIP gene is associated with dark hair and eyes; however, little is known about its role in inter-individual variation in skin color. Here we examine the relationship between the ASIP g.8818A>G SNP and skin color (M index) as assessed by reflectometry in 234 African Americans. Analyses of variance (ANOVA) were performed to evaluate the effects of ASIP genotypes, age, individual ancestry, and sex on skin color variation. Significant effects on M index variation were observed for ASIP genotypes (F(2,236)=4.37, P=0.01), ancestry (F(1,243)=37.2, P<0.001), and sex (F(1,244)=4.08, P=0.05). Subsequent analyses revealed a strong effect on M index from ASIP genotypes in African American females (P<0.001). Our study suggests that the ASIP G>A polymorphism exhibits a dominant effect leading to lighter skin color and that variation in the ASIP gene may have been one of several factors contributing to reductions in pigmentation in some populations. Further study is needed to reveal how interactions between ASIP and several other genes, such as MC1R and P, predict human pigmentation.

• Chai BX et al.
• Receptor-antagonist interactions in the complexes of agouti and agouti-related protein with human melanocortin 1 and 4 receptors.
• Biochemistry. 2005; 44: 3418-31
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• The molecular interactions between human melanocortin receptor-1 and -4 (hMC1R and hMC4R) and their endogenous antagonists, agouti signaling protein (ASIP) and agouti-related protein (AGRP), were assessed by studying the effects of site-directed mutations on the binding affinity of (125)I-ASIP[90-132(L89Y)] and (125)I-AGRP(86-132). Mutations of homologous residues from transmembrane helices (TMHs) 3 and 6 and extracellular loop (EL) 3 (D121A, T124A, F257A, and F277M in hMC1R and D126A, I129A F261A, and M281F in hMC4R) impaired binding of both antagonists to hMC4R and binding of the ASIP fragment to hMC1R. However, the mutations in TMH2 (E94A in hMC1R and E100A in hMC4R), TMH7 (F280A in hMC1R and F284A in hMC4R), and EL2 (Y183S, H184S, and D184H in hMC1R) only significantly affected binding of the ASIP fragment. The dependence of agonist binding on the dithiothreitol concentration followed a monophasic curve for wild-type hMC4R and its C40A, C271A, and C279A mutants and a biphasic curve for hMC1R, suggesting the presence of at least one structurally and functionally essential disulfide bond in both wild-type receptors and the hMC4R mutants. Models of complexes of both receptors with the ASIP fragment and hMC4R with the AGRP fragment were calculated using constraints from the experimental structures of rhodopsin and AGRP fragments, a set of deduced hydrogen bonds, supplemented by two proposed disulfide bridges and receptor-ligand contacts, derived from our mutagenesis data. In the models of the ASIP fragment complexed with both receptors, the core ligand tripeptide, Arg-Phe-Phe, positioned between TMHs 3 and 6, is shifted toward TMHs 2 and 7 relative to its position in the AGRP-hMC4R model, while the N-terminal loop and two central disulfides of the antagonists interact with EL2 of the receptors.

• Berryere TG, Kerns JA, Barsh GS, Schmutz SM
• Association of an Agouti allele with fawn or sable coat color in domestic dogs.
• Mamm Genome. 2005; 16: 262-72
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• The type of pigment synthesized in mammalian hair, yellow-red pheomelanin or black-brown eumelanin, depends on the interaction between Agouti protein and the Melanocortin 1 receptor. Although the genetics of pigmentation is broadly conserved across most mammalian species, pigment type-switching in domestic dogs is unusual because a yellow-tan coat with variable amounts of dark hair is thought to be caused by an allele of the Agouti locus referred to as fawn or sable (a(y)). In a large survey covering thirty seven breeds, we identified an Agouti allele with two missense alterations, A82S and R83H, which was present (heterozygous or homozygous) in 41 dogs (22 breeds) with a fawn or sable coat, but was absent from 16 dogs (8 breeds) with a black-and-tan or tricolor phenotype. In an additional 33 dogs (14 breeds) with a eumelanic coat, 8 (German Shepherd Dogs, Groenendaels, Schipperkes, or Shetland Sheepdogs) were homozygous for a previously reported mutation, non-agouti R96C; the remainder are likely to have carried dominant black, which is independent of and epistatic to Agouti. This work resolves some of the complexity in dog coat color genetics and provides diagnostic opportunities and practical guidelines for breeders.

• Sanchez-Mas J, Guillo LA, Zanna P, Jimenez-Cervantes C, Garcia-Borron JC
• Role of G protein-coupled receptor kinases in the homologous desensitization of the human and mouse melanocortin 1 receptors.
• Mol Endocrinol. 2005; 19: 1035-48
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• The melanocortin 1 receptor, a G protein-coupled receptor positively coupled to adenylyl cyclase, is a key regulator of epidermal melanocyte proliferation and differentiation and a determinant of human skin phototype and skin cancer risk. Despite its potential importance for regulation of pigmentation, no information is available on homologous desensitization of this receptor. We found that the human melanocortin 1 receptor (MC1R) and its mouse ortholog (Mc1r) undergo homologous desensitization in melanoma cells. Desensitization is not dependent on protein kinase A, protein kinase C, calcium mobilization, or MAPKs, but is agonist dose-dependent. Both melanoma cells and normal melanocytes express two members of the G protein-coupled receptor kinase (GRK) family, GRK2 and GRK6. Cotransfection of the receptor and GRK2 or GRK6 genes in heterologous cells demonstrated that GRK2 and GRK6 impair agonist-dependent signaling by MC1R or Mc1r. However, GRK6, but not GRK2, was able to inhibit MC1R agonist-independent constitutive signaling. Expression of a dominant negative GRK2 mutant in melanoma cells increased their cAMP response to agonists. Agonist-stimulated cAMP production decreased in melanoma cells enriched with GRK6 after stable transfection. Therefore, GRK2 and GRK6 seem to be key regulators of melanocortin 1 receptor signaling and may be important determinants of skin pigmentation.

• Sanchez-Mas J, Hahmann C, Gerritsen I, Garcia-Borron JC, Jimenez-Cervantes C
• Agonist-independent, high constitutive activity of the human melanocortin 1 receptor.
• Pigment Cell Res. 2004; 17: 386-95
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• The melanocortins (alpha-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.

• Candille SI et al.
• Dorsoventral patterning of the mouse coat by Tbx15.
• PLoS Biol. 2004; 2: 3-3
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• Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (de(H)), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (a(t)) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the de(H) mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in de(H) that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in de(H)/de(H) animals. Construction of a targeted allele of Tbx15 confirmed that the de(H) phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.

• Kuklin AI et al.
• Liver-specific expression of the agouti gene in transgenic mice promotes liver carcinogenesis in the absence of obesity and diabetes.
• Mol Cancer. 2004; 3: 17-17
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• BACKGROUND: The agouti protein is a paracrine factor that is normally present in the skin of many species of mammals. Agouti regulates the switch between black and yellow hair pigmentation by signalling through the melanocortin 1 receptor (Mc1r) on melanocytes. Lethal yellow (Ay) and viable yellow (Avy) are dominant regulatory mutations in the mouse agouti gene that cause the wild-type protein to be produced at abnormally high levels throughout the body. Mice harboring these mutations exhibit a pleiotropic syndrome characterized by yellow coat color, obesity, hyperglycemia, hyperinsulinemia, and increased susceptibility to hyperplasia and carcinogenesis in numerous tissues, including the liver. The goal of this research was to determine if ectopic expression of the agouti gene in the liver alone is sufficient to recapitulate any aspect of this syndrome. For this purpose, we generated lines of transgenic mice expressing high levels of agouti in the liver under the regulatory control of the albumin promoter. Expression levels of the agouti transgene in the liver were quantified by Northern blot analysis. Functional agouti protein in the liver of transgenic mice was assayed by its ability to inhibit binding of the alpha-melanocyte stimulating hormone (alphaMSH) to the Mc1r. Body weight, plasma insulin and blood glucose levels were analyzed in control and transgenic mice. Control and transgenic male mice were given a single intraperitoneal injection (10 mg/kg) of the hepatocellular carcinogen, diethylnitrosamine (DEN), at 15 days of age. Mice were euthanized at 36 or 40 weeks after DEN injection and the number of tumors per liver and total liver weights were recorded. RESULTS: The albumin-agouti transgene was expressed at high levels in the livers of mice and produced a functional agouti protein. Albumin-agouti transgenic mice had normal body weights and normal levels of blood glucose and plasma insulin, but responded to chemical initiation of the liver with an increased number of liver tumors compared to non-transgenic control mice. CONCLUSIONS: The data demonstrate that liver-specific expression of the agouti gene is not sufficient to induce obesity or diabetes, but, in the absence of these factors, agouti continues to promote hepatocellular carcinogenesis.

• Kerns JA et al.
• Characterization of the dog Agouti gene and a nonagoutimutation in German Shepherd Dogs.
• Mamm Genome. 2004; 15: 798-808
• Display abstract

• The interaction between two genes, Agouti and Melanocortin-1 receptor ( Mc1r), produces diverse pigment patterns in mammals by regulating the type, amount, and distribution pattern of the two pigment types found in mammalian hair: eumelanin (brown/black) and pheomelanin (yellow/red). In domestic dogs ( Canis familiaris), there is a tremendous variation in coat color patterns between and within breeds; however, previous studies suggest that the molecular genetics of pigment-type switching in dogs may differ from that of other mammals. Here we report the identification and characterization of the Agouti gene from domestic dogs, predicted to encode a 131-amino-acid secreted protein 98% identical to the fox homolog, and which maps to chromosome CFA24 in a region of conserved linkage. Comparative analysis of the Doberman Pinscher Agouti cDNA, the fox cDNA, and 180 kb of Doberman Pinscher genomic DNA suggests that, as with laboratory mice, different pigment-type-switching patterns in the canine family are controlled by alternative usage of different promoters and untranslated first exons. A small survey of Labrador Retrievers, Greyhounds, Australian Shepherds, and German Shepherd Dogs did not uncover any polymorphisms, but we identified a single nucleotide variant in black German Shepherd Dogs predicted to cause an Arg-to-Cys substitution at codon 96, which is likely to account for recessive inheritance of a uniform black coat.

• Yang CH et al.
• Seeing the gene therapy: application of gene gun technique to transfect and decolour pigmented rat skin with human agouti signalling protein cDNA.
• Gene Ther. 2004; 11: 1033-9
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• We developed a gene gun method for the transfer of human agouti signalling protein (ASP) cDNA to alter rat skin colour in vivo. Human ASP cDNA was cloned into a modified cytomegalovirus plasmid and delivered to the skin of Long-Evans rats by gene gun bombardment. Skin pigmentation, body weight and blood sugar of ASP cDNA-transfected rats were recorded against the control group, which were injected with plasmids encoding for green fluorescent protein. The treated skin showed lighter skin colour after 3 days of ASP gene transfection. This depigmentation effect was most prominent on day 14 and the skin gradually returned to its original pigmentation by day 28. Successful transfection of ASP gene in skin and hair follicles, as well as downregulation of melanocortin-1 receptor (MC1R) and tyrosinase expression upon treatment, was confirmed using immunohistochemistry and Western blot analysis. Body weight and blood sugar in the treated rats did not show statistically significant differences as compared to control groups. These observations demonstrate that gene transfer using the gene gun method can induce high cutaneous ASP production and facilitate a switch from dark to fair colour without systemic pleiotropic effects. Such a colour switch may be that ASP is acting in a paracrine fashion. In addition, this study verifies that ASP exerts its functions by acting as an independent ligand that downregulates the melanocyte MC1R and tyrosinase protein in an in vivo system. Our result offers new, interesting insights about the effect of ASP on pigmentation, providing a novel approach to study the molecular mechanisms underlying skin melanogenesis.

