Genetic and biochemical studies of the Agouti-attractin system

Pleiotropic effects of melanocortin signaling were first described nearly 100 years ago when mice carrying the lethal yellow (A(y)) allele of the Agouti coat color gene were recognized to develop increased growth and adiposity. Work from our laboratory and others over the last several years has demonstrated that the non-pigmentary effects of A(y) are caused by ectopic expression of Agouti protein, a paracrine signaling molecule whose normal function is to inhibit signaling through the melanocortin 1 receptor (Mc1r), but which can mimic the effects of Agouti-related protein (Agrp), a homologous neuropeptide produced in the medial portion of the arcuate nucleus that acts as a potent antagonist of the Mc3r and Mc4r. Recently we have used the genetics of pigmentation as an in vivo screening system to analyze other mutations in the Agouti-melanocortin pathway, leading to the identification of Attractin (Atrn), a widely expressed type I transmembrane protein that serves as an accessory receptor for Agouti protein. Surprisingly, homologs of Atrn are found in fruitflies and nematodes, even though Agouti and/or Agouti-related protein are found only in vertebrates. Insight into this apparent paradox now comes from studies of different Atrn alleles, in which we find hyperactivity, abnormal myelination, and widespread CNS vacuolation. We suggest that the neurodegenerative phenotype reflects the ancestral function of Atrn to facilitate and/or maintain cell-cell interactions in the nervous system. Expression in neurectodermal cells during vertebrate evolution may have allowed Atrn to be recruited by the Agouti-melanocortin system to control coat color.     

Genetic Studies of the Mouse Mutations Mahogany and Mahoganoid

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 switch 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 α-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, Mc1r(e)) or a gain-of-function Agouti mutation (lethal yellow, A(y)). We found that the Mc1r(e) mutation suppressed the effects of md and mg, but that md and mg suppressed the effects of A(y) on both coat color and obesity. Plasma levels of α-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.     

Melanocortin 1 receptor variation in the domestic dog

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.     

Exclusion of melanocortin-1 receptor (mc1r) and agouti as candidates for dominant black in dogs

The domestic dog exhibits a variety of coat colors that encompass a wide range of variation among different breeds. Very little is known about the molecular biology of dog pigmentation; current understanding is based mostly on traditional breeding experiments, which in some cases have suggested genetic interactions that are different from those reported in other mammals. We have examined the molecular genetics of dominant black, a uniform coat color characteristic of black Labrador retrievers or Newfoundlands that has been proposed to be caused by either variation in the melanocortin-1 receptor gene (Mc1r) or by variation in the Agouti gene (A). We identified several coding polymorphisms within Mc1r and several simple sequence repeat polymorphisms closely linked to A, and examined their inheritance in a Labrador retriever x greyhound cross that segregates dominant black. No single Mc1r allele was found consistently in animals carrying dominant black, and neither Mc1r nor A cosegregated with dominant black. These results refine our understanding of mammalian coat color inheritance and suggest that dominant black coat color in dogs is caused by a gene not previously implicated in pigment type switching.     

Agouti: from mouse to man, from skin to fat

The agouti protein regulates pigmentation in the mouse hair follicle producing a black hair with a subapical yellow band. Its effect on pigmentation is achieved by antagonizing the binding of alpha-melanocyte stimulating hormone (alpha-MSH) to melanocortin 1 receptor (Mc1r), switching melanin synthesis from eumelanin (black/brown) to phaeomelanin (red/yellow). Dominant mutations in the non-coding region of mouse agouti cause yellow coat colour and ectopic expression also results in obesity, type 11 diabetes, increased somatic growth and tumourigenesis. At least some of these pleiotropic effects can be explained by antagonism of other members of the melanocortin receptor family by agouti protein. The yellow coat colour is the result of agouti chronically antagonizing the binding of alpha-MSH to Mc1r and the obese phenotype results from agouti protein antagonizing the binding of alpha-MSH to Mc3r and/or Mc4r. Despite the existence of a highly homologous agouti protein in humans, agouti signal protein (ASIP), its role has yet to be defined. However it is known that human ASIP is expressed at highest levels in adipose tissue where it may antagonize one of the melanocortin receptors. The conserved nature of the agouti protein combined with the diverse phenotypic effects of agouti mutations in mouse and the different expression patterns of human and mouse agouti, suggest ASIP may play a role in human energy homeostasis and possibly human pigmentation.     

