Antioxidative and melanogenesis-inhibitory activities of caffeoylquinic acids and other compounds from moxa.

The MeOH extract of moxa, the processed leaves of Artemisia princeps PAMP. (Asteraceae), exhibited potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and melanogenesis-inhibitory activity in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells. Eight caffeoylquinic acids, 1 and 6-12, five flavonoids, 13-17, two benzoic acid derivatives, 18 and 19, three coumarin derivatives, 20-22, four steroids, 23-26, and six triterpenoids, 27-32, were isolated from the MeOH extract. Upon evaluation of compounds 1, 6-23, and four semisynthetic caffeoylquinic acid esters, 2-5, for their DPPH radical-scavenging activity, 15 compounds, 1-13, 17, and 19, showed potent activities (IC(50) 3.1-16.8 µM). The 15 compounds exhibited, moreover, potent inhibitory activities (51.1-92.5% inhibition) against peroxidation of linoleic acid emulsion at 10 µg/ml concentration. In addition, when 27 compounds, 1-8, 10, 12, 13, 15-18, 20-25, and 27-32, were evaluated for their inhibitory activity against melanogenesis in α-MSH-stimulated B16 melanoma cells, five caffeoylquinic acids, i.e., chlorogenic acid (1), ethyl chlorogenate (3), propyl chlorogenate (4), isopropyl chlorogenate (5), and butyl chlorogenate (6), along with homoorientin (17) and vanillic acid (18), exhibited inhibitory activities with 33-62% reduction of melanin content at 100 µM concentration with no or almost no toxicity to the cells (89-114% of cell viability at 100 µM). Western blot analysis showed that compound 6 reduced the protein levels of microphtalmia-associated transcription factor (MITF), tyrosinase, tyrosine-related protein 1 (TRP-1), and TRP-2 mostly in a concentration-dependent manner, suggesting that this compound inhibits melanogenesis on α-MSH-stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP-1, and TRP-2. Furthermore, four compounds, 13, 15, 16, and 30, exhibited cytotoxicities against HL60 human leukemia cell line (IC(50) 7.0-11.1 µM), and nine compounds, 14-16, 23, 26-28, 31, and 32, showed inhibitory effects (IC(50) 272-382 mol ratio/32 pmol 12-O-tetradecanoylphohrbol-13-acetate (TPA)) against Epstein-Barr virus early antigen (EBV-EA) activation induced by TPA in Raji cells.

ß-Cryptoxanthin suppresses UVB-induced melanogenesis in mouse: involvement of the inhibition of prostaglandin E2 and melanocyte-stimulating hormone pathways.

OBJECTIVE: ß-cryptoxanthin (ß-CPX) is a carotenoid that is widely contained in the fruits of citrus plants. We evaluated the effect of ß-CPX on UVB-induced pigmentation and mRNA expression related to melanogenesis in mouse skin. In addition, changes in melanogenic molecules were evaluated in cultured melanocytes stimulated with prostaglandin (PG) E(2), melanocyte-stimulating hormone (MSH) and endothelin (ET)-1. METHODS: Mice were irradiated with UVB and were given ß-CPX (0.1, 1 and 10 mg/kg) orally for 14 days. Pigmentation was evaluated by skin colour change and microscopic observation. Total RNA was obtained from the skin and the expression of melanogenic mRNA was evaluated by RT-PCR. In cell culture studies, human melanocytes were cultured with ß-CPX and melanogenic stimulants (PGE(2), MSH and ET-1) for 6-10 days. Melanin contents, dendricity, melanogenic mRNA and phosphorylation of cyclic AMP response element-binding protein (CREB) were evaluated. KEY FINDINGS: ß-CPX (10 mg/kg) significantly suppressed skin pigmentation and mRNA expression of cyclooxygenase-2, ET-1 receptors, low-affinity neurotrophin receptor, PGE(2) receptor (EP1), melanocortin 1 receptor (MC1R), tyrosinase (Tyr), tyrosinase-related protein (Tyrp) 1 and microphthalmia transcription factor. ß-CPX (10 µg/ml) suppressed melanogenesis induced by PGE(2), MSH and ET-1. In the PGE(2)-stimulated melanocytes, mRNA expressions of EP-1, Tyr and Tyrp1 and phosphorylation of CREB protein were suppressed. In the ET-1-stimulated cells, only expression of CREB protein was suppressed. In the MSH-induced cells, mRNA expression of MC1R and Tyrp1 and protein expression of CREB were suppressed. CONCLUSION: Oral administration of ß-CPX was found to suppress UVB-induced melanogenesis. Suppression of melanogenic enzymes, receptors of melanogenic stimulators, expression and phosphorylation of CREB are thought to be involved in the mechanism.