• John PR, Makova K, Li WH, Jenkins T, Ramsay M
• DNA polymorphism and selection at the melanocortin-1 receptor gene in normally pigmented southern African individuals.
• Ann N Y Acad Sci. 2003; 994: 299-306
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• Skin pigmentation is a polygenic multifactorial trait determined by the cumulative effects of multiple genetic variants and environmental factors. Melanocortin-1 receptor (MC1R) is one of the genes involved in pigmentation, and has been implicated in the red hair and pale skin phenotype in human Caucasoid individuals. The present study was undertaken to identify variation at the MC1R locus in normally pigmented individuals in two African populations, sub-Saharan Negroids (22 unrelated individuals) and the San (17 unrelated individuals). The study showed considerable MC1R gene sequence variation with the detection of eight synonymous and three nonsynonymous mutations. This is the first report of nonsynonymous mutations in African individuals in the MC1R gene: L99I was found in a single San individual, S47I was detected in a single Negroid individual, and F196L was detected in five Negroid individuals (5/44; 0.11). The functional significance of these mutations is not known. Three of the eight synonymous mutations found, L106L (CTG --> CTA), F300F (TTC --> TTT), and T314T (ACA --> ACG) (also known as A942G), have been reported previously. T314T was the only variant that showed a significant difference between the Negroid and San populations (0.477 and 0.059, respectively; P = 1.6 x 10(-5)). Its low frequency in the San may be the result of random genetic drift in a population of small size, or selection. Several tests of neutrality of the MC1R coding region in these and other African populations were significant, suggesting that purifying selection (functional constraint) had occurred at this gene locus in Africans. This demonstrates that although some nonsynonymous MC1R mutations are tolerated in individuals with dark skin, this gene has likely played a significant role in the maintenance of dark pigmentation in Africans and normal pigment variation in non-African populations.

• Sturm RA et al.
• Genetic association and cellular function of MC1R variant alleles in human pigmentation.
• Ann N Y Acad Sci. 2003; 994: 348-58
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• We have examined MC1R variant allele frequencies in the general population of South East Queensland and in a collection of adolescent dizygotic and monozygotic twins and family members to define statistical associations with hair and skin color, freckling, and mole count. Results of these studies are consistent with a linear recessive allelic model with multiplicative penetrance in the inheritance of red hair. Four alleles, D84E, R151C, R160W, and D294H, are strongly associated with red hair and fair skin with multinomial regression analysis showing odds ratios of 63, 118, 50, and 94, respectively. An additional three low-penetrance alleles V60L, V92M, and R163Q have odds ratios 6, 5, and 2 relative to the wild-type allele. To address the cellular effects of MC1R variant alleles in signal transduction, we expressed these receptors in permanently transfected HEK293 cells. Measurement of receptor activity via induction of a cAMP-responsive luciferase reporter gene found that the R151C and R160W receptors were active in the presence of NDP-MSH ligand, but at much reduced levels compared with that seen with the wild-type receptor. The ability to stimulate phosphorylation of the cAMP response element binding protein (CREB) transcription factor was also apparent in all stimulated MC1R variant allele-expressing HEK293 cell extracts as assessed by immunoblotting. In contrast, human melanoma cell lines showed wide variation in the their ability to undergo cAMP-mediated CREB phosphorylation. Culture of human melanocytes of known MC1R genotype may provide the best experimental approach to examine the functional consequences for each MC1R variant allele. With this objective, we have established more than 300 melanocyte cell strains of defined MC1R genotype.

• He L, Eldridge AG, Jackson PK, Gunn TM, Barsh GS
• Accessory proteins for melanocortin signaling: attractin and mahogunin.
• Ann N Y Acad Sci. 2003; 994: 288-98
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• Switching from eumelanin to pheomelanin synthesis during hair growth is accomplished by transient synthesis of Agouti protein, an inverse agonist for the melanocortin-1 receptor (Mc1r). The coat color mutations mahogany and mahoganoid prevent hair follicle melanocytes from responding to Agouti protein. The gene mutated in mahogany, which is also known as Attractin (Atrn), encodes a type I transmembrane protein that functions as an accessory receptor for Agouti protein. We have recently determined that the gene mutated in mahoganoid, which is also known as Mahogunin (Mgrn1), encodes an E3 ubiquitin ligase. Like Attractin, Mahogunin is conserved in invertebrate genomes, and its absence causes a pleiotropic phenotype that includes spongiform neurodegeneration.

• Voisey J, Kelly G, Van Daal A
• Agouti signal protein regulation in human melanoma cells.
• Pigment Cell Res. 2003; 16: 65-71
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• Production of the pigment eumelanin is controlled by alpha-melanocyte stimulating hormone (alpha-MSH) stimulation of melanocortin 1 receptor (Mc1r), whereas production of pheomelanin results from agouti antagonism of alpha-MSH signalling through Mc1r. The role of agouti in mouse pigmentation has been extensively investigated but a role for agouti signalling protein (ASIP) in human pigmentation has not been determined. To determine whether ASIP regulates known melanogenic genes in humans, ASIP was over-expressed in a human melanoma cell line. Levels of mRNA and protein were measured in genes known to be up or down-regulated by agouti in the mouse, namely microphthalmia (Mitf), tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), dopachrome tautomerase (Dct), Mc1r, silver, initiation transcription factor 2 (Itf2) and mini chromosome maintenance protein 6 (Mcm6). These melanogenic genes were not found to be significantly up or down-regulated by ASIP at the transcriptional level in human melanoma cells. However, ASIP down-regulation of tyrp1 was observed at the translational level. To identify novel genes that may be regulated by ASIP in melanoma cells, microarrays were used to determine differences in gene expression between the control and ASIP transfected melanoma cells. The expression level of human RNAs were determined by microarray analysis using a 19,200 cDNA and a 19,200 oligonucleotide array representing 13,000 and 18,864 individual genes, respectively. Genes observed to be modulated by ASIP were confirmed by quantitative real-time polymerase chain reaction. Results identify five genes, namely PPARbeta, eIF-4B, RRM2, MINOR and EVI2B that are down-regulated by ASIP, indicating a likely role for ASIP in human melanogenesis.

• Kadekaro AL, Kanto H, Kavanagh R, Abdel-Malek ZA
• Significance of the melanocortin 1 receptor in regulating human melanocyte pigmentation, proliferation, and survival.
• Ann N Y Acad Sci. 2003; 994: 359-65
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• The characterization of the melanocortin 1 receptor (MC1R) expressed on human melanocytes and the findings that certain mutations in the POMC gene or the MC1R gene result in red hair phenotype underscore the significance of melanocortins and MC1R in regulating human pigmentation. We demonstrated that human melanocytes respond to alpha-melanocortin (alpha-MSH) or ACTH with increased proliferation and melanogenesis, and to agouti signaling protein by abrogation of these effects. alpha-MSH and ACTH were equipotent and more potent than beta-MSH, and gamma-MSH was the least potent in activating the MC1R and stimulating melanogenesis and proliferation of human melanocytes. We characterized the MC1R genotype in a panel of human melanocyte cultures and identified three cultures that were homozygous for Arg160Trp, heterozygous for Arg151Cys and Asp294His, and heterozygous for Arg160Trp and Asp294His substitutions, respectively. Those cultures failed to respond to alpha-MSH with increase in cAMP levels, tyrosinase activity, or proliferation and had an exaggerated response to the cytotoxic effect of ultraviolet (UV) radiation. These loss-of-function mutations have been associated with red hair phenotype and increased risk for skin cancer. Melanocytes homozygous for Val29Met substitution in MC1R responded normally to alpha-MSH and UVB, suggesting that this variant is a polymorphism. We observed that alpha-MSH promotes human melanocyte survival by inhibiting the UV-induced apoptosis independently of melanin synthesis. This effect was absent in human melanocytes with loss of function MC1R mutations. We predict that the survival effect of alpha-MSH is caused by reduction of UV-induced DNA damage and contributes to the prevention of melanoma.

• Mas JS, Gerritsen I, Hahmann C, Jimenez-Cervantes C, Garcia-Borron JC
• Rate limiting factors in melanocortin 1 receptor signalling through the cAMP pathway.
• Pigment Cell Res. 2003; 16: 540-7
• Display abstract

• The melanotropic actions of alpha-melanocyte-stimulating hormone (alpha-MSH) and other melanocortins are mediated by activation of the melanocortin 1 receptor (MC1R). This G protein-coupled receptor is positively coupled to Gs and triggers the cyclic adenosine mono-phosphate (cAMP) pathway. Mutations of the MC1R gene are associated with skin type and pigmentation phenotypes, and with increased risk of skin cancers. Genetic studies have demonstrated an heterozygote carrier effect for these associations, suggesting the importance of variant allele dosage. This could be accounted for, at least partially, if the number of MC1R molecules, rather than the Gs protein or the effector enzyme, adenylyl cyclase, is limiting for the activation of the signalling pathway. However, the nature of the limiting factor(s) in MC1R signalling has not been investigated. We addressed this question by comparing the cAMP output of clones of human melanoma cell lines enriched in MC1R by stable transfection. We also analysed heterologous cell systems widely used for functional studies of MC1R. We show that cAMP production in clones of Chinese hamster ovary cells stably expressing the MC1R is a linear function of receptor number up to high, supraphysiological levels of approximately 50,000 alpha-MSH binding sites per cell. Enrichment of human melanoma cell lines with MC1R also results in increased cAMP levels, with a small leftward shift of the agonist dose-response curves. Therefore, at physiological expression levels second-messenger generation is dependent on receptor density. Within melanoma cells and also likely in normal melanocytes, MC1R appears the limiting factor controlling the output of the cAMP signalling pathway.

• Wolff GL
• Regulation of yellow pigment formation in mice: a historical perspective.
• Pigment Cell Res. 2003; 16: 2-15
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• Pigment synthesis by hair follicle melanocytes is modulated by a large number of environmental and genetic factors, many of which are discussed in this review. Eumelanic (non-yellow) pigment is produced by hair follicle melanocytes following the binding of alpha-melanocyte stimulating hormone to melanocortin receptor 1. Binding of this hormone to the melanocyte membrane is blocked by agouti signaling protein (ASP) which is encoded by the agouti locus and results in the synthesis of yellow pigment, instead of non-yellow (black/brown) pigment. The cyclical release of ASP by hair follicle cells results in a black/brown hair with a subapical yellow band. This is the wild-type coat color pattern of many mammals and is called agouti. Several dominant mutations at the agouti locus in mice, induced by retrotransposon-like intracisternal A particles, result in ectopic over-expression of ASP and animals with much higher proportions of all-yellow hairs. This abnormal presence of ASP in essentially all body cells results in the 'yellow agouti obese mouse syndrome.' The obesity has been associated with binding of ASP to melanocortin receptor 4 inactivating the latter. The syndrome also includes hyperinsulinemia, increased somatic growth, and increased susceptibility to hyperplasia and carcinogenesis. The physiologic and molecular bases for these syndrome components have not yet been elucidated. This historically orientated review is subdivided, where applicable, into pre- and post-1992 subsections to emphasize the impact of the cloning of the agouti and extension loci and their protein products on the identification of the molecular and physiological pathways modulating the manifold aspects of pheomelanogenesis.

• Lu XM, Shi P, Zhang YP
• [Genotype, melanocortin 1 receptor and ultraviolet radiation]
• Yi Chuan. 2002; 24: 563-70
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• Recent work on the melanocortin 1 receptor (MC1R) suggests that MC1R plays a central role in regulation of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) synthesis within the mammalian melanocyte.In the mouse,genetic studies show that the MC1R appears to be a unique, bifunctionally controlled receptor, activated by alpha-MSH and antagonized by agouti,both of which contribute to the variability seen in mammalian coat color. Variants of this receptor are associated with different animal's coat,human skin and hair colors. In addition, cutaneous pigmentation is a major determinant of the cutaneous response to ultraviolet radiation,and consequently of the risk of developing skin cancer. MC1R variants are a risk factor for melanoma susceptibility.

• Sanchez Mas J et al.
• Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor function.
• Eur J Biochem. 2002; 269: 6133-41
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• The alpha-melanocyte-stimulating hormone (alphaMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of alphaMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent alphaMSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships.

• Robinson SJ, Healy E
• Human melanocortin 1 receptor (MC1R) gene variants alter melanoma cell growth and adhesion to extracellular matrix.
• Oncogene. 2002; 21: 8037-46
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• Pigmentation is a significant determinant of individual susceptibility to cutaneous melanoma, with fair skinned subjects at highest risk of developing this neoplasm. Melanocortin 1 receptor (MC1R) gene variants alter pigment synthesis in vivo, and are causally associated with red hair and fair skin in humans. MC1R variants are more frequent in subjects with melanoma, and increase the risk of developing this tumour in sporadic and familial cases. MC1R variants may predispose to melanoma as a result of alterations in skin pigmentation (which affords less protection against incident ultraviolet radiation). However, melanoma cells synthesize and release alpha-melanocyte stimulating hormone (alphaMSH, the ligand for MC1R), therefore MC1R variants could alter the autocrine effects of alphaMSH on melanoma cell behaviour, thereby affecting early melanoma development and progression via non-pigmentary mechanisms. B16G4F melanoma cells, which are functionally null at Mc1r, were stably transfected with wild type and variant (Arg151Cys, Arg160Trp, and Asp294His) human MC1R. At similar MC1 receptor numbers per cell, alphaMSH increased intracellular cAMP in wild type MC1R transfected melanoma cells, but the cAMP response was compromised in the variant MC1R transfected clones. In growth inhibition experiments, alphaMSH significantly reduced growth of wild type MC1R transfected cells, but had no effect on cells transfected with variant MC1R. In addition, binding to fibronectin was significantly reduced by alphaMSH in the wild type transfectants whereas this was not observed in the variant transfected clones; binding to laminin was not affected by alphaMSH in this cell line. These results provide evidence for differences in melanoma cell behaviour secondary to MC1R variants, and suggest an alternative non-pigmentary mechanism whereby MC1R variants could modify melanoma susceptibility or progression.

