The mahoganoid mutation (Mgrn1md) improves insulin sensitivity in mice with mutations in the melanocortin signaling pathway independently of effects on adiposity

Mahoganoid (Mgrn1(md)) is a mutation of the mahogunin (Mgrn1) gene. The hypomorphic allele suppresses the yellow pigmentation and obesity of the A(y) mouse that ubiquitously overexpresses agouti signaling protein (ASP). To assess the physiological effects of MGRN1 on energy and glucose homeostasis, we generated animals doubly mutant for Mgrn1(md) and A(y), Lep(ob), or a null allele of Mc4r, and diet-induced obesity (DIO) mice segregating for Mgrn1(md). Mgrn1(md) suppressed the obesity, hyperglycemia, and hyperinsulinemia of A(y) mice. Mgrn1(md) suppressed A(y)-induced obesity by reducing food intake, and reduced adiposity in Lep(ob)/Lep(ob) females, but did not alter the body weight or body composition of mice fed a high-fat diet. There was no effect of Mgrn1(md) on weight gain, body composition, energy intake, or energy expenditure in Mc4r-null animals. Mgrn1(md) reduced circulating insulin concentrations in DIO, A(y), and Mc4r-null but not Lep(ob)/Lep(ob) mice. The effect of Mgrn1(md) on circulating insulin concentrations was not due primarily to reductions in fat mass, since the plasma insulin concentrations of Mgrn1(md) mice segregating for either A(y) or Mc4r-null alleles, adjusted for fat mass and plasma glucose, were reduced compared with A(y) and Mc4r mice, respectively. The effect of Mgrn1(md) on insulin sensitivity of Mc4r-null mice suggests that Mgrn1(md) may be increasing insulin sensitivity via the hypothalamic melanocortin-3 receptor pathway.     

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.     

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.     

Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of α-melanocyte-stimulating hormone (α-MSH) to the α-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro site-directed mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors.     

Linkage and segregation analysis of black and brindle coat color in domestic dogs

Mutations of pigment type switching have provided basic insight into melanocortin physiology and evolutionary adaptation. In all vertebrates that have been studied to date, two key genes, Agouti and Melanocortin 1 receptor (Mc1r), encode a ligand-receptor system that controls the switch between synthesis of red-yellow pheomelanin vs. black-brown eumelanin. However, in domestic dogs, historical studies based on pedigree and segregation analysis have suggested that the pigment type-switching system is more complicated and fundamentally different from other mammals. Using a genomewide linkage scan on a Labrador x greyhound cross segregating for black, yellow, and brindle coat colors, we demonstrate that pigment type switching is controlled by an additional gene, the K locus. Our results reveal three alleles with a dominance order of black (K(B)) > brindle (k(br)) > yellow (k(y)), whose genetic map position on dog chromosome 16 is distinct from the predicted location of other pigmentation genes. Interaction studies reveal that Mc1r is epistatic to variation at Agouti or K and that the epistatic relationship between Agouti and K depends on the alleles being tested. These findings suggest a molecular model for a new component of the melanocortin signaling pathway and reveal how coat-color patterns and pigmentary diversity have been shaped by recent selection.     

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.     

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.     

Independent regulation of hair and skin color by two G protein‐coupled pathways

Hair color and skin color are frequently coordinated in mammalian species. To explore this, we have studied mutations in two different G protein coupled pathways, each of which affects the darkness of both hair and skin color. In each mouse mutant (Gnaq^Dsk1, Gna11^Dsk7, and Mc1r^e), we analyzed the melanocyte density and the concentrations of eumelanin (black pigment) and pheomelanin (yellow pigment) in the hair or skin to determine the mechanisms regulating pigmentation. Surprisingly, we discovered that each mutation affects hair and skin color differently. Furthermore, we have found that in the epidermis, the melanocortin signaling pathway does not couple the synthesis of eumelanin with pheomelanin, as it does in hair follicles. Even by shared signaling pathways, hair and skin melanocytes are regulated quite independently.     

Adaptive variation in beach mice produced by two interacting pigmentation genes

Little is known about the genetic basis of ecologically important morphological variation such as the diverse color patterns of mammals. Here we identify genetic changes contributing to an adaptive difference in color pattern between two subspecies of oldfield mice (Peromyscus polionotus). One mainland subspecies has a cryptic dark brown dorsal coat, while a younger beach-dwelling subspecies has a lighter coat produced by natural selection for camouflage on pale coastal sand dunes. Using genome-wide linkage mapping, we identified three chromosomal regions (two of major and one of minor effect) associated with differences in pigmentation traits. Two candidate genes, the melanocortin-1 receptor (Mc1r) and its antagonist, the Agouti signaling protein (Agouti), map to independent regions that together are responsible for most of the difference in pigmentation between subspecies. A derived mutation in the coding region of Mc1r, rather than change in its expression level, contributes to light pigmentation. Conversely, beach mice have a derived increase in Agouti mRNA expression but no changes in protein sequence. These two genes also interact epistatically: the phenotypic effects of Mc1r are visible only in genetic backgrounds containing the derived Agouti allele. These results demonstrate that cryptic coloration can be based largely on a few interacting genes of major effect.     