Efficacy of a novel hydroquinone-free skin-brightening cream in patients with melasma.

BACKGROUND: For treating irregular skin pigmentation, a hallmark of premature skin aging, safe and effective alternatives to hydroquinone and kojic acid are being researched. AIMS: Four skin-brightening actives targeting melanin formation at multiple levels, namely disodium glycerophosphate, L-leucine, phenylethyl resorcinol, and undecylenoyl phenylalanine, in an oil-in-water emulsion cream were evaluated. PATIENTS/METHODS: Twenty female patients with mild-to-moderate epidermal melasma were included. After a washout period of 1 month with a sunscreen, they continued using the sunscreen and added the novel skin-brightening cream for 12 weeks twice daily to entire face. RESULTS: Whereas there was no significant change in skin pigmentation during the washout, signs for uneven skin tone including melasma area and severity and appearance of lentigines significantly decreased by up to 43% after the 12-week treatment period with the skin-brightening cream. The skin-brightening complex is well tolerated, which should allow its continued use over a prolonged period of time, in particular, when comparing skin-brightening approaches with exfoliating or peeling agents. CONCLUSIONS: When used with a daily sunscreen, the skin-brightening complex represents a valuable alternative to hydroquinone products and can be used for maintenance or adjunct skin care with lightening therapies.

Melanocortins protect against multiple organ dysfunction syndrome in mice.

BACKGROUND AND PURPOSE: Melanocortins reverse circulatory shock and improve survival by counteracting the systemic inflammatory response, and through the activation of the vagus nerve-mediated cholinergic anti-inflammatory pathway. To gain insight into the potential therapeutic value of melanocortins against multiple organ damage following systemic inflammatory response, here we investigated the effects of the melanocortin analogue [Nle4 D-Phe7]α-MSH (NDP-α-MSH) in a widely used murine model of multiple organ dysfunction syndrome (MODS). EXPERIMENTAL APPROACH: MODS was induced in mice by a single intraperitoneal injection of lipopolysaccharide followed, 6 days later (= day 0), by zymosan. After MODS or sham MODS induction, animals were randomized to receive intraperitoneally NDP-α-MSH (340 µg·kg⁻¹ day) or saline for up to 16 days. Additional groups of MODS mice were concomitantly treated with the melanocortin MC4 receptor antagonist HS024, or the nicotinic acetylcholine receptor antagonist chlorisondamine, and NDP-α-MSH. KEY RESULTS: At day 7, in the liver and lung NDP-α-MSH, significantly reduced mRNA expression of tumour necrosis factor-α (TNF-α), increased mRNA expression of interleukin-10 and improved the histological picture, as well as reduced TNF-α plasma levels; furthermore, NDP-α-MSH dose-dependently increased survival rate, as assessed throughout the 16 day observation period. HS024 and chlorisondamine prevented all the beneficial effects of NDP-α-MSH in MODS mice. CONCLUSIONS AND IMPLICATIONS: These data indicate that NDP-α-MSH protects against experimental MODS by counteracting the systemic inflammatory response, probably through brain MC4 receptor-triggered activation of the cholinergic anti-inflammatory pathway. These findings reveal previously undescribed effects of melanocortins and could have clinical relevance in the MODS setting.

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.

Constitutive receptor activity series: endogenous inverse agonists and constitutive receptor activity in the melanocortin system.

The recent discovery of melanocortin receptor mutations that selectively decrease constitutive receptor activity in obese individuals supports the physiological relevance of constitutive melanocortin receptor activity and its control by an endogenous inverse agonist. Furthermore, studies using mice that lack endogenous melanocortin receptor agonists show that differences in coat color are caused by different degrees of constitutive melanocortin receptor signaling regulated by an endogenous inverse agonist. Thus, the regulation of constitutive activity of melanocortin receptors is important for the normal control of pigmentation and body-weight homeostasis.

Blockade of melanocortin transmission inhibits cocaine reward.