• Scott MC et al.
• Human melanocortin 1 receptor variants, receptor function and melanocyte response to UV radiation.
• J Cell Sci. 2002; 115: 2349-55
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• Cutaneous pigmentation is determined by the amounts of eumelanin and pheomelanin synthesized by epidermal melanocytes and is known to protect against sun-induced DNA damage. The synthesis of eumelanin is stimulated by the binding of alpha-melanotropin (alpha-melanocyte-stimulating hormone) to the functional melanocortin 1 receptor (MC1R) expressed on melanocytes. The human MC1R gene is highly polymorphic and certain allelic variants of the gene are associated with red hair phenotype, melanoma and non-melanoma skin cancer. The importance of the MC1R gene in determining skin cancer risk led us to examine the impact of specific polymorphisms in this gene on the responses of human melanocytes to alpha-melanotropin and UV radiation. We compared the ability of human melanocyte cultures, each derived from a single donor, to respond to alpha-melanotropin with dose-dependent stimulation of cAMP formation, tyrosinase activity and proliferation. In each of those cultures the MC1R gene was sequenced, and the eumelanin and pheomelanin contents were determined. Human melanocytes homozygous for Arg160Trp, heterozygous for Arg160Trp and Asp294His, or for Arg151Cys and Asp294His substitutions, but not melanocytes homozygous for Val92Met substitution, in the MC1R demonstrated a significantly reduced response to alpha-melanotropin. Additionally, melanocytes with a non-functional MC1R demonstrated a pronounced increase in their sensitivity to the cytotoxic effect of UV radiation compared with melanocytes expressing functional MC1R. We conclude that loss-of-function mutations in the MC1R gene sensitize human melanocytes to the DNA damaging effects of UV radiation, which may increase skin cancer risk.

• Yang YK, Dickinson C, Lai YM, Li JY, Gantz I
• Functional properties of an agouti signaling protein variant and characteristics of its cognate radioligand.
• Am J Physiol Regul Integr Comp Physiol. 2001; 281: 187786-187786
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• Agouti signaling protein (ASIP), the human (h) homolog of agouti, is an endogenous melanocortin peptide antagonist. To date, characterization of this protein has been performed with recombinant protein only and without the availability of an ASIP/agouti radioligand. In this report we describe the functional characteristics of a chemically synthesized truncated ASIP variant, ASIP-[90-132 (L89Y)], and the binding characteristics of its cognate radioligand, (125)I-ASIP-[90-132 (L89Y)]. Similar to full-length recombinant ASIP/agouti, ASIP-[90-132 (L89Y)] was a potent inhibitor of alpha-melanocyte-stimulating hormone cAMP generation at the cloned human melanocortin receptor (hMCR) subtypes hMC1R and hMC4R. It also displayed a lesser degree of inhibition at the hMC3R and hMC5R. However, ASIP-[90-132 (L89Y)] was found to be less potent than full-length recombinant ASIP and, surprisingly, only exhibited weak inhibitory activity at the hMC2R. In competition binding assays with the radioligand (125)I-ASIP-[90-132 (L89Y)], ASIP-[90-132 (L89Y)] displayed a hierarchy of binding affinity that roughly paralleled its rank order of inhibitory potency at the various MCR subtypes, i.e., hMC1R approximately hMC4R > hMC3R approximately hMC5R > hMC2R. Structure-activity studies revealed that ASIP-[90-132 (L89Y)] possessed greater pharmacological potency than either the further truncated ASIP variants ASIP-(116-132) or cyclo(CRFFRSAC). Interestingly, the latter molecules were both weak agonists at the hMC1R. These studies further support the concept that ASIP/agouti inhibits melanocortin action by directly binding to target MCRs and provide additional insight into the structural requirements for maximal inhibitory potency.

• Healy E, Jordan SA, Budd PS, Suffolk R, Rees JL, Jackson IJ
• Functional variation of MC1R alleles from red-haired individuals.
• Hum Mol Genet. 2001; 10: 2397-402
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• Red hair in humans is associated with variant alleles of the alphaMSH receptor gene, MC1R. Loss of MC1R function in other mammals results in red or yellow hair pigmentation. We show that a mouse bacterial artificial chromosome (BAC) which contains Mc1r will efficiently rescue loss of Mc1r in transgenic mice, and that overexpression of the receptor suppresses the effect of the endogenous antagonist, agouti protein. We engineered the BAC to replace the mouse coding region with the human MC1R sequence and used this in the transgenic assay. The human receptor also efficiently rescued Mc1r deficiency, and in addition, appeared to be completely resistant to the effects of agouti, suggesting agouti protein may not play a role in human pigmentary variation. Three human variant alleles account for 60% of all cases of red hair. We engineered each of these in turn into the BAC and find that they have reduced, but not completely absent, function in transgenic mice. Comparison of the phenotypes of alphaMSH-deficient mice and humans in conjunction with this data suggests that red hair may not be the null phenotype of MC1R.

• Ritland K, Newton C, Marshall HD
• Inheritance and population structure of the white-phased "Kermode" black bear.
• Curr Biol. 2001; 11: 1468-72
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• We report that a single nucleotide replacement in the melanocortin 1 receptor gene [1] (mc1r) is responsible for the white coat color of the "Kermode" bear [2], a color phase of the black bear (Ursus americanus Pallus) found in the rainforests along the north coast of British Columbia. In a sample of 220 bears, of which 22 were white, there was complete association of a recessive Tyr-to-Cys replacement at codon 298 with the white phase. This variant has not been yet been reported in other mammals, and it also is the lightest-colored variant yet found at mc1r. Also, we found that heterozygotes, which act as a hidden reservoir for the allele among black bears, were infrequent outside of the three islands where Kermodes are common and that, within these three islands, heterozygotes were less frequent than expected under random mating. Immigration of black bears into Kermode populations can depress the occurrence of the white phase, and management practices should be designed to avoid facilitating higher immigration rates.

• Voisey J, Box NF, van Daal A
• A polymorphism study of the human Agouti gene and its association with MC1R.
• Pigment Cell Res. 2001; 14: 264-7
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• To determine whether the Agouti Signalling Protein (ASP) gene is associated with skin and hair coloration in humans, the complete coding region of ASP was screened for polymorphisms. Analysis of ASP in Caucasian, African-American, Spanish Basque, Hispanic, Apache and Australian Aboriginal populations revealed no amino acid substitutions. A single polymorphism in the 3' untranslated region occurred at a frequency of 0.2 in African-Americans. Variants of the Melanocortin 1 Receptor (MC1R) gene have been found to be associated with red hair and fair skin in humans. Red hair individuals are usually compound heterozygotes or homozygous for one of a number of MC1R polymorphisms associated with red hair. Some individuals however are heterozygous for only one of these polymorphisms and dizygotic twins can be concordant for MC1R variants but discordant for hair colour. A recent study has also identified rare redheads carrying no MC1R variants indicating that polymorphisms of the human MC1R gene are required but not sufficient for the red hair phenotype. To address the question of whether ASP also contributes to the red hair phenotype, individuals previously identified as having unexpected MC1R genotypes were screened for polymorphisms at the ASP locus. No polymorphisms were found in any of these individuals. Results indicate that the human ASP gene is unlikely to function in normal human pigmentation in the same way as MC1R.

• He L et al.
• A biochemical function for attractin in agouti-induced pigmentation and obesity.
• Nat Genet. 2001; 27: 40-7
• Display abstract

• Agouti protein, a paracrine signaling molecule normally limited to skin, is ectopically expressed in lethal yellow (A(y)) mice, and causes obesity by mimicking agouti-related protein (Agrp), found primarily in the hypothalamus. Mouse attractin (Atrn) is a widely expressed transmembrane protein whose loss of function in mahogany (Atrn(mg-3J)/ Atrn(mg-3J)) mutant mice blocks the pleiotropic effects of A(y). Here we demonstrate in transgenic, biochemical and genetic-interaction experiments that attractin is a low-affinity receptor for agouti protein, but not Agrp, in vitro and in vivo. Additional histopathologic abnormalities in Atrn(mg-3J)/Atrn(mg-3J) mice and cross-species genomic comparisons indicate that Atrn has multiple functions distinct from both a physiologic and an evolutionary perspective.

• Sviderskaya EV, Hill SP, Balachandar D, Barsh GS, Bennett DC
• Agouti signaling protein and other factors modulating differentiation and proliferation of immortal melanoblasts.
• Dev Dyn. 2001; 221: 373-9
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• The melanocyte lineage potentially forms an attractive model system for studies in cell differentiation, developmental genetics, cell signaling, and melanoma, because differentiated cells produce the visible pigment melanin. Immortal lines of murine melanoblasts (melanocyte precursors) have been described previously, but induction of differentiation involved a complex culture system with keratinocyte feeder cells. Here we describe conditions for both growth and induced differentiation of the melanoblast line melb-a, without feeder cells, and analyze factors that directly control proliferation and differentiation of these pure melanoblasts. Several active factors are products of developmental and other coat color genes, including stem cell factor (SCF), melanocyte-stimulating hormone (alphaMSH), and agouti signaling protein (ASP), a natural antagonist at the MSH receptor (melanocortin 1 receptor, MC1R) encoded by the agouti gene. A stable analog of alphaMSH (NDP-MSH) stimulated differentiation and inhibited growth. ASP in excess inhibited both effects of NDP-MSH, that is, ASP could inhibit pigmentation and stimulate growth. These effects provide an explanation for the interactions in mice of melanocyte developmental mutations with yellow agouti and Mc1r alleles, and a role for embryonic expression patterns of ASP.

• Abdel-Malek ZA et al.
• The melanocortin 1 receptor is the principal mediator of the effects of agouti signaling protein on mammalian melanocytes.
• J Cell Sci. 2001; 114: 1019-24
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• The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with (&agr;)-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E(+)/E(+), e/e, or E(so)/E(so) mice to (alpha)-MSH and/or ASP. E(+)/E(+) melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and E(so)/E(so) is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to (alpha)-melanocyte stimulating hormone. Mouse E(+)/E(+) melanocytes, but not e/e or E(so)/E(so) melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E(+)/E(+) melanocytes does agouti signaling protein abrogate the stimulatory effects of (alpha)-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.

• Ha T, Rees JL
• Melanocortin 1 receptor: what's red got to do with it?
• J Am Acad Dermatol. 2001; 45: 961-4

• Ito S et al.
• Cyclic oscillations in melanin composition within hairs of baboons.
• Pigment Cell Res. 2001; 14: 180-4
• Display abstract

• The wild-type agouti-banding pattern for hair is well characterized in lower mammals such as mice. The switch between eumelanin and pheomelanin in bands in the hair results from the interaction of alpha-melanocyte stimulating hormone and agouti signal protein through the melanocortin 1 receptor on melanocytes. However, such banding patterns have not been described to date in higher mammals. We now report such 'agouti'-banding patterns that occur in several subspecies of baboons, and characterize those hairs using chemical and immunohistochemical methods. Hair and skin samples were obtained from the dorsa of adult male baboons of different subspecies (Papio cynocephalus hamadryas (PCH) and Papio cynocephalus anubis (PCA)). The hairs were excised with scissors into the gray and the white bands of the PCH subspecies and into the black and the yellow bands of the PCA subspecies, and were analyzed for total melanin, eumelanin, and pheomelanin by spectrophotometric and chemical methods. Hairs in the PCA subspecies oscillate between a eumelanic band (with high melanin content) and a pheomelanic band, while hairs in the PCH subspecies oscillate between a eumelanic band (with low melanin content) and a non-pigmented band. Those chemical data are consistent with the histological appearance of the hair bulbs stained by the Fontana-Masson technique. The difference in the melanin content between PCH and PCA subspecies is most likely related to tyrosinase levels, as suggested by the presence of unpigmented muzzle in the PCH subspecies compared with the black muzzle in the PCA subspecies.

