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.     

A polymorphism in the agouti signaling protein gene is associated with human pigmentation

In mice and humans, binding of alpha-melanocyte--stimulating hormone to the melanocyte-stimulating--hormone receptor (MSHR), the protein product of melanocortin-1 receptor (MC1R) gene, leads to the synthesis of eumelanin. In the mouse, ligation of MSHR by agouti signaling protein (ASP) results in the production of pheomelanin. The role of ASP in humans is unclear. We sought to characterize the agouti signaling protein gene (ASIP) in a group of white subjects, to assess whether ASIP was a determinant of human pigmentation and whether this gene may be associated with increased melanoma risk. We found no evidence of coding-region sequence variation in ASIP, but detected a g.8818A-->G polymorphism in the 3' untranslated region. We genotyped 746 participants in a study of melanoma susceptibility for g.8818A-->G, by means of polymerase chain reaction and restriction fragment--length polymorphism analysis. Among the 147 healthy controls, the frequency of the G allele was.12. Carriage of the G allele was significantly associated with dark hair (odds ratio 1.8; 95% confidence interval [CI] 1.2--2.8) and brown eyes (odds ratio 1.9; 95% CI 1.3--2.8) after adjusting for age, gender, and disease status. ASIP g.8818A-->G was not associated independently with disease status. This is the first report of an association of ASIP with specific human pigmentation characteristics. It remains to be investigated whether the interaction of MC1R and ASIP can enhance prediction of human pigmentation and melanoma risk.     

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.     

Loss‐of‐function mutations in the melanocortin 1 receptor cause disruption of dorso‐ventral countershading in teleost fish

The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in vertebrate pigmentation. Mutations in this gene cause variations in coat coloration in amniotes. Additionally, in mammals MC1R is the main receptor for agouti‐signaling protein (ASIP), making it the critical receptor for the establishment of dorsal‐ventral countershading. In fish, Mc1r is also involved in pigmentation, but it has been almost exclusively studied in relation to melanosome dispersion activity and as a putative genetic factor involved in dark/light adaptation. However, its role as the crucial component for the Asip1‐dependent control of dorsal‐ventral pigmentation remains unexplored. Using CRISPR/Cas9, we created mc1r homozygous knockout zebrafish and found that loss‐of‐function of mc1r causes a reduction of countershading and a general paling of the animals. We find ectopic development of melanophores and xanthophores, accompanied by a decrease in iridophore numbers in the ventral region of mc1r mutants. We also reveal subtle differences in the role of mc1r in repressing pigment cell development between the skin and scale niches in ventral regions.     

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

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

Human hair melanins: what we have learned and have not learned from mouse coat color pigmentation

Hair pigmentation is one of the most conspicuous phenotypes in humans. Melanocytes produce two distinct types of melanin pigment: brown to black, indolic eumelanin and yellow to reddish brown, sulfur‐containing pheomelanin. Biochemically, the precursor tyrosine and the key enzyme tyrosinase and the tyrosinase‐related proteins are involved in eumelanogenesis, while only the additional presence of cysteine is necessary for pheomelanogenesis. Other important proteins involved in melanogenesis include P protein, MATP protein, α‐MSH, agouti signaling protein (ASIP), MC1R (the receptor for MSH and ASIP), and SLC7A11, a cystine transporter. Many studies have examined the effects of loss‐of‐function mutations of those proteins on mouse coat color pigmentation. In contrast, much less is known regarding the effects of mutations of the corresponding proteins on human hair pigmentation except for MC1R polymorphisms that lead to pheomelanogenesis. This perspective will discuss what we have/have not learned from mouse coat color pigmentation, with special emphasis on the significant roles of pH and the level of cysteine in melanosomes in controlling melanogenesis. Based on these data, a hypothesis is proposed to explain the diversity of human hair pigmentation.     

Agouti signalling protein is an inverse agonist to the wildtype and agonist to the melanic variant of the melanocortin-1 receptor in the grey squirrel (Sciurus carolinensis)

The melanocortin-1 receptor (MC1R) is a key regulator of mammalian pigmentation. Melanism in the grey squirrel is associated with an eight amino acid deletion in the mutant melanocortin-1 receptor with 24 base pair deletion (MC1RΔ24) variant. We demonstrate that the MC1RΔ24 exhibits a higher basal activity than the wildtype MC1R (MC1R-wt). We demonstrate that agouti signalling protein (ASIP) is an inverse agonist to the MC1R-wt but is an agonist to the MC1RΔ24. We conclude that the deletion in the MC1RΔ24 leads to a receptor with a high basal activity which is further activated by ASIP. This is the first report of ASIP acting as an agonist to MC1R.     

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.     

Interaction of melanin-concentrating hormone (MCH), neuropeptide E-I (NEI), neuropeptide G-E (NGE), and alpha-MSH with melanocortin and MCH receptors on mouse B16 melanoma cells.

Melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between alpha-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-Rpc) and the different melanocortin (MC) receptors. Radioreceptor assays using [125I]MCH, [125l]alpha-MSH and [125I]NEI as radioligands and bioassays were performed with MCI-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC50s of alpha-MSH and NEI or NGE for [125I]MCH displacement from mouse MCH-Rpc were 80-fold and, respectively, >300-fold higher than that of MCH, and the IC50s for MCH and NEI or NGE for [125I]alpha-MSH displacement from mouse MC1-R were 50,000-fold and >200,000-fold higher than that of alpha-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [1251]alpha-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 microM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.     

Role of G protein-coupled receptor kinases in the homologous desensitization of the human and mouse melanocortin 1 receptors

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.     

Antagonist and agonist activities of the mouse agouti protein fragment (91-131) at the melanocortin-1 receptor.

Antagonist and agonist activities of chemically synthetized mouse agouti protein fragment (91-131) (AP91-131) at the melanocortin type-1 receptor (MC1-R) were assessed using B 16-F1 mouse melanoma cells in vitro and the following assay systems: (i) receptor binding, (ii) adenylate cyclase, (iii) tyrosinase, (iv) melanin production, and (v) cell proliferation. In competition binding studies AP91-131 was about 3-fold less potent than the natural agonist alpha-melanocyte-stimulating hormone (alpha-MSH) in displacing the radioligand [125I]-[Nle4, D-Phe7]-alpha-MSH (Ki 6.5 +/- 0.8 nmol/l). Alpha-MSH-induced tyrosinase activation and melanin production were completely inhibited by a 100-fold higher concentration of AP9 l -131; the IC50 values for AP91-131 in thetwo assay systems were 91 +/- 22 nM and 95 +/- 15 nM respectively. Basal melanin production and adenylate cyclase activity in the absence of agonist were decreased by AP91-131 with IC50 values of 9.6+/-1.8 nM and 5.0+/-2.4 nM, respectively. This indicates inverse agonist activity of AP91-131 similar to that of native AP. The presence of 10 nM melanin-concentrating hormone (MCH) slightly potentiated the inhibitory activity of AP91-131 in the adenylate cyclase and melanin assays. On the other hand, AP91-131 inhibited cell growth similar to alpha-MSH (IC50 11.0 +/- 2.1 nM; maximal inhibition 1.8-fold higher than that of alpha-MSH). Furthermore, MC1-R was down-regulated by AP91-131 with about the same potency and time-course as with alpha-MSH. These results demonstrate that AP91-131 displays both agonist and antagonist activities at the MC1-R and hence that it is the cysteine-rich region of agouti protein which inhibits and mimics the different alpha-MSH functions, most likely by simultaneous modulation of different intracellular signalling pathways.     

Differences in the expression of the ASIP gene are involved in the recessive black coat colour pattern in sheep: evidence from the rare Xalda sheep breed

Here we have tested the hypothesis of association between different levels of agouti signalling peptide (ASIP) mRNA and the recessive black coat colour in the rare Xalda breed of sheep. To deal with this task, we first tested the possible action of both the dominant black extension allele (E(D)) and a 5-bp deletion (X99692:c.100_104del; A(del)) in the ovine ASIP coding sequence on the black coat colour pattern in 188 Xalda individuals. The E(D) allele was not present in the sample and only 11 individuals were homozygous for the A(del)ASIP allele. All Xalda individuals carrying the A(del)/A(del) genotype were phenotypically black. However, most black-coated individuals (109 out of 120) were not homozygous for the 5-bp deletion, thus rejecting the A(del)/A(del) genotype as the sole cause of recessive black coat colour in sheep. Differences in expression of ASIP mRNA were assessed via RT-PCR in 14 black-coated and 10 white-coated Xalda individuals showing different ASIP genotypes (A(wt)/A(wt), A(wt)/A(del) and A(del)/A(del)). Levels of expression in black animals were significantly (P < 0.0001) lower than those assessed for white-coated individuals. However, the ASIP genotype did not influence the ASIP mRNA level of expression. The consistency of these findings with those recently reported in humans is discussed, and the need to isolate the promoter region of ovine ASIP to obtain further evidence for a role of ASIP in recessive black ovine pigmentation is pointed out.     

Chemical disulfide mapping identifies an inhibitor cystine knot in the agouti signaling protein

The agouti signaling protein (ASIP) and its homolog, the agouti-related protein (AgRP), act as inverse agonists that control, respectively, pigmentation and metabolic function in mammals. NMR investigations find that the C-terminal domains of these proteins adopt a fold consistent with an inhibitor cystine knot (ICK), previously identified in invertebrate toxins. Although these structural studies suggest that ASIP and AgRP define a new mammalian protein fold class, the results with ASIP are inconclusive. Here, we apply direct chemical mapping to determine the complete set of disulfide linkages in ASIP. The results demonstrate unequivocally that ASIP adopts the ICK fold and thereby supports a recent evolution structure function analysis, which proposes that ASIP and AgRP arose from a common antagonist ligand.     