Skin layer-specific transcriptional profiles in normal and recessive yellow (Mc1re/Mc1re) mice

The melanocortin 1 receptor (Mc1r) plays a central role in cutaneous biology, but is expressed at very low levels by a small fraction of cells in the skin. In humans, loss-of-function MC1R mutations cause fair skin, freckling, red hair, and increased predisposition to melanoma; in mice, Mc1r loss-of-function is responsible for the recessive yellow mutation, associated with pheomelanic hair and a decreased number of epidermal melanocytes. To better understand how Mc1r signaling affects different cutaneous phenotypes, we examined large-scale patterns of gene expression in different skin components (whole epidermal sheets, basal epidermal cells and whole skins) of neonatal (P2.5) normal and recessive yellow mice, starting with a 26K mouse cDNA microarray. From c. 17 000 genes whose levels could be accurately measured in neonatal skin, we identified 883, 2097 and 552 genes that were uniquely expressed in the suprabasal epidermis, basal epidermis and dermis, respectively; specific biologic roles could be assigned for each class. Comparison of normal and recessive yellow mice revealed 69 differentially expressed genes, of which the majority had not been previously implicated in Mc1r signaling. Surprisingly, many of the Mc1r-dependent genes are expressed in cells other than melanocytes, even though Mc1r expression in the skin is confined almost exclusively to epidermal melanocytes. These results reveal new targets for Mc1r signaling, and point to a previously unappreciated role for a Mc1r-dependent paracrine effect of melanocytes on other components of the skin.     

GENETIC AND BIOCHEMICAL STUDIES OF THE AGOUTI–ATTRACTIN SYSTEM

ABSTRACT

Pleiotropic effects of melanocortin signaling were first described nearly 100 years ago when mice carrying the lethal yellow (A^y) allele of the Agouti coat color gene were recognized to develop increased growth and adiposity. Work from our laboratory and others over the last several years has demonstrated that the non-pigmentary effects of A^?y are caused by ectopic expression of Agouti protein, a paracrine signaling molecule whose normal function is to inhibit signaling through the melanocortin 1 receptor (Mc1r), but which can mimic the effects of Agouti-related protein (Agrp), a homologous neuropeptide produced in the medial portion of the arcuate nucleus that acts as a potent antagonist of the Mc3r and Mc4r. Recently we have used the genetics of pigmentation as an in vivo screening system to analyze other mutations in the Agouti–melanocortin pathway, leading to the identification of Attractin (Atrn), a widely expressed type I transmembrane protein that serves as an accessory receptor for Agouti protein. Surprisingly, homologs of Atrn are found in fruitflies and nematodes, even though Agouti and/or Agouti-related protein are found only in vertebrates. Insight into this apparent paradox now comes from studies of different Atrn alleles, in which we find hyperactivity, abnormal myelination, and widespread CNS vacuolation. We suggest that the neurodegenerative phenotype reflects the ancestral function of Atrn to facilitate and/or maintain cell–cell interactions in the nervous system. Expression in neurectodermal cells during vertebrate evolution may have allowed Atrn to be recruited by the Agouti–melanocortin system to control coat color.     

Excess tyrosine rescues the reduced activity of proliferation and differentiation of cultured recessive yellow melanocytes derived from neonatal mouse epidermis

The murine recessive yellow (Mc1r(e)) is a loss-of-function mutation in the receptor for alpha-melanocyte-stimulating hormone, melanocortin receptor 1 (Mc1r) and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. However, it is not known whether the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes. In this study, the proliferation and differentiation of recessive yellow epidermal melanocytes cultured in dibutyryl cyclic AMP-supplemented serum-free medium were investigated in detail. The melanocytes produced mainly eumelanin in this culture system. The proliferation of recessive yellow melanocytes was decreased compared with that of wild-type at the e-locus, black melanocytes. The differentiation of melanocytes was also delayed and inhibited in recessive yellow mice. Tyrosinase (TYR) activity and TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT) expressions were decreased and, in addition, the maturation of stage IV melanosomes was inhibited. Excess l-tyrosine (l-Tyr) added to the culture media rescued the reduced activity of proliferation of melanocytes. l-Tyr also stimulated TYR activity and TRP1 and TRP2 expressions as well as the maturation of stage IV melanosomes and pigmentation. These results suggest that the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes and l-Tyr rescues the reduced proliferative and differentiative activities by stimulating TYR activity and TRP1 and TRP2 expressions as well as melanosome maturation.     