• Grimes EA, Noake PJ, Dixon L, Urquhart A
• Sequence polymorphism in the human melanocortin 1 receptor gene as an indicator of the red hair phenotype.
• Forensic Sci Int. 2001; 122: 124-9
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• We describe a minisequencing protocol for screening DNA samples for the presence of 12 mutations in the human melanocortin 1 receptor gene (MC1R), eight of which are associated with the red hair phenotype. A minisequencing profile which shows homozygosity for one of these mutations or the presence of two different mutations would strongly indicate that the sample donor is red haired. The absence of any red hair causing mutations would indicate that the sample donor does not have red hair. We report the frequencies of MC1R variants in the British red haired population.

• Lamoreux ML, Wakamatsu K, Ito S
• Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin.
• Pigment Cell Res. 2001; 14: 23-31
• Display abstract

• Melanocytes produce two chemically distinct types of melanin pigments, eumelanin and pheomelanin. These pigments can be quantitatively analyzed by acidic permanganate oxidation or reductive hydrolysis with hydriodic acid to form pyrrole-2,3,5-tricarboxylic acid or aminohydroxyphenylalanine, respectively. About 30 coat color genes in mice have been cloned, and functions of many of those genes have been elucidated. However, little is known about the interacting functions of these loci. In this study, we used congenic mice to eliminate genetic variability, and analyzed eumelanin and pheomelanin contents of hairs from mice mutant at one or more of the major pigment loci, i.e., the albino (C) locus that encodes tyrosinase, the slaty (Slt) locus that encodes tyrosinase-related protein 2 (TRP2 also known as dopachrome tautomerase, DCT), the brown (B) locus that encodes TRP1, the silver (Si) locus that encodes a melanosomal silver protein, the agouti (A) locus that encodes agouti signaling protein (ASP), the extension (E) locus that encodes melanocortin-1 receptor, and the mahogany (Mg) locus that encodes attractin. We also measured total melanin contents after solubilization of hairs in hot Soluene-350 plus water. Hairs were shaved from 2-3-month-old congenic C57BL/6J mice. The chinchilla (c(ch)) allele is known to encode tyrosinase, whose activity is about one third that of wild type (C). Phenotypes of chinchilla (c(ch)/c(ch)) mice that are wild type or mutant at the brown and/or slaty, loci indicate that functioning TRP2 and TRP1 are necessary, in addition to high levels of tyrosinase, for a full production of eumelanin. The chinchilla allele was found to reduce the amount of pheomelanin in lethal yellow and recessive yellow mice to less than one fifth of that in congenic yellow mice that were wild type at the albino locus. This indicates that reduction in tyrosinase activity affects pheomelanogenesis more profoundly compared with eumelanogenesis. Hairs homozygous for mutation at the slaty locus contain 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-poor melanin, and this chemical phenotype was retained in hairs that were mutant at both the brown locus and the slaty locus. Hair from mice mutant at the brown locus, but not at the slaty locus, do not contain DHICA-poor melanin. This indicates that the proportion of DHICA in eumelanin is determined by TRP2, but not by TRP1. Mutation at the slaty locus (Slt(lt)) was found to have no effect on pheomelanogenesis, supporting a role of TRP2 only in eumelanogenesis. The mutation at silver (si) locus showed an effect similar to brown, a partial suppression of eumelanogenesis. The mutation at mahogany (mg) locus partially suppressed the effect of lethal yellow (Ay) on pheomelanogenesis, supporting a role of mahogany in interfering with agouti signaling. These results show that combination of double mutation study of congenic mice with chemical analysis of melanins is useful in evaluating the interaction of pigment gene functions.

• Schaffer JV, Bolognia JL
• The melanocortin-1 receptor: red hair and beyond.
• Arch Dermatol. 2001; 137: 1477-85
• Display abstract

• Although human pigmentation is genetically complex, to date polymorphism at only 1 locus, the melanocortin-1 receptor (MC1-R), has been associated with physiologic variation in hair and skin color. The MC1-R, a G protein-coupled receptor with 7 transmembrane-spanning domains, plays a key role in determining the type of melanin (eumelanin vs pheomelanin) that is produced within melanocytes. This article begins with an overview of melanocortin receptors, proopiomelanocortin-derived ligands, and the agouti antagonist, with particular focus on their functions in regulating eumelanin and pheomelanin synthesis, including UV-induced melanogenesis. A brief description of mouse-coat-color genetics is then followed by a discussion of human MC1-R variants, which are present in approximately 50% of white populations. We review the increasing evidence that loss-of-function MC1-R mutations largely account for the red hair phenotype in humans (which approximates an autosomal recessive trait) and also have a strong association with fair skin and a decreased ability to tan, with a significant heterozygote effect in individuals without red hair. Finally, we examine recent work showing that loss-of-function MC1-R variants may increase the risk of developing melanoma and nonmelanoma skin cancer beyond their effects on pigmentation phenotype.

• Sturm RA, Teasdale RD, Box NF
• Human pigmentation genes: identification, structure and consequences of polymorphic variation.
• Gene. 2001; 277: 49-62
• Display abstract

• The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones alpha-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation.

• Rieder S, Taourit S, Mariat D, Langlois B, Guerin G
• Mutations in the agouti (ASIP), the extension (MC1R), and the brown (TYRP1) loci and their association to coat color phenotypes in horses (Equus caballus).
• Mamm Genome. 2001; 12: 450-5
• Display abstract

• Coat color genetics, when successfully adapted and applied to different mammalian species, provides a good demonstration of the powerful concept of comparative genetics. Using cross-species techniques, we have cloned, sequenced, and characterized equine melanocortin-1-receptor (MC1R) and agouti-signaling-protein (ASIP), and completed a partial sequence of tyrosinase-related protein 1 (TYRP1). The coding sequences and parts of the flanking regions of those genes were systematically analyzed in 40 horses and mutations typed in a total of 120 horses. Our panel represented 22 different horse breeds, including 11 different coat colors of Equus caballus. The comparison of a 1721-bp genomic fragment of MC1R among the 11 coat color phenotypes revealed no sequence difference apart from the known chestnut allele (C901T). In particular, no dominant black (ED) mutation was found. In a 4994-bp genomic fragment covering the three putative exons, two introns and parts of the 5'- and 3'-UTRs of ASIP, two intronic base substitutions (SNP-A845G and C2374A), a point mutation in the 3'-UTRs (A4734G), and an 11-bp deletion in exon 2 (ADEx2) were detected. The deletion was found to be homozygous and completely associated with horse recessive black coat color (Aa/Aa) in 24 black horses out of 9 different breeds from our panel. The frameshift initiated by ADEx2 is believed to alter the regular coding sequence, acting as a loss-of-function ASIP mutation. In TYRP1 a base substitution was detected in exon 2 (C189T), causing a threonine to methionine change of yet unknown function, and an SNP (A1188G) was found in intron 2.

• Harding RM et al.
• Evidence for variable selective pressures at MC1R.
• Am J Hum Genet. 2000; 66: 1351-61
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• It is widely assumed that genes that influence variation in skin and hair pigmentation are under selection. To date, the melanocortin 1 receptor (MC1R) is the only gene identified that explains substantial phenotypic variance in human pigmentation. Here we investigate MC1R polymorphism in several populations, for evidence of selection. We conclude that MC1R is under strong functional constraint in Africa, where any diversion from eumelanin production (black pigmentation) appears to be evolutionarily deleterious. Although many of the MC1R amino acid variants observed in non-African populations do affect MC1R function and contribute to high levels of MC1R diversity in Europeans, we found no evidence, in either the magnitude or the patterns of diversity, for its enhancement by selection; rather, our analyses show that levels of MC1R polymorphism simply reflect neutral expectations under relaxation of strong functional constraint outside Africa.

• Jordan S, Beermann F
• Nomenclature for identified pigmentation genes in the mouse.
• Pigment Cell Res. 2000; 13: 70-1
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• More than 90 different loci influence pigmentation in the mouse. During the past few years, an increasing number of genes have been identified, and assigned to the corresponding coat color loci and pigmentation mutants. As a consequence, different names have been used in publications for loci, genes and corresponding proteins. In the following article, we present the rules and guidelines for gene nomenclature, and provide the current nomenclature for pigmentation mutants in the mouse.

• Leeb T, Doppe A, Kriegesmann B, Brenig B
• Genomic structure and nucleotide polymorphisms of the porcine agouti signalling protein gene (ASIP).
• Anim Genet. 2000; 31: 335-6

• Flanagan N et al.
• Pleiotropic effects of the melanocortin 1 receptor (MC1R) gene on human pigmentation.
• Hum Mol Genet. 2000; 9: 2531-7
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• Variants of the melanocortin 1 receptor (MC1R) gene are common in individuals with red hair and fair skin, but the relative contribution to these pigmentary traits in heterozygotes, homozygotes and compound heterozygotes for variants at this locus from the multiple alleles present in Caucasian populations is unclear. We have investigated 174 individuals from 11 large kindreds with a preponderance of red hair and an additional 99 unrelated redheads, for MC1R variants and have confirmed that red hair is usually inherited as a recessive characteristic with the R151C, R160W, D294H, R142H, 86insA and 537insC alleles at this locus. The V60L variant, which is common in the population may act as a partially penetrant recessive allele. These individuals plus 167 randomly ascertained Caucasians demonstrate that heterozygotes for two alleles, R151C and 537insC, have a significantly elevated risk of red hair. The shade of red hair frequently differs in heterozygotes from that in homozygotes/compound heterozygotes and there is also evidence for a heterozygote effect on beard hair colour, skin type and freckling. The data provide evidence for a dosage effect of MC1R variants on hair as well as skin colour.

• Matsunaga N et al.
• In situ localization of agouti signal protein in murine skin using immunohistochemistry with an ASP-specific antibody.
• Biochem Biophys Res Commun. 2000; 270: 176-82
• Display abstract

• Switching between production of eumelanin or pheomelanin in follicular melanocytes is responsible for hair color in mammals; in mice, this switch is controlled by the agouti locus, which encodes agouti signal protein (ASP) through the action of melanocortin receptor 1. To study expression and processing patterns of ASP in the skin and its regulation of pigment production in hair follicles, we have generated a rabbit antibody (termed alphaPEP16) against a synthetic peptide that corresponds to the carboxyl terminus of ASP. The specificity of that antibody was measured by ELISA and was confirmed by Western blot analysis. Using immunohistochemistry, we characterized the expression of ASP in the skin of newborn mice at 3, 6, and 9 days postnatally. Expression in nonagouti (a/a) black mouse skin was negative at all times examined, as expected, and high expression of ASP was observed in 6 day newborn agouti (A/+) and in 6 and 9 day newborn lethal yellow (A(y)/a) mouse skin. In lethal yellow (pheomelanogenic) mice, ASP expression increased day by day as the hair color became more yellow. These expression patterns suggest that ASP is delivered quickly and efficiently to melanocytes and to hair matrix cells in the hair bulbs where it regulates melanin production.

• Abdel-Malek Z et al.
• The melanocortin-1 receptor is a key regulator of human cutaneous pigmentation.
• Pigment Cell Res. 2000; 13: 156-62
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• The cloning and characterization of the human melanocortin-1 receptor (MC1R) and the demonstration that normal human melanocytes respond to the melanocortins, alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotrophic hormone (ACTH), with increased proliferation and eumelanogenesis had put an end to a long-standing controversy about the role of melanocortins in regulating human cutaneous pigmentation. We have shown that alpha-MSH and ACTH bind the human MC1R with equal affinity, and are equipotent in their mitogenic and melanogenic effects on human melanocytes. We also showed that the activation of the MC1R is important for the melanogenic response of human melanocytes to ultraviolet radiation (UVR). The MC1R is also the principal mediator of the inhibitory effects of agouti signaling protein (ASP) on melanogenesis. Expression of the MC1R is subject to regulation by its own ligands alpha-MSH and ACTH, as well as by UVR and endothelin-1. Recent studies that we conducted on the expression of MC1R variants by human melanocytes and the implications of these variants on the function of the MC1R revealed the following. Human melanocytes homozygous for Arg160Trp mutation in the MC1R demonstrated a significantly reduced response to alpha-MSH. Also, this culture responded poorly to ASP and exhibited an exaggerated cytotoxic response to UVR. Another culture, which was homozygous for Val92Met mutation in the MC1R, demonstrated a normal response to alpha-MSH. Heterozygous mutations that are frequently expressed in various melanocyte cultures did not disrupt MC1R function. These results begin to elucidate the significance of MC1R variants in the function of the receptor. Our data emphasize the significance of a normally functioning MC1R in the response of melanocytes to melanocortins, ASP, and UVR.