Agouti protein,mahogunin, and attractin in pheomelanogenesis and melanoblast‐like alteration of melanocytes: a cAMP‐independent pathway

Melanocortin‐1 receptor (MC1R) and its ligands, α‐melanocyte stimulating hormone (αMSH) and agouti signaling protein (ASIP), regulate switching between eumelanin and pheomelanin synthesis in melanocytes. Here we investigated biological effects and signaling pathways of ASIP. Melan‐a non agouti (a/a) mouse melanocytes produce mainly eumelanin, but ASIP combined with phenylthiourea and extra cysteine could induce over 200‐fold increases in the pheomelanin to eumelanin ratio, and a tan‐yellow color in pelletted cells. Moreover, ASIP‐treated cells showed reduced proliferation and a melanoblast‐like appearance, seen also in melanocyte lines from yellow (A^y/a and Mc1r^e/ Mc1r^e) mice. However ASIP‐YY, a C‐terminal fragment of ASIP, induced neither biological nor pigmentary changes. As, like ASIP, ASIP‐YY inhibited the cAMP rise induced by αMSH analog NDP‐MSH, and reduced cAMP level without added MSH, the morphological changes and depigmentation seemed independent of cAMP signaling. Melanocytes genetically null for ASIP mediators attractin or mahogunin (Atrn^{mg‐3J/mg‐3J} or Mgrn1^{md‐nc/md‐nc}) also responded to both ASIP and ASIP‐YY in cAMP level, while only ASIP altered their proliferation and (in part) shape. Thus, ASIP–MC1R signaling includes a cAMP‐independent pathway through attractin and mahogunin, while the known cAMP‐dependent component requires neither attractin nor mahogunin.     

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.     

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.     

Distribution of Mahogany/Attractin mRNA in the rat central nervous system

The Mahogany/Attractin gene (Atrn) has been proposed as a downstream mediator of Agouti signaling because yellow hair color and obesity in lethal yellow (A(y)) mice are suppressed by the mahogany (Atrn(mg)) mutation. The present study examined the distribution of Atrn mRNA in the brain and spinal cord by in situ hybridization. Atrn mRNA was found widely distributed throughout the central nervous system, with high levels in regions of the olfactory system, some limbic structures, regions of the brainstem, cerebellum and spinal cord. In the hypothalamus, Atrn mRNA was found in specific nuclei including the suprachiasmatic nucleus, the supraoptic nucleus, the medial preoptic nucleus, the paraventricular hypothalamic nucleus, the ventromedial hypothalamic nucleus, and the arcuate nucleus. These results suggest a broad spectrum of physiological functions for the Atrn gene product.     

A first genotyping assay of French cattle breeds based on a new allele of the extension gene encoding the melanocortin-1 receptor (Mc1r)

The seven transmembrane domain melanocortin-1 receptor (Mc1r) encoded by the coat color extension gene (E) plays a key role in the signaling pathway of melanin synthesis. Upon the binding of agonist (melanocortin hormone, α-MSH) or antagonist (Agouti protein) ligands, the melanosomal synthesis of eumelanin and/or phaeomelanin pigments is stimulated or inhibited, respectively. Different alleles of the extension gene were cloned from unrelated animals belonging to French cattle breeds and sequenced. The wild type E allele was mainly present in Normande cattle, the dominant E^D allele in animals with black color (i.e. Holstein), whereas the recessive e allele was identified in homozygous animals exhibiting a more or less strong red coat color (Blonde d''Aquitaine, Charolaise, Limousine and Salers). A new allele, named E¹, was found in either homozygous (E¹/E¹) or heterozygous (E¹/E) individuals in Aubrac and Gasconne breeds. This allele displayed a 4 amino acid duplication (12 nucleotides) located within the third cytoplasmic loop of the receptor, a region known to interact with G proteins. A first genotyping assay of the main French cattle breeds is described based on these four extension alleles.     

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.     

Agonist-independent, high constitutive activity of the human melanocortin 1 receptor

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.     

Point mutations in the melanocortin-4 receptor cause variable obesity in mice

Mutations in the melanocortin-4 receptor (MC4R) are associated with early-onset obesity in humans. Furthermore, a null Mc4r allele in mice leads to severe obesity due to hyperphagia and decreased energy expenditure. As part of independent N-ethyl- N-nitrosourea (ENU) mutagenesis screens, two obesity mutants, Fatboy and Southbeach, were isolated. Mapping revealed linkage to the melanocortin-4 receptor (Mc4r) and sequencing found single amino acid changes in Mc4r for each line. Expression of the mutant receptors in HEK 293 cells revealed defects in receptor signaling. The mutated Fatboy receptor (I194T) shows an increase in the effective concentration necessary for 50% of maximal signaling (EC₅₀) when stimulated with α-MSH. Based on competitive binding, I194T is expressed on the cell surface at lower levels than the nonmutated receptor. In contrast, Southbeach (L300P) displays minimal receptor signaling when stimulated with the natural ligand α-MSH or the synthetic agonist NDP-α-MSH. Cell surface binding is absent, which usually indicates a lack of cell surface expression. However, antibody binding to Flag-tagged receptors by flow cytometry analysis and immunofluorescence demonstrates that L300P is translocated to the plasma membrane at a level comparable to the wild-type receptor. These results indicate a correlation with remaining receptor activity and the severity of the obesity in the mice homozygous for the mutations. Southbeach has less receptor activity and becomes more obese. These mutants will serve as good models for the variability in phenotype in humans carrying mutations in the MC4R gene.