A Transgenic Mouse Assay for Agouti Protein Activity

The mouse agouti gene encodes an 131 amino acid paracrine signaling molecule that instructs hair follicle melanocytes to switch from making black to yellow pigment. Expression of agouti during the middle part of the hair growth cycle in wild-type mice produces a yellow band on an otherwise black hair. The ubiquitous unregulated expression of agouti in mice carrying dominant yellow alleles is associated with pleiotropic effects including increased yellow pigment in the coat, obesity, diabetes and increased tumor susceptibility. Agouti shows no significant homology to known genes, and the molecular analysis of agouti alleles has shed little new light on the important functional elements of the agouti protein. In this paper, we show that agouti expression driven by the human β-ACTIN promoter produces obese yellow transgenic mice and that this can be used as an assay for agouti activity. We used this assay to evaluate a point mutation associated with the a(16H) allele within the region encoding agouti's putative signal sequence and our results suggest that this mutation is sufficient to cause the a(16H) phenotype. Thus, in vitro mutagenesis followed by the generation of transgenic mice should allow us to identify important functional elements of the agouti protein.     

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a G_s-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection ¹. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.     

Coat colours in the Massese sheep breed are associated with mutations in the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes

Massese is an Italian dairy sheep breed characterized by animals with black skin and horns and black or apparent grey hairs. Owing to the presence of these two coat colour types, this breed can be considered an interesting model to evaluate the effects of coat colour gene polymorphisms on this phenotypic trait. Two main loci have been already shown to affect coat colour in sheep: Agouti and Extension coding for the agouti signalling protein (ASIP) and melanocortin 1 receptor (MC1R) genes, respectively. The Agouti locus is affected by a large duplication including the ASIP gene that may determine the Agouti white and tan allele (A(Wt)). Other disrupting or partially inactivating mutations have been identified in exon 2 (a deletion of 5 bp, D(5); and a deletion of 9 bp, D(9)) and in exon 4 (g.5172T>A, p.C126S) of the ASIP gene. Three missense mutations in the sheep MC1R gene cause the dominant black E(D) allele (p.M73K and p.D121N) and the putative recessive e allele (p.R67C). Here, we analysed these ASIP and MC1R mutations in 161 Massese sheep collected from four flocks. The presence of one duplicated copy allele including the ASIP gene was associated with grey coat colour (P = 9.4E-30). Almost all animals with a duplicated copy allele (37 out of 41) showed uniform apparent grey hair and almost all animals without a duplicated allele (117 out of 120) were completely black. Different forms of duplicated alleles were identified in Massese sheep including, in almost all cases, copies with exon 2 disrupting or partially inactivating mutations making these alleles different from the A(Wt) allele. A few exceptions were observed in the association between ASIP polymorphisms and coat colour: three grey sheep did not carry any duplicated copy allele and four black animals carried a duplicated copy allele. Of the latter four sheep, two carried the E(D) allele of the MC1R gene that may be the cause of their black coat colour. The coat colour of all other black animals may be determined by non-functional ASIP alleles (non-agouti alleles, A(a)) and in a few cases by the E(D) Extension allele. At least three frequent ASIP haplotypes ([D(5):g.5172T], [N:g.5172A] and [D(5):g.5172A]) were detected (organized into six different diplotypes). In conclusion, the results indicated that coat colours in the Massese sheep breed are mainly derived by combining ASIP and MC1R mutations.     

The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity

The yellow mouse obesity syndrome is due to dominant mutations at the Agouti locus, which is characterized by obesity, hyperinsulinemia, insulin resistance, hyperglycemia, hyperleptinemia, increased linear growth, and yellow coat color. This syndrome is caused by ectopic expression of Agouti in multiple tissues. Mechanisms of Agouti action in obesity seem to involve, at least in part, competitive melanocortin antagonism. Both central and peripheral effects have been implicated in Agouti-induced obesity. An Agouti-Related Protein (AGRP) has been described recently. It has been shown to be expressed in mice hypothalamus and to act similarly to agouti as a potent antagonist to central melanocortin receptor MC4-R, suggesting that AGRP is an endogenous MC4-R ligand. Mice lacking MC4-R become hyperphagic and develop obesity, implying that agouti may lead to obesity by interfering with MC4-R signaling in the brain and consequently regulating food intake. Furthermore, food intake is inhibited by intracerebro-ventricular injection of a potent melanocortin agonist and was reversed by administration of an MC4-R antagonist. The direct cellular actions of Agouti include stimulation of fatty acid and triglyceride synthesis via a Ca²⁺-dependent mechanism. Agouti and insulin act in an additive manner to increase lipogenesis. This additive effect of agouti and insulin is demonstrated by the necessity of insulin in eliciting weight gain in transgenic mice expressing agouti specifically in adipose tissue. This suggests that agouti expression in adipose tissue combined with hyperinsulinemia may lead to increased adiposity. The roles of melanocortin receptors or agouti-specific receptor(s) in agouti regulation of adipocyte metabolism and other peripheral effects remain to be determined. In conclusion, both central and peripheral actions of agouti contribute to the yellow mouse obesity syndrome and this action is mediated at least in part by antagonism with melanocortin receptors and/or regulation of intracellular calcium.     