• Barsh G, Gunn T, He L, Schlossman S, Duke-Cohan J
• Biochemical and genetic studies of pigment-type switching.
• Pigment Cell Res. 2000; 13: 48-53
• Display abstract

• Mutations that affect the balance between the synthesis of eumelanin and pheomelanin provide a powerful set of tools with which to understand general aspects of cell signaling. Previous work from our laboratory has demonstrated that pheomelanin synthesis is triggered by the ability of Agouti protein to inhibit signaling through the Melanocortin 1 receptor (Mc1r). In a bioassay based on the Xenopus Mc1r, Agouti protein has two effects, competitive inhibition of receptor occupancy by alpha-MSH and down-regulation of receptor signaling, which are mediated separately by domains in the amino- and carboxy-terminal regions of Agouti protein, respectively. Recently, we have used the genetics of pigmentation as an in vivo system to screen for and analyze other mutations in the Agouti-melanocortin pathway. The pigmentary effects of Agouti are suppressed by the previously existing coat-color mutations mahogany (mg), mahoganoid (md), and Umbrous (U). Double mutant studies, with animals deficient for the Mc1r or those which carry Ay, indicate that mg and md are genetically upstream of the Mc1r, and can suppress the effects of Ay on both pigmentation and body weight. Positional cloning has recently identified the gene mutated in mahogany as a single transmembrane-spanning protein whose ectodomain is orthologous to human Attractin (Atrn).

• Rees JL
• The melanocortin 1 receptor (MC1R): more than just red hair.
• Pigment Cell Res. 2000; 13: 135-40
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• The melanocortin 1 receptor, a seven pass transmembrane G protein coupled receptor, is a key control point in melanogenesis. Loss-of-function mutations at the MC1R are associated with a switch from eumelanin to phaeomelanin production, resulting in a red or yellow coat colour. Activating mutations, in animals at least, lead to enhanced eumelanin synthesis. In man, a number of loss-of-function mutations in the MC1R have been described. The majority of red-heads (red-haired persons) are compound heterozygotes or homozygotes for up to five frequent loss-of-function mutations. A minority of redheads are, however, only heterozygote. The MC1R is, therefore, a major determinant of sun sensitivity and a genetic risk factor for melanoma and non-melanoma skin cancer. Recent work suggests that the MC1R also shows a clear heterozygote effect on skin type, with up to 30% of the population harbouring loss-of-function mutations. Activating mutations of the MC1R in man have not been described. The MC1R is particularly informative and a tractable gene for studies of human evolution and migration. In particular, study of the MC1R may provide insights into the lightening of skin colour observed in most European populations. The world wide pattern of MC1R diversity is compatible with functional constraint operating in Africa, whereas the greater allelic diversity seen in non-African populations is consistent with neutral predictions rather than selection. Whether this conclusion is as a result of weakness in the statistical testing procedures applied, or whether it will be seen in other pigment genes will be of great interest for studies of human skin colour evolution.

• Newton JM et al.
• Melanocortin 1 receptor variation in the domestic dog.
• Mamm Genome. 2000; 11: 24-30
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• The melanocortin 1 receptor (Mc1r) is encoded by the Extension locus in many different mammals, where a loss-of-function causes exclusive production of red/yellow pheomelanin, and a constitutively activating mutation causes exclusive production of black/brown eumelanin. In the domestic dog, breeds with a wild-type E allele, e. g., the Doberman, can produce either pigment type, whereas breeds with the e allele, e.g., the Golden Retriever, produce exclusively yellow pigment. However, a black coat color in the Newfoundland and similar breeds is thought to be caused by an unusual allele of Agouti, which encodes the physiologic ligand for the Mc1r. Here we report that the predicted dog Mc1r is 317 residues in length and 96% identical to the fox Mc1r. Comparison of the Doberman, Newfoundland, Black Labrador, Yellow Labrador, Flat-coated Retriever, Irish Setter, and Golden Retriever revealed six sequence variants, of which two, S90G and R306ter, partially correlated with a black/brown coat and red/yellow coat, respectively. R306ter was found in the Yellow Labrador, Golden Retriever, and Irish Setter; the latter two had identical haplotypes but differed from the Yellow Labrador at three positions other than R306ter. In a larger survey of 194 dogs and 19 breeds, R306ter and a red/yellow coat were completely concordant except for the Red Chow. These results indicate that the e allele is caused by a common Mc1r loss-of-function mutation that either reoccurred or was subject to gene conversion during recent evolutionary history, and suggest that the allelic and locus relationships for dog coat color genes may be more analogous to those found in other mammals than previously thought.

• Suzuki I et al.
• Participation of the melanocortin-1 receptor in the UV control of pigmentation.
• J Investig Dermatol Symp Proc. 1999; 4: 29-34
• Display abstract

• The cloning of the melanocortin-1 receptor (MC1R) gene from human melanocytes and the demonstration that these cells respond to the melanocortins alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) with increased proliferation and melanogenesis have renewed the interest in investigation the physiological role of these hormones in regulating human pigmentation. Alpha-melanocyte stimulating hormone and ACTH are both synthesized in the human epidermis, and their synthesis is upregulated by exposure to ultraviolet radiation (UVR). Activation of the MC1R by ligand binding results in stimulation of cAMP formation, which is a principal mechanism for inducing melanogenesis. The increase in cAMP is required for the pigmentary response of human melanocytes to UVR, and for allowing them to overcome the UVR-induced G1 arrest. Treatment of human melanocytes with alpha-MSH increases eumelanin synthesis, an effect that is expected to enhance photoprotection of the skin. Population studies have revealed more than 20 allelic variants of the MC1R gene. Some of these variants are overexpressed in individuals with skin type I or II, red hair, and poor tanning ability. Future studies will aim at further exploration of the role of these variants in MC1R function, and in determining constitutive human pigmentation, the response to sun exposure, and possibly the susceptibility to skin cancer.

• Norman RA, Permana P, Tanizawa Y, Ravussin E
• Absence of genetic variation in some obesity candidate genes (GLP1R, ASIP, MC4R, MC5R) among Pima indians.
• Int J Obes Relat Metab Disord. 1999; 23: 163-5
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• OBJECTIVE: To examine the obesity candidate genes glucagon-like-peptide receptor (GLP1R), agouti signaling protein (ASIP) and the melanocortin receptors 4 and 5 (MC4R and MC5R) for DNA polymorphisms in their coding regions. SUBJECTS: Unrelated, non-diabetic Pima Indians (8 to 12 from each extreme of body fat). MEASUREMENTS: DNA sequencing within the coding regions of each gene. RESULT: Only one variant was detected, a silent substitution in exon 6 of GLP1R. CONCLUSION: The exclusion of any common amino-acid polymorphisms (allele frequency > or = 0.20). implies that structural variants of these genes do not contribute to variation in the high level of obesity observed among the Pima Indians.

• Jackson IJ
• The mahogany mouse mutation: further links between pigmentation, obesity and the immune system.
• Trends Genet. 1999; 15: 429-31
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• Completely different lines of experimentation have identified attractin, a protein that seems to have multiple roles in regulating physiological processes. It affects the balance between agonist and antagonist at receptors on melanocytes, modifies behaviour and basal metabolic rate, and mediates an interaction between activated T cells and macrophages. It may well be a target for development of drugs to treat obesity.

• Parsons YM, Fleet MR, Cooper DW
• Isolation of the ovine agouti coding sequence.
• Pigment Cell Res. 1999; 12: 394-7

• Rana BK et al.
• High polymorphism at the human melanocortin 1 receptor locus.
• Genetics. 1999; 151: 1547-57
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• Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.

• Abdel-Malek Z, Suzuki I, Tada A, Im S, Akcali C
• The melanocortin-1 receptor and human pigmentation.
• Ann N Y Acad Sci. 1999; 885: 117-33
• Display abstract

• alpha-Melanocyte stimulating hormone (alpha-MSH) is known to be the main physiologic regulator for integumental pigmentation of various vertebrate species. However, the role of alpha-MSH and related melanocortins in the regulation of human cutaneous pigmentation is only beginning to be understood. Cloning of the melanocortin-1 receptor (MC1R), and the feasibility of establishing normal human epidermal melanocyte cultures have made it possible to demonstrate direct and specific biological effects of alpha-MSH on these cells. It is now recognized that both alpha-MSH and ACTH have similar mitogenic and melanogenic effects on human epidermal melanocytes. These effects are mediated by binding of these hormones to the specific MC1R that recognizes them both with similar affinity. Human MC1R is homologous to its mouse counterpart in that its activation leads to stimulation of eumelanin synthesis. MC1R is also the binding site for agouti signaling protein (ASP), the product of the agouti locus. Human epidermal melanocytes respond to purified recombinant mouse or human ASP, with a reduction in basal tyrosinase activity, and complete abrogation of the mitogenic and melanogenic effects of alpha-MSH. These results suggest that ASP induces pheomelanin synthesis by competing with alpha-MSH for binding to the MC1R. This receptor seems to be subject to regulation by a variety of paracrine and/or autocrine factors that are synthesized in response to exposure of the skin to ultraviolet radiation (UVR). Activation of MC1R seems to be pivotal for UV-induced melanogenesis, since stimulation of the cAMP pathway plays a key role in the melanogenic response of human epidermal melanocytes. The melanogenic response to UVR might be influenced by the presence of allelic variants of the MC1R gene. Allelic variants have been identified and shown to be associated with red hair, poor tanning ability, and possibly melanoma. The possible influence of these variants on the function of the MC1R needs to be investigated, in order to understand the physiological consequence of these mutations. Also, the interaction of alpha-MSH with other factors that are known to affect pigmentation needs to be better understood in order to define the role possible of this hormone and its receptor in acquired human cutaneous hyper- or hypopigmentation.

• Wada A, Okumoto M, Tsudzuki M
• Tawny: a novel light coat color mutation found in a wild population of Mus musculus molossinus, a new allele at the melanocortin 1 receptor (Mc1r) locus.
• Exp Anim. 1999; 48: 73-8
• Display abstract

• We found a new coat color mutant in a population of Japanese wild mice (Mus musculus molossinus) and called the trait tawny. The tawny mutant is characterized by a light yellowish brown coat color. The tawny hair has a so-called agouti pattern, but the yellow band is greatly lengthened. There are no differences between the tawny and wildtype hairs in size and the number of melanosomes. Genetic analyses revealed that the tawny trait is an autosomal recessive and its gene is located in the distal region on Chromosome 8 between the microsatellite markers D8Mit87 and D8Mit122. An allelism test indicated the tawny mutant gene to be a new allele at the Mc1r locus and dominant to the recessive yellow (Mc1re). The proposed gene symbol for the tawny is Mc1rtaw.

• Wolff GL, Roberts DW, Mountjoy KG
• Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome.
• Physiol Genomics. 1999; 1: 151-63
• Display abstract

• This review summarizes primary and downstream phenotypic manifestations, with emphasis on altered responsiveness to environmental stimuli, of dominant yellow mutations at the mouse agouti locus. Obvious effects include hyperinsulinemia, obesity, stimulation of somatic growth and tumorigenesis, and coat color. Downstream influences of hyperinsulinemia and obesity on the individual's physiology determine important components of the obese yellow agouti mouse syndrome. Collectively, the phenotypic aberrations described support the concept that identical genomes are expressed in a spectrum of physiological phenotypes that reflect the complex interdependence of gene-regulated physiological pathways and processes in the organism throughout extended, but temporally ordered, periods of fetal and neonatal development and aging. This summary identifies important areas for additional research and provides integrated information required for a systematic approach to the development of interventions for common adult human health problems.

• Barsh GS, Ollmann MM, Wilson BD, Miller KA, Gunn TM
• Molecular pharmacology of Agouti protein in vitro and in vivo.
• Ann N Y Acad Sci. 1999; 885: 143-52
• Display abstract

• Agouti protein and Agouti-related protein (Agrp) are paracrine signaling molecules that act by antagonizing the effects of melanocortins, and several alternatives have been proposed to explain their mechanisms of action. Genetic crosses in a sensitized background uncover a phenotypic difference between overexpression of Agouti and loss of Mc1r function, demonstrate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. In vitro, Agouti protein inhibits melanocortin action by two mechanisms: competitive antagonism that depends on the carboxyterminus of the protein, and downregulation of melanocortin receptor signaling that depends on the aminoterminus. Our findings provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.