Involvement of phospholipase D1 in melanogenesis of mouse B16 melanoma cells

In response to alpha-melanocyte-stimulating hormone (alpha-MSH) or cAMP-elevating agents (forskolin and isobutylmethylxanthine), mouse B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. However, the mechanism(s) underlying the regulation of melanogenesis during differentiation has not yet been clearly understood. Phospholipase D (PLD) has been reported to be involved in differentiation. This enzyme cleaves phosphatidylcholine upon stimulation with stimuli to generate phosphatidic acid. In the current study, the involvement of PLD in the regulation of melanogenesis characteristic of differentiation was examined using mouse B16 melanoma cells. Treatment of B16 cells with alpha-MSH was found to cause marked decreases in the PLD1 activity concurrent with its reduced protein level. Moreover, treatment of exogenous bacterial PLD also inhibited alpha-MSH-induced melanogenesis. To further investigate the role of PLD1 in the regulation of melanogenesis, we examined the effects of overexpression of PLD1 on melanogenesis in B16 melanoma cells. The B16 cells overexpressing PLD were prepared by transfection with the vector containing the cDNA encoding PLD1. The melanin contents in PLD1-overexpressing cells (B16/PLD1) were observed to be lower compared with those in the vector control cells (B16/Vec), concomitant with the decreases in both activity and protein level of tyrosinase, a key regulatory enzyme in melanogenesis. Moreover, overexpression of PLD1 resulted in a marked inhibition of melanogenesis induced by alpha-MSH. The inhibition of melanogenesis was well correlated with the decrease in the tyrosinase activity associated with its expression. These results indicated that PLD1 negatively regulated the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells.     

A new role for epidermal cell-derived thymocyte activating factor/IL-1 as an antagonist for distinct epidermal cell function

It is known that many immunologic responses to IL-1 are antagonized by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH). This led us to investigate the possible reciprocal effects of IL-1 and the functionally related epidermal cytokines, epidermal cell-derived thymocyte activating factor (ETAF) and IL-6, on the melanogenic effect of alpha-MSH on murine Cloudman melanoma cells. When these cells were treated with ETAF in combination with alpha-MSH or its potent analog [Nle4,D-Phe7]-alpha-MSH, the melanotropin induced increase in tyrosinase activity, and thus melanin synthesis, was abrogated. This inhibitory effect of ETAF was not mediated by competitive binding to the melanotropin receptor, because ETAF also blocked the melanogenic response of melanoma cells to isobutyl methylxanthine (IBMX) and to PGE1 and PGE2. ETAF had no effect on cellular proliferation. Inhibition of the stimulated tyrosinase activity by ETAF was not due to diminished cAMP synthesis or increased cAMP degradation. Cells treated concomitantly with ETAF and alpha-MSH, IBMX, or PGE1 had the same cAMP levels as cells treated with alpha-MSH, IBMX, or PGE1 alone. In contrast to ETAF, human rIL-1 alpha or IL-1 beta alone or in combination did not have an inhibitory effect on melanogenesis. IL-6 significantly inhibited the basal level of tyrosinase and partially abrogated the alpha-MSH-induced tyrosinase activity. IL-6 also stimulated cellular proliferation when added alone or in combination with alpha-MSH. Granulocyte-macrophage colony stimulating factor (GM-CSF) did not alter either the tyrosinase activity or cellular replication at the concentrations tested. IL-1 alpha, GM-CSF, and IL-6 or IL-1 alpha and GM-CSF added together did not significantly affect the MSH-induced tyrosinase activity. These results ascribe a new potential function for ETAF and IL-6 as modulators of the melanogenic response of pigment cells.     

Population differences in the frequency of the agouti signaling protein g.8818a>G polymorphism

The role of agouti signaling protein (ASIP) in human pigmentation pathways is not definitively understood although its murine homologue regulates, in part, pheomelanogenesis. We have reported an association of a polymorphism in the 3'-untranslated region of ASIP (g.8818A>G) with dark hair and eye color among a group of European-Americans (Am J Hum Genet 2002 March;70:770). Among 147 healthy control subjects, the frequency of the G-allele was 0.12. We hypothesized that this polymorphism would occur at different frequencies among different population groups. Using PCR-RFLP, we genotyped 25 East Asian, 86 African-American, and 207 West African individuals for the ASIP g.8818A>G polymorphism. The g.8818G-allele was present in the West African sample at a frequency of 0.80, in the African-American sample at a frequency of 0.62, and in the East Asian sample at 0.28. The difference in allele frequency among population groups was statistically significant (P < 0.0001). Although the effect of the g.8818A>G polymorphism upon ASIP function is unknown, the large difference in allele frequency between our West African and European-American sample populations lends support to the notion that this gene may be important in human pigmentation.