Melanocortins and the melanocortin-4 receptor (MC4-R) are enriched in the nucleus accumbens, a brain region that has been implicated in the rewarding action of cocaine and other drugs of abuse. In the present study we use a number of rat behavioral models to show that infusion of a melanocortin peptide antagonist into the nucleus accumbens blocks the reinforcing, incentive motivational, and locomotor sensitizing effects of cocaine. We also show that locomotor responses to repeated cocaine exposure are completely blocked in MC4-R null mutant mice and reduced in Agouti mice that overexpress an endogenous inhibitor of melanocortins in the brain. The results also demonstrate that cocaine administration increases the expression of MC4-R in the nucleus accumbens and striatum, and that MC4-R is co-localized with prodynorphin in medium spiny neurons in the nucleus accumbens. Together, these findings indicate that the behavioral actions of cocaine are dependent on activation of MC4-R, and suggest that upregulation of this receptor by drug exposure may contribute to sensitization of these behavioral responses. Modulation of cocaine reward is a novel action of the melanocortin-MC4-R system and could be targeted for the development of new medications for cocaine addiction.

Dynamic regulation of hypothalamic neuropeptide gene expression and food intake by melanocortin analogues and reversal with melanocortin-4 receptor antagonist.

Melanocortins are known to be involved in the inhibition of food intake and energy metabolism. Acute and chronic intracerebroventricular administration of several different analogues of alpha-MSH, such as alpha-MSH, NDP-MSH, alpha-MSH-ND, [Gln(6)]alpha-MSH-ND, and [Lys(6)]alpha-MSH-ND, which were substituted in the position of His(6) with Gln and Lys, and cyclic16k-MSH to C57J/BL6 mice resulted in a significant inhibition of both time course food intake and body weight gain, compared to the saline-administered control. However, [Gln(6)]alpha-MSH-ND(6-10), the truncated form of [Gln(6)]alpha-MSH-ND, had no inhibitory effects on food intake. In situ hybridization analysis revealed that the expression levels of AGRP and NPY in the hypothalamus were significantly and rapidly diminished while POMC expression was strongly induced by [Gln(6)]alpha-MSH-ND. Administration of JKC-363, a selective MC4R-specific antagonist, coupled with [Gln(6)]alpha-MSH-ND, specifically reversed the [Gln(6)]alpha-MSH-ND-induced inhibition of food intake, but also reversed the hypothalamic expression levels of neuropeptides such as AGRP, NPY, MCH, and POMC, which suggests [Gln(6)]alpha-MSH-ND can function as a selective MC4R agonist.

Effects of the cocaine- and amphetamine-regulated transcript peptide on the turnover of dopamine in tuberoinfundibular neurons and serum prolactin levels: studies using estrogen, melanin concentrating hormone, and melanocortin.

Effects of the cocaine- and amphetamine-regulated transcript (CART) peptide on tuberoinfundibular dopaminergic (TIDA) neurons were examined in female and male Sprague-Dawley rats in the morning and afternoon. We also examined the blocking effects of melanin concentrating hormone (MCH) and the antagonists of alpha-melanocyte stimulating hormone (alpha-MSH), SHU9119 and HS014, on stimulation induced by the CART peptide in TIDA systems. Intracerebroventricular administration of 1 mug CART peptide (55-102) at 45 min, either in the morning or afternoon, produced an increase in the median eminence (ME) DOPAC (3,4-dihydroxyphenylacetic acid) level and a corresponding decrease in serum prolactin (PRL) levels. This resulted from stimulation of TIDA neurons regardless of castration, and whether or not male and female rats were estrogen-primed. The stimulatory effects of the CART peptide on ME DOPAC levels were similar in the morning and afternoon in both male and female rats. Central treatment with 1 microg SHU9119, HS014, or MCH significantly decreased the ME DOPAC levels and elevated serum PRL levels in female rats. However, only MCH prevented the stimulatory effect of the CART peptide on TIDA neurons. These results indicate that stimulation by the CART peptide on TIDA neurons is gender-independent; and this stimulatory effect can be blocked by MCH, but not the antagonists of alpha-MSH.

Pre-obese and obese agouti mice are sensitive to the anorectic effects of peptide YY(3-36) but resistant to ghrelin.