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

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

Shades of meaning: the pigment-type switching system as a tool for discovery

The pigment-type switching system, which controls whether melanocytes produce black/brown eumelanin or yellow/red pheomelanin, is responsible for many familiar coat coloration patterns in both domestic and wild mammals. In conjunction with the accessory proteins attractin and mahogunin ring finger 1, endogenous agonists and antagonists modulate signaling by the melanocortin 1 receptor to determine pigment type. Mutations in pigment-type switching genes can cause a variety of pleiotropic phenotypes, and these are often similar between mutants at different loci because the proteins encoded by these genes act together as part of conserved molecular pathways that are deployed in multiple biological contexts. When this is the case, pigment-type switching provides a powerful model system for elucidating the shared molecular mechanisms underlying the pigmentary and non-pigmentary phenotypes. This review outlines the current understanding of the pigment-type switching pathway and discusses the opportunities that exist for exploring the molecular basis of pleiotropic phenotypes using this model system.     

Skin layer‐specific transcriptional profiles in normal and recessive yellow (Mc1re/Mc1re) mice

The melanocortin 1 receptor (Mc1r) plays a central role in cutaneous biology, but is expressed at very low levels by a small fraction of cells in the skin. In humans, loss‐of‐function MC1R mutations cause fair skin, freckling, red hair, and increased predisposition to melanoma; in mice, Mc1r loss‐of‐function is responsible for the recessive yellow mutation, associated with pheomelanic hair and a decreased number of epidermal melanocytes. To better understand how Mc1r signaling affects different cutaneous phenotypes, we examined large‐scale patterns of gene expression in different skin components (whole epidermal sheets, basal epidermal cells and whole skins) of neonatal (P2.5) normal and recessive yellow mice, starting with a 26K mouse cDNA microarray. From c. 17 000 genes whose levels could be accurately measured in neonatal skin, we identified 883, 2097 and 552 genes that were uniquely expressed in the suprabasal epidermis, basal epidermis and dermis, respectively; specific biologic roles could be assigned for each class. Comparison of normal and recessive yellow mice revealed 69 differentially expressed genes, of which the majority had not been previously implicated in Mc1r signaling. Surprisingly, many of the Mc1r‐dependent genes are expressed in cells other than melanocytes, even though Mc1r expression in the skin is confined almost exclusively to epidermal melanocytes. These results reveal new targets for Mc1r signaling, and point to a previously unappreciated role for a Mc1r‐dependent paracrine effect of melanocytes on other components of the skin.     

Female Fertility and Mating Type Effects on Effective Population Size and Evolution in Filamentous Fungi

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 (A(hvy)) and demonstrated that A(hvy) 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 A(hvy) mutants. Agouti expression levels are positively correlated with the degree of yellow pigmentation in individual A(hvy) mice, consistent with results from other dominant yellow agouti mutations. Sequencing of 5' RACE and genomic PCR products revealed that A(hvy) 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 A(hvy) 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 A(hvy) offspring is influenced in part by the phenotype of their A(hvy) female parent.     

Tissue microenvironment and the genetic control of hair pigment patterns in mice

This study was conducted to assess microenvironmental variability within integumental tissue of genetically identical mice with respect to a specific cellular response: cyclic synthesis of yellow and black pigment by hair bulb melanocytes. Crosses were performed within and between inbred strains of mice that were isogenic with the exception of a single gene substitution at the agouti locus. Agouti locus genes included the A^vy, A^w, A, a^td, a^t, a^x, a^m, and a alleles. The pigment patterns of dorsal, flank, and ventral hairs of the first and third hair generations and of hairs growing in special integumentary areas such as the pinna, tail, and hind foot were studied. It was found that the amount of yellow pigment synthesized by hair bulb melanocytes within genetically identical mice is both agedependent and conditioned by the integumentary environment. Furthermore, the special integumentary regions produce hairs with a variety of pigment patterns in which the distribution and relative amounts of black and yellow pigments do not necessarily conform to dominance relationships expected among agouti locus alleles as judged by their effects on the pigmentation of the dorsal pelage. We conclude that within genetically uniform integumental tissues, microenvironmental differences occur and are reflected as alterations in the metabolic pattern of differentiated cells.