• Vage DI, Klungland H, Lu D, Cone RD
• Molecular and pharmacological characterization of dominant black coat color in sheep.
• Mamm Genome. 1999; 10: 39-43
• Display abstract

• Dominant black coat color in sheep is predicted to be caused by an allele ED at the extension locus. Recent studies have shown that this gene encodes the melanocyte stimulating hormone receptor (MC1-R). In mouse and fox, naturally occurring mutations in the coding region of MC1-R produce a constitutively activated receptor that switches the synthesis from phaeomelanin to eumelanin within the melanocyte, explaining the black coat color observed phenotypically. In the sheep, we have identified a Met-->Lys mutation in position 73 (M73K) together with a Asp --> Asn change at position 121 (D121N) showing complete cosegregation with dominant black coat color in a family lineage. Only the M73K mutation showed constitutive activation when introduced into the corresponding mouse receptor (mMC1-R) for pharmacological analysis; however, the position corresponding to D121 in the mouse receptor is required for high affinity ligand binding. The pharmacological profile of the M73K change is unique compared to the constitutively active E92K mutation in the sombre mouse and C123R mutation in the Alaska silver fox, indicating that the M73K change activates the receptor via a mechanism distinct from these previously characterized mutations.

• Miltenberger RJ, Mynatt RL, Bruce BD, Wilkison WO, Woychik RP, Michaud EJ
• An agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice.
• Proc Natl Acad Sci U S A. 1999; 96: 8579-84
• Display abstract

• Chronic antagonism of melanocortin receptors by the paracrine-acting agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type agouti. Functional analysis of agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the agouti basic domain, the entire 29-aa region was deleted from the agouti cDNA, and the resulting mutation (agoutiDeltabasic) was expressed in transgenic mice under the control of the beta-actin promoter (BAPaDeltabasic). Three independent lines of BAPaDeltabasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the agoutiDeltabasic protein was functional in vivo. However, none of the BAPaDeltabasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of agoutiDeltabasic expression in relevant tissues exceeded the level of agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the agoutiDeltabasic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPaDeltabasic transgenic mice. These findings implicate a functional role for the agouti basic domain in vivo, possibly influencing the biogenesis of secreted agouti protein or modulating protein-protein interactions that contribute to effective antagonism of melanocortin receptors.

• Morgan HD, Sutherland HG, Martin DI, Whitelaw E
• Epigenetic inheritance at the agouti locus in the mouse.
• Nat Genet. 1999; 23: 314-8
• Display abstract

• Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( A(vy)/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene (A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A(vy) dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.

• Rees JL, Birch-Machin M, Flanagan N, Healy E, Phillips S, Todd C
• Genetic studies of the human melanocortin-1 receptor.
• Ann N Y Acad Sci. 1999; 885: 134-42
• Display abstract

• Genetic approaches have suggested a critical role for the melanocortin-1 receptor in the control of pigmentation. We showed that this gene is unusually polymorphic in European populations and that, of the many variants, three in particular appear to be associated with red hair or fair skin. Family studies suggest these are inherited as an autosomal recessive trait (or at least approximate to this in many families). To date all individuals with two of these three changes (homozygote or compound heterozygote) have red hair. Early functional studies are in keeping with defective signalling through MC1R. An interested and perhaps unexpected question relates to the evolutionary factors that have given rise to such variants. Two models can be proposed, that are based on multiple alleles with minor changes in function or genetic hitch-hicking.

• Monroe DG, Wipf LP, Diggins MR, Matthees DP, Granholm NH
• Agouti-related maturation and tissue distribution of alpha-Melanocyte Stimulating Hormone in wild-type (AwJ/AwJ) and mutant (Ay/a,a/a) mice.
• Pigment Cell Res. 1998; 11: 310-3
• Display abstract

• Because of ectopic overproduction of agouti protein, yellow alleles (A(y) and A(vy)) of the murine agouti gene may secondarily modulate the synthesis, maturation (i.e., acetylation), and/or tissue deployment of alpha-Melanocyte Stimulating Hormone (MSH). We used HPLC to test the hypothesis that A(y)/a mice exhibit altered concentrations of desacetyl-, monoacetyl-, and diacetyl-alpha-MSH in pituitaries, sera, and telogen hair bulbs when compared to black (a/a) mice. We also used RIA to measure total MSH in those same tissues of A(y)a,a/a, and white-bellied agouti (A(wJ)/A(wJ)) mice (Strain C57BL/6J). We found no evidence that A(y)/a mice possessed an imbalance of des-, mono-, and diacetylated alpha-MSH species. However, radioimmunoassay (RIA) analyses of total MSH suggest that wild-type agouti mice (A(wJ)/A(wJ)) exhibited significantly decreased (P < 0.05) tissue levels of total alpha-MSH in pituitaries, sera, and regenerating hair bulbs when compared to those of mutant A(y)/a and a/a mice.

• Wolff GL, Kodell RL, Moore SR, Cooney CA
• Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice.
• FASEB J. 1998; 12: 949-57
• Display abstract

• 'Viable yellow' (Avy/a) mice are larger, obese, hyperinsulinemic, more susceptible to cancer, and, on average, shorter lived than their non-yellow siblings. They are epigenetic mosaics ranging from a yellow phenotype with maximum ectopic agouti overexpression, through a continuum of mottled agouti/yellow phenotypes with partial agouti overexpression, to a pseudoagouti phenotype with minimal ectopic expression. Pseudoagouti Avy/a mice are lean, healthy, and longer lived than their yellow siblings. Here we report that feeding pregnant black a/a dams methyl-supplemented diets alters epigenetic regulation of agouti expression in their offspring, as indicated by increased agouti/black mottling in the direction of the pseudoagouti phenotype. We also present confirmatory evidence that epigenetic phenotypes are maternally heritable. Thus Avy expression, already known to be modulated by imprinting, strain-specific modification, and maternal epigenetic inheritance, is also modulated by maternal diet. These observations suggest, at least in this special case, that maternal dietary supplementation may positively affect health and longevity of the offspring. Therefore, this experimental system should be useful for identifying maternal factors that modulate epigenetic mechanisms, especially DNA methylation, in developing embryos.

• Furumura M, Sakai C, Potterf SB, Vieira WD, Barsh GS, Hearing VJ
• Characterization of genes modulated during pheomelanogenesis using differential display.
• Proc Natl Acad Sci U S A. 1998; 95: 7374-8
• Display abstract

• Molecular and biochemical mechanisms that modulate the production of eumelanin or pheomelanin pigments involve the opposing effects of two intercellular signaling molecules, alpha-melanocyte stimulating hormone (MSH) and agouti signal protein (ASP). ASP is an antagonist of MSH signaling through the melanocyte-specific MSH receptor, although its mechanism(s) of action is controversial. We previously have reported significant down-regulation of all known melanogenic genes during the eumelanin to pheomelanin switch in murine hair follicle melanocytes and in cultured melanocytes treated with recombinant ASP. To identify factors that might be involved in the switch to pheomelanogenesis, we screened ASP-treated melanocytes by using differential display and identified three up-regulated genes: a DNA replication control protein, a basic helix-loop-helix transcription factor, and a novel gene. We have simultaneously identified six down-regulated genes in ASP-treated melanocytes; two of those encode tyrosinase and TRP2, melanogenic genes known to be down-regulated during pheomelanogenesis, which provide good internal controls for this approach. These results suggest that there are complex mechanisms involved in the switch to pheomelanin production, and that these modulated genes might be involved in the pleiotropic changes seen in yellow mice, including the change in coat color.

• Jordan SA, Jackson IJ
• Melanocortin receptors and antagonists regulate pigmentation and body weight.
• Bioessays. 1998; 20: 603-6
• Display abstract

• The action two genetic loci--agouti and the melanocortin receptor-1 (Mc1r)-- have opposing effects in the control of mammalian pigmentation and ultimately determine the color of the pigment produced. In a recent paper, Ollmann et al. confirmed that the agouti protein acts via the Mc1r. They show that high-affinity binding of the agouti protein to Mc1r expressed in mammalian cells can be inhibited by the receptor's natural ligand, alpha-melanocyte-stimulating hormone (alpha-MSH). In addition, genetic studies using mice carrying mutations at the Mc1r and agouti loci on a sensitized background of low tyrosinase expression confirm that a functional Mc1r is required for the maximum pigmentary effect of agouti. Thus, the Mc1r appears to be a unique, bifunctionally controlled receptor, activated by alpha-MSH and antagonized by agouti, both of which contribute to the variability seen in mammalian coat color.

• Smith R et al.
• Melanocortin 1 receptor variants in an Irish population.
• J Invest Dermatol. 1998; 111: 119-22
• Display abstract

• The identification of an association between variants in the human melanocortin 1 receptor (MC1R) gene and red hair and fair skin, as well as the relation between variants of this gene and coat color in animals, suggests that the MC1R is an integral control point in the normal pigmentation phenotype. In order to further define the contribution of MC1R variants to pigmentation in a normal population, we have looked for alterations in this gene in series of individuals from a general Irish population, in whom there is a preponderance of individuals with fair skin type. Seventy-five per cent contained a variant in the MC1R gene, with 30% containing two variants. The Arg151Cys, Arg160Trp, and Asp294His variants were significantly associated with red hair (p = 0.0015, p < 0.001, and p < 0.005, respectively). Importantly, no individuals harboring two of these three variants did not have red hair, although some red-haired individuals only showed one alteration. The same three variants were also over-represented in individuals with light skin type as assessed using a modified Fitzpatrick scale. Despite these associations many subjects with dark hair/darker skin type harbored MC1R variants, but there was no evidence of any particular association of variants with the darker phenotype. The Asp294His variant was similarly associated with red hair in a Dutch population, but was infrequent in red-headed subjects from Sweden. The Asp294His variant was also significantly associated with nonmelanoma skin cancer in a U.K. population. The results show that the Arg151Cys, Arg160Trp, and Asp294His variants are of key significance in determining the pigmentary phenotype and response to ultraviolet radiation, and suggest that in many cases the red-haired component and in some cases fair skin type are inherited as a Mendelian recessive.

• Rees JL, Healy E
• Melanocortin receptors, red hair, and skin cancer.
• J Investig Dermatol Symp Proc. 1997; 2: 94-8
• Display abstract

• Cutaneous pigmentation is a major determinant of the cutaneous response to ultraviolet radiation, and consequently of the risk of developing skin cancer. Over the past 10 years, several genes involved in melanogenesis have been identified, including the melanocortin 1 receptor gene. Recent work on the melanocortin 1 receptor suggests that it is a key player in determining whether eumelanin or pheomelanin is predominantly produced both in vitro and in vivo. In the mouse, variants of this receptor, which differ in their ability to activate adenylyl cyclase, are associated with different coat colors. In humans, melanocortin 1 receptor variants are associated with red hair and fair skin, and work in progress from our laboratory suggests that certain melanocortin 1 receptor variants may preferentially be associated with hair color rather than skin type. In addition, melanocortin 1 receptor variants are a risk factor, possibly independent of skin type, for melanoma susceptibility.

• Miller KA, Gunn TM, Carrasquillo MM, Lamoreux ML, Galbraith DB, Barsh GS
• Genetic studies of the mouse mutations mahogany and mahoganoid.
• Genetics. 1997; 146: 1407-15
• Display abstract

• The mouse mutations mahogany (mg) and mahoganoid (md) are negative modifiers of the Agouti coat color gene, which encodes a paracrine signaling molecule that induces a swithc in melanin synthesis from eumelanin to pheomelanin. Animals mutant for md or mg synthesize very little or no pheomelanin depending on Agouti gene background. The Agouti protein is normally expressed in the skin and acts as an antagonist of the melanocyte receptor for alpha-MSH (Mc1r); however, ectopic expression of Agouti causes obesity, possibly by antagonizing melanocortin receptors expressed in the brain. To investigate where md and mg lie in a genetic pathway with regard to Agouti and Mc1r signaling, we determined the effects of these mutations in animals that carried either a loss-of-function Mc1r mutation (recessive yellow, Mc1re) or a gain-of-function Agouti mutation (lethal yellow, Ay). We found that the Mc1re mutation suppressed the effects of md and mg, but that md and mg suppressed the effects of Ay on both coat color and obesity. Plasma levels of alpha-MSH and of ACTH were unaffected by md or mg. These results suggest that md and mg interfere directly with Agouti signaling, possibly at the level of protein production or receptor regulation.