OBJECTIVE: The role of the melanocortin system in the feeding effects of peripheral peptide YY(3-36) (PYY(3-36)) and ghrelin was investigated using the agouti (A(y)/a) mouse as a model of abnormal melanocortin signalling. Furthermore, we examined whether the ectopic expression of agouti protein in A(y)/a mice results in complete MC4-R inhibition, by studying the effects of peripheral alpha-melanocyte-stimulating hormone (alpha-MSH) and leptin on food intake. DESIGN: Adult A(y)/a mice were studied in the pre-obese state (7-8 weeks) and obese state (14-15 weeks). Animals received PYY(3-36) (0.02 micromol/kg), NDP-alpha-MSH (0.2 micromol/kg), leptin (2 micromol/kg) (all 24 h fasted state) and ghrelin (0.2 micromol/kg) (fed state) by intraperitoneal (i.p.) injection. Age-matched A(y)/a controls received i.p. saline. A separate cohort of wild-type (WT), age-matched controls received the same peptide dose or saline. Food intake was measured at 1, 2, 4, 8 and 24 h post-injection and compared in all four groups. Plasma leptin-, ghrelin- and PYY-like immunoreactivity (IR) were measured using radioimmunoassay (RIA). RESULTS: At 2 h post-injection, PYY(3-36) reduced food intake in pre-obese and obese A(y)/a mice, whereas ghrelin had no effect. Plasma ghrelin levels were significantly reduced in pre-obese and obese A(y)/a mice compared to WT controls. Peripheral administration of NDP-alpha-MSH and leptin acutely suppressed feeding (0-2 h) in pre-obese and obese A(y)/a mice. CONCLUSIONS: Responsiveness of pre-obese and obese A(y)/a mice to PYY(3-36) suggests that the melanocortin system may not be essential for the anorectic effects of this peptide. Melanocortinergic antagonism by agouti protein in A(y)/a mice may be sufficient to block the effects of endogenous, but not exogenous PYY(3-36), alpha-MSH and leptin. The mechanism underlying ghrelin resistance in A(y)/a mice may result from antagonism of hypothalamic melanocortin receptors-4 by agouti protein, supporting a role for the melanocortin system in mediating ghrelin's actions.

Biological and conformational study of beta-substituted prolines in MT-II template: steric effects leading to human MC5 receptor selectivity.

To investigate the molecular basis for the interaction of the chi-constrained conformation of melanotropin peptide with the human melanocortin receptors, a series of beta-substituted proline analogs were synthesized and incorporated into the Ac-Nle-C[Asp-His-D-Phe-Arg-Trp-Lys]-NH2 (MT-II) template at the His6 and D-Phe7 positions. It was found that the binding affinities generally diminished as the steric bulk of the p-substituents of the 3-phenylproline residues increased. From (2S, 3R)-3-phenyl-Pro6 to (2S, 3R)-3-(p-methoxyphenyl)-Pro6 analogs the binding affinity decreased 23-fold at the human melanocortin-3 receptor (hMC3R), 17-fold at the hMC4R, and eight-fold at the hMC5R, but selectivity for the hMC5R increased. In addition, the substitution of the D-Phe7 residue with a (2R, 3S)-3-phenyl-Pro resulted in greatly reduced binding affinity (10(3)-10(5)) at these melanocortin receptors. Macromodel's Large Scale Low Mode (LLMOD) with OPLS-AA force field simulations revealed that both MT-II and SHU-9119 share a similar backbone conformation and topography with the exception of the orientation of the side chains of D-Phe7/D-Nal (2')7 in chi space. Introduction of the dihedrally constrained phenylproline analogs into the His6 position (analogs 2-6) caused topographical changes that might be responsible for the lower binding affinities. Our findings indicate that hMC3 and hMC4 receptors are more sensitive to steric effects and conformational constraints than the hMC5 receptor. This is the first example for melanocortin receptor selectivity where the propensity of steric interactions in chi space of beta-modified Pro6 analogs of MT-II has been shown to play a critical role for binding as well as bioefficacy of melanotropins at hMC3 and hMC4 receptors, but not at the hMC5 receptor.

[MC1R--the key gene in mammalian melanin synthesis].

The study of the molecular regulation mechanism of melanin synthesis during animal development has become a new focus recently . The synthesis and production of melanin during animal development are regulated by many genes. This paper summarized the molecular function mechanism of melanocortin-1-receptor (MC1R) gene and the relationship between the consequences of polymorphic variation of the gene and melanin traits, in addition to summarized the identification and mutation of MC1R gene in birds. Furthermore, the melanin synthesis mechanism in birds is also discussed here.

Melanocortins and their receptors and antagonists.

The melanocortins are a group of small protein hormones derived by post-translational cleavage of the proopiomelanocortin (POMC) gene product. The known melanocortin hormones include alpha-melanocyte stimulating hormone (MSH), beta-MSH, gamma-MSH and adrenocorticotropic hormone (ACTH). Five melanocortin receptors (MCIR through to MC5R) have been identified and most of these show tissue-specific expression patterns, as well as different binding affinities for each of the melanocortin hormones. The central melanocortin system consists of alpha-MSH, agouti-related protein (AGRP), MC3R and MC4R. AGRP and alpha-MSH are believed