• Suzuki I et al.
• Agouti signaling protein inhibits melanogenesis and the response of human melanocytes to alpha-melanotropin.
• J Invest Dermatol. 1997; 108: 838-42
• Display abstract

• In mouse follicular melanocytes, the switch between eumelanin and pheomelanin synthesis is regulated by the extension locus, which encodes the melanocortin-1 receptor (MC1R) and the agouti locus, which encodes a novel paracrine-signaling molecule that inhibits binding of melanocortins to the MC1R. Human melanocytes express the MC1R and respond to melanotropins with increased proliferation and eumelanogenesis, but a potential role for the human homolog of agouti-signaling protein, ASIP, in human pigmentation has not been investigated. Here we report that ASIP blocked the binding of alpha-melanocyte-stimulating hormone (alpha-MSH) to the MC1R and inhibited the effects of alpha-MSH on human melanocytes. Treatment of human melanocytes with 1 nM-10 nM recombinant mouse or human ASIP blocked the stimulatory effects of alpha-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation. In the absence of exogenous alpha-MSH, ASIP inhibited basal levels of tyrosinase activity and cell proliferation and reduced the level of immunoreactive tyrosinase-related protein-1 (TRP-1) without significantly altering the level of immunoreactive tyrosinase. In addition, ASIP blocked the stimulatory effects of forskolin or dibutyryl cAMP, agents that act downstream from the MC1R, on tyrosinase activity and cell proliferation. These results demonstrate that the functional relationship between the agouti and MC1R gene products is similar in mice and humans and suggest a potential physiologic role for ASIP in regulation of human pigmentation.

• Hayssen V
• Effects of the nonagouti coat-color allele on behavior of deer mice (Peromyscus maniculatus): a comparison with Norway rats (Rattus norvegicus).
• J Comp Psychol. 1997; 111: 419-23
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• The agouti locus influences coat color by antagonizing melanocyte-stimulating hormone (MSH) at its receptor on pigment cells and may antagonize MSH in neural tissue. This study replicates work on rats to assess whether behavioral (neural) effects of the agouti locus are as similar across mammals as those on coat color. Handling, open-field, platform jump, and food-novelty tests were conducted on agouti and nonagouti deer mice (Peromyscus maniculatus) following protocols in C. A. Cottle and E. O. Price (1987). As with rats, nonagouti deer mice were less aggressive, less active, and easier to handle compared with their agouti counterparts. Nonagouti deer mice also groomed more than agouti subjects. Thus, behavioral effects of the agouti locus are conservative, and agouti may be an important modulator of melanocortins in neural as well as integumentary tissue.

• Miltenberger RJ, Mynatt RL, Wilkinson JE, Woychik RP
• The role of the agouti gene in the yellow obese syndrome.
• J Nutr. 1997; 127: 19021907-19021907
• Display abstract

• The yellow obese syndrome in mice encompasses many pleiotropic effects including yellow fur, maturity-onset obesity, hyperinsulinemia, insulin resistance, hyperglycemia, increased skeletal length and lean body mass, and increased susceptibility to neoplasia. The molecular basis of this syndrome is beginning to be unraveled and may have implications for human obesity and diabetes. Normally, the agouti gene is expressed during the hair-growth cycle in the neonatal skin where it functions as a paracrine regulator of pigmentation. The secreted agouti protein antagonizes the binding of the alpha-melanocyte-stimulating hormone to its receptor (melanocortin 1 receptor) on the surface of hair bulb melanocytes, causing alterations in intracellular cAMP levels. Widespread, ectopic expression of the mouse agouti gene is central to the yellow obese phenotype, as demonstrated by the molecular cloning of several dominant agouti mutations and the ubiquitous expression of the wild-type agouti gene in transgenic mice. Recent experiments have revealed that the hypothalamus and adipose tissue are biologically active target sites for agouti in the yellow obese mutant lines.

• Vage DI, Lu D, Klungland H, Lien S, Adalsteinsson S, Cone RD
• A non-epistatic interaction of agouti and extension in the fox, Vulpes vulpes.
• Nat Genet. 1997; 15: 311-5
• Display abstract

• Agouti and extension are two genes that control the production of yellow-red (phaeomelanin) and brown-black (eumelanin) pigments in the mammalian coat. Extension encodes the melanocyte-stimulating hormone receptor (MC1R) while agouti encodes a peptide antagonist of the receptor. In the mouse, extension is epistatic to agouti, hence dominant mutants of the MC1R encoding constitutively active receptors are not inhibited by the agouti antagonist, and animals with dominant alleles of both loci remain darkly pigmented. In the fox the proposed extension locus is not epistatic to the agouti locus. We have cloned and characterized the MC1R and the agouti gene in coat colour variants of the fox (Vulpes vulpes). A constitutively activating C125R mutation in the MC1R was found specifically in darkly pigmented animals carrying the Alaska Silver allele (EA). A deletion in the first coding exon of the agouti gene was found associated with the proposed recessive allele of agouti in the darkly pigmented Standard Silver fox (aa). Thus, as in the mouse, dark pigmentation can be caused by a constitutively active MC1R, or homozygous recessive status at the agouti locus. Our results, demonstrating the presence of dominant extension alleles in foxes with significant red coat colouration, suggest the ability of the fox agouti protein to counteract the signalling activity of a constitutively active fox MC1R.

• Yang YK et al.
• Effects of recombinant agouti-signaling protein on melanocortin action.
• Mol Endocrinol. 1997; 11: 274-80
• Display abstract

• Mouse agouti protein is a paracrine signaling molecule that has previously been demonstrated to be an antagonist of melanocortin action at several cloned rodent and human melanocortin receptors. In this study we report the effects of agouti-signaling protein (ASIP), the human homolog of mouse agouti, on the action of alpha-MSH or ACTH at the five known human melanocortin receptor subtypes (hMCR 1-5). When stably expressed in L cells (hMC1R, hMC3R, hMC4R, hMC5R) or in the adrenocortical cell line OS3 (hMC1R, hMC2R, hMC4R), purified recombinant ASIP inhibits the generation of cAMP stimulated by alpha-MSH (hMC1R, hMC3R, hMC4R, hMC5R) or by ACTH (hMC2R). However, dose-response and Schild analysis indicated that the degree of ASIP inhibition varied significantly among the receptor subtypes; ASIP is a potent inhibitor of the hMC1R, hMC2R, and hMC4R, but has relatively weak effects at the hMC3R and hMC5R. These analyses also indicated that the apparent mechanism of ASIP antagonism varied among receptor subtypes, with characteristics consistent with competitive antagonism observed only at the hMC1R, and more complex behavior observed at the other receptors. ASIP inhibition at these latter receptors, nonetheless, can be classified as surmountable (hMC3R, hMC4R and hMC5R) or nonsurmountable (hMC2R). Recombinant ASIP also inhibited binding of radiolabeled melanocortins, [125I-Nle4, D-Phe7] alpha-MSH and [125I-Phe2, Nle4]ACTH 1-24, to the hMCR 1-5 receptors, with a relative efficacy that paralleled the ability of ASIP to inhibit cAMP accumulation at the hMC1R, hMC2R, hMC3R, and hMC4R. These results provide new insight into the biochemical mechanism of ASIP action and suggest that ASIP may play an important role in modulating melanocortin signaling in humans.

• Jackson IJ
• Homologous pigmentation mutations in human, mouse and other model organisms.
• Hum Mol Genet. 1997; 6: 1613-24
• Display abstract

• Mouse coat colour genes have long been studied as a paradigm for genetic interactions in development. A number of these genes have been cloned and most correspond to human genetic disease loci. The proteins encoded by these genes include transcription factors, receptor tyrosine kinases and growth factors, G-protein coupled receptors and their ligands, membrane proteins, structural proteins and enzymes. Many of the mutations have pleiotropic effects, indicating that these proteins play a wider role in developmental or cellular processes. In this review I tabulate the available data on all pigmentation genes cloned from mouse or human, and I focus on three particular systems. One family of genes, including LYST and HPS/ep, shows the relationship between melanosomes and lysosomes. The G-protein coupled receptor, endothelin receptor-B, and its ligand, endothelin-3, are required for the development of both melanocytes and enteric neurons. The melanocortin-1 receptor is expressed only on melanocytes, but mutations that cause overexpression of agouti protein, an antagonist of the receptor, result in obesity, and highlight a role of melanocortins in weight homoeostasis.

• Furumura M, Sakai C, Abdel-Malek Z, Barsh GS, Hearing VJ
• The interaction of agouti signal protein and melanocyte stimulating hormone to regulate melanin formation in mammals.
• Pigment Cell Res. 1996; 9: 191-203
• Display abstract

• Important regulatory controls of melanogenesis that operate at the subcellular level to modulate the structural and/or the functional nature of the melanins and melanin granules produced in melanocytes are reviewed. Melanocyte stimulating hormone and agouti signal protein have antagonistic roles and possibly opposing mechanisms of action in the melanocyte. In the mouse, melanocyte stimulating hormone promotes melanogenic enzyme function and elicits increases in the amount of eumelanins produced, while agouti signal protein reduces total melanin production and elicits the synthesis of pheomelanin rather than eumelanin. We are now beginning to understand the complex controls involved in regulating this switch at the molecular and biochemical levels. The quality and quantity of melanins produced by melanocytes have important physiological consequences for melanocyte function and undoubtedly play important roles in the various functions of the melanins per se, including hair and skin coloration and photoprotection.

• Granholm DE, Reese RN, Granholm NH
• Agouti alleles alter cysteine and glutathione concentrations in hair follicles and serum of mice (A y/a, A wJ/A wJ, and a/a).
• J Invest Dermatol. 1996; 106: 559-63
• Display abstract

• Ectopic overexpression of the agouti protein in the lethal yellow (A y/a) mouse causes a yellow coat as well as the lethal yellow syndrome. Presence of thiols like glutathione (GSH) or cysteine (Cys) may regulate the conversion of dopaquinone to phaeomelanin in hair follicle melanocytes. GSH also plays important roles in cellular health and maintenance. Cys and GSH were measured using high-performance liquid chromatography in hair follicles and serum of A wJ/A wJ (agouti), A y/a (yellow), and a/a (black) mice over a 20-d hair growth regeneration period. Agouti alleles modulate thiol concentrations. A y/a hair follicles exhibited higher total thiol levels and an increased ratio of Cys to GSH. A wJ/A wJ mice showed intermediate levels, while a/a mice had lowest total thiol concentrations and a decreased ratio of Cys to GSH. Hair follicle cysteine concentrations showed yellow > agouti > black (p < 0.01). In all genotypes, unplucked skin and day 0 hair follicles showed GSH as the major thiol, but a shift to predominantly Cys on peak melanogenic days was seen. Presence of high concentrations of free cysteine support the hypothesis of phaeomelanin synthesis via cysteinyldopas. The A y/a mouse had the most dramatic follicular thiol changes as well as a depression in serum thiols. An altered thiol metabolism in these and other A y/a tissues might impair normal cell functioning to contribute to the lethal yellow syndrome.

• Barsh GS
• The genetics of pigmentation: from fancy genes to complex traits.
• Trends Genet. 1996; 12: 299-305
• Display abstract

• Genes that control mammalian pigmentation interact with each other in intricate networks that have been studied for decades using mouse coat color mutations. Molecular isolation of the affected genes and the ability to study their effects in a defined genetic background have led to surprising new insights into the potential interaction between tyrosine kinase and G-protein-coupled signaling pathways. Recent developments show that homologous genes in humans are responsible not only for rare diseases, such as albinism and piebaldism, but also for common phenotypic variations, such as red hair and fair skin.

• Argeson AC, Nelson KK, Siracusa LD
• Molecular basis of the pleiotropic phenotype of mice carrying the hypervariable yellow (Ahvy) mutation at the agouti locus.
• Genetics. 1996; 142: 557-67
• Display abstract

• The murine agouti locus regulates a switch in pigment synthesis between eumelanin (black/brown pigment) and phaeomelanin (yellow/red pigment) by hair bulb melanocytes. We recently described a spontaneous mutation, hypervariable yellow (Ahvy) and demonstrated that Ahvy is responsible for the largest range of phenotypes yet identified at the agouti locus, producing mice that are obese with yellow coats to mice that are of normal weight with black coats. Here, we show that agouti expression is altered both temporally and spatially in Ahvy mutants. Agouti expression levels are positively correlated with the degree of yellow pigmentation in individual Ahvy mice, consistent with results from other dominant yellow agouti mutations. Sequencing of 5' RACE and genomic PCR products revealed that Ahvy resulted from the integration of an intracisternal A particle (IAP) in an antisense orientation within the 5' untranslated agouti exon 1C. This retrovirus-like element is responsible for deregulating agouti expression in Ahvy mice; agouti expression is correlated with the methylation state of CpG residues in the IAP long terminal repeat as well as in host genomic DNA. In addition, the data suggest that the variable phenotype of Ahvy offspring is influenced in part by the phenotype of their Ahvy female parent.

• Hunt G, Thody AJ
• Agouti protein can act independently of melanocyte-stimulating hormone to inhibit melanogenesis.
• J Endocrinol. 1995; 147: 14-14
• Display abstract

• In animals, the coat-darkening effects of alpha-melanocyte stimulating hormone (alpha-MSH) are opposed by agouti protein. Although agouti protein has been shown to be a competitive antagonist of the melanocyte-associated MC-1 melanocortin receptor, the possibility that agouti protein can affect melanogenesis independently of its ability to antagonise melanocortin activity cannot be excluded. This study demonstrates that murine agouti protein causes both a time- and concentration-dependent suppression of melanogenesis in B16 F1 murine melanoma cells. In addition, human agouti protein decreases melanogenesis in cultured human epidermal melanocytes. However, agouti protein has little effect on the ability of alpha-MSH to stimulate melanogenesis. These observations raise fundamental questions about the mode of action of agouti protein in regulating melanogenesis.

• Wilson BD, Ollmann MM, Kang L, Stoffel M, Bell GI, Barsh GS
• Structure and function of ASP, the human homolog of the mouse agouti gene.
• Hum Mol Genet. 1995; 4: 223-30
• Display abstract

• The mouse agouti coat color gene encodes a novel paracrine signaling molecule whose pulsatile expression produces a characteristic pattern of banded pigment in individual hairs. Several spontaneous agouti alleles produce adult-onset obesity and diabetes, and have provided important single-gene animal models for alterations in energy metabolism. Utilizing linkage groups conserved between mice and humans, we have cloned the human homolog of the mouse agouti gene from a human chromosome 20 yeast artificial chromosome known to contain S-adenosyl homocysteine hydrolase (AHCY). The human agouti gene, named Agouti Signaling Protein (ASP), encodes a 132 amino acid protein, the mRNA for which is expressed in testis, ovary, and heart, and at lower levels in liver, kidney, and foreskin. As predicted by the interactions of mouse agouti with the extension gene (which encodes the melanocyte receptor for alpha-melanocyte stimulating hormone [alpha-MSH]), expression of ASP in transgenic mice produces a yellow coat, and expression of ASP in cell culture blocks the alpha-MSH-stimulated accumulation of cAMP in mouse melanoma cells. The localization of ASP relative to other loci on chromosome 20 excludes it as a candidate for the MODY1 locus, a gene responsible for one form of early-onset non-insulin-dependent diabetes mellitus or maturity-onset diabetes of the young. The expression of ASP in human tissues suggests a function for agouti homologs in species that do not exhibit the characteristic phenotype of banded hairs.

• Valverde P, Healy E, Jackson I, Rees JL, Thody AJ
• Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair and fair skin in humans.
• Nat Genet. 1995; 11: 328-30
• Display abstract

• Melanin pigmentation protects the skin from the damaging effects of ultraviolet radiation (UVR). There are two types of melanin, the red phaeomelanin and the black eumelanin, both of which are present in human skin. Eumelanin is photoprotective whereas phaeomelanin, because of its potential to generate free radicals in response to UVR, may contribute to UV-induced skin damage. Individuals with red hair have a predominance of phaeomelain in hair and skin and/or a reduced ability to produce eumelanin, which may explain why they fail to tan and are at risk from UVR. In mammals the relative proportions of phaeomelanin and eumelanin are regulated by melanocyte stimulating hormone (MSH), which acts via its receptor (MC1R), on melanocytes, to increase the synthesis of eumelanin and the product of the agouti locus which antagonises this action. In mice, mutations at either the MC1R gene or agouti affect the pattern of melanogenesis resulting in changes in coat colour. We now report the presence of MC1R gene sequence variants in humans. These were found in over 80% of individuals with red hair and/or fair skin that tans poorly but in fewer than 20% of individuals with brown or black hair and in less than 4% of those who showed a good tanning response. Our findings suggest that in humans, as in other mammals, the MC1R is a control point in the regulation of pigmentation phenotype and, more importantly, that variations in this protein are associated with a poor tanning response.

• Takeuchi T, Tamate H, Saijoh Y
• A pleiotropic gene which controls coat color and lethality in early development in the mouse.
• Zoolog Sci. 1995; 12: 675-81

• Granholm DE, Reese RN, Granholm NH
• Agouti alleles influence thiol concentrations in hair follicles and extrafollicular tissues of mice (Ay/a, AwJ/AwJ, a/a).
• Pigment Cell Res. 1995; 8: 302-6
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• Agouti protein (AP) expression in the wild-type agouti mouse (AwJ/AwJ) coincides with a switch in hair follicle melanogenesis from black (eumelanin) to yellow (pheomelanin). Ectopic overexpression of AP in the lethal yellow (Ay/a) mouse cause a pure yellow coat and the lethal yellow syndrome. Thiol concentrations may control the conversion of dopaquinone to pheomelanin in hair follicle melanocytes. Glutathione (GSH) also plays important roles in cellular health and protection. Using HPLC, cysteine and GSH were measured in 1) hair follicles, liver and serum of Ay/a, AwJ/AwJ, and a/a (black) mice, and 2) adipose and spleen tissues of Ay/a and a/a mice on day 9 of regenerating hair growth (late pheomelanin phase). Agouti locus alleles influence thiol metabolism in hair follicles and in other systemic tissues. Ay/a hair follicles and serum showed highest cysteine and lowest GSH levels. AwJ/AwJ mice showed intermediate levels, while a/a hair follicles and serum had lowest cysteine and highest GSH concentrations. In the hair follicle, cysteine (likely derived from enzymatic degradation of GSH) appears to be the primary pheomelanogenic thiol. Agouti locus alleles may also directly or indirectly affect thiol concentrations in systemic tissues like liver and spleen. Cysteine in spleen extracts showed Ay/a > a/a (P < 0.01). An Ay-induced imbalance of thiol metabolism (altering GSH concentrations in multiple tissues) may contribute to the pleiotropic defects of the lethal yellow syndrome.

• Duhl DM, Vrieling H, Miller KA, Wolff GL, Barsh GS
• Neomorphic agouti mutations in obese yellow mice.
• Nat Genet. 1994; 8: 59-65
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• Several dominant mutations of the mouse agouti coat colour gene have pleiotropic effects that include obesity and a yellow coat. The Ay allele is caused by a large deletion that affects the expression of several contiguous genes. We show that three other obesity-associated agouti mutations, Aiy, Asy and Avy, are due to different molecular alterations that result in ubiquitous expression of a chimaeric RNA that encodes a normal agouti protein. The Aiy and Avy alleles are caused by insertion of an intracisternal A particle element 1 kb or 100 kb, respectively, upstream of agouti coding sequences. These results provide a model for other genes that show allele-specific imprinting, and demonstrate that molecular mechanisms typically responsible for activation of proto-oncogenes can also lead to other disease phenotypes.

• Duhl DM et al.
• Pleiotropic effects of the mouse lethal yellow (Ay) mutation explained by deletion of a maternally expressed gene and the simultaneous production of agouti fusion RNAs.
• Development. 1994; 120: 1695-708
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• Heterozygosity for the mouse lethal yellow (Ay) mutation leads to obesity, increased tumor susceptibility and increased activity of the agouti coat color gene; homozygosity for Ay results in embryonic death around the time of implantation. Although these pleiotropic effects have not been separated by recombination, previous studies have suggested that the dominant and recessive effects result from distinct genetic lesions. Here we use a combination of genomic and cDNA cloning experiments to demonstrate that the Ay mutation is caused by a 120 kb deletion which lies centromere-proximal to the agouti coat color gene. The deletion removes coding but not 5' untranslated sequences for a ubiquitously expressed gene predicted to encode a protein similar in sequence to an RNA-binding protein, which we named Merc, for maternally expressed hnRNP C-related gene, but have renamed Raly, since the gene is nearly identical to one reported recently by Michaud et al. (Gene Dev. 7, 1203-1213, 1993). The Ay deletion results in the splicing of Merc/Raly 5' untranslated sequences to agouti protein-coding sequences, which suggests that ectopic expression of the normal agouti protein by the Ay fusion RNA is responsible for the pleiotropic effects associated with heterozygosity for Ay. We find that Merc/Raly RNA is present in the unfertilized egg and is also transcribed in preimplantation embryos. Using a PCR-based assay to determine the genotype of individual embryos from an Ay/a x Ay/a intercross, we show that, in the absence of zygotic Merc/Raly expression, Ay/Ay embryos develop to the blastocyst stage, but do not hatch from the zona pellucida or form trophoblastic outgrowths. Injection of a Merc/Raly antisense oligonucleotide into non-mutant embryos blocks development prior to the blastocyst stage, and can be rescued by coinjection of a Merc/Raly transgene. These results suggest that maternal expression of Merc/Raly plays an important role in preimplantation development and that its deletion of is sufficient to explain Ay-associated embryonic lethality.

• Siracusa LD
• The agouti gene: turned on to yellow.
• Trends Genet. 1994; 10: 423-8
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• The agouti locus was first identified as a result of its effects on the type and temporal deposition of coat color pigments in mammals. Many mutations at the murine agouti locus have now been found, some of which not only affect coat color, but also interfere with diverse biological processes leading to diabetes, obesity, tumor susceptibility and embryonic lethality. Correlations between the genotype and phenotype of agouti mutants, as well as reasons for the pleiotropy of effects caused by agouti mutations, have begun to unfold with the molecular cloning of the agouti gene and its surrounding genomic region.

• Vrieling H, Duhl DM, Millar SE, Miller KA, Barsh GS
• Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene.
• Proc Natl Acad Sci U S A. 1994; 91: 5667-71
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• The agouti coat color gene encodes a paracrine signaling molecule that controls the production of yellow and black pigment by melanocytes within hair follicles. Some agouti alleles affect the dorsum and ventrum independently, which has provided the basis for speculation that agouti gene action in different regions of the body is controlled by distinct genetic loci that are closely linked. Using a combination of cDNA cloning and RNA expression studies, we find that alternative isoforms of agouti mRNA contain different noncoding first exons located 100 kb apart, whose patterns of expression indicate independent control by regulatory elements that are either ventral specific or hair cycle specific. These results demonstrate that the apparent genetic complexity of the agouti locus is explained by the existence of multiple regulatory elements exerting control over a single coding sequence and provide a conceptual basis for understanding differences in dorsal and ventral hair coloration in many mammalian species. The ventral-specific agouti isoform represents an example of a transcript whose expression is restricted to ventral skin and provide an approach to investigate the mechanisms by which dorsal-ventral differences in gene expression are established and maintained.

• Prasolova LA, Bazhan NM, Vsevolodov EB, Latypov IF
• [Verification of the color genotype based on morphological analysis of fur pigmentation of brown and black water voles Arvicola terrestris L.]
• Genetika. 1991; 27: 1423-30
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• Qualitative and quantitative analysis of fur pigmentation in brown and black water voles (Arvicola terrestris L.) was performed. Morphology of pigment granules, their distribution along the hair layers and histology of hair bulbs were studied. Morphological data and the results of the analysis of segregation in the progeny, when brown voles were self-crossed and crossed with black ones, led to conclusion that fur colour of water voles is mediated by genes from the agouti series, precisely, black colour is determined by the extreme non-agouti allele (aeae genotype), and in brown voles which are homo- or heterozygous the colour is determined by the agouti allele (AA or Aae genotypes).

• Matsuzaki T, Yasuda Y, Nonaka S
• The genetics of coat colors in the mongolian gerbil (Meriones unguiculatus).
• Jikken Dobutsu. 1989; 38: 337-41
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• Genetic studies demonstrated three loci controlling coat colors in the Mongolian gerbil. F1 hybrids of white gerbils with red eyes and agouti gerbils with wild coat color had the agouti coat color. The segregating ratio of agouti and white in the F2 generation was 3:1. In the backcross (BC) generation (white x F1), the ratio of the agouti and white coat colors was 1:1. Next, inheritance of the agouti coat color was investigated. Matings between agouti and non-agouti (black) gerbils produced only agouti gerbils. In the F2 generation, the ratio of agouti to non-agouti (black) was 3:1. There was no distortion in the sex ratios within each coat color in the F1, F2 and BC generations. This indicated that the white coat color of gerbils is governed by an autosomal recessive gene which should be named the c allele of the c (albino) locus controlling pigmentation, and the agouti coat color is controlled by an autosomal dominant gene which might be named the A allele of the A (agouti) locus controlling pigmentation patterns in the hair. The occurrence of the black gerbil demonstrated clearly the existence of the b (brown) locus, and it clearly indicated that the coat colors of gerbils can basically be explained by a, b, and c loci as in mice and rats.

• Robinson R
• The agouti alleles of Peromyscus.
• J Hered. 1981; 72: 132-132

• Galbraith DB
• Expression of genes at the agouti locus and mitotic activity of the hair bulb of the mouse.
• Genetics. 1971; 67: 559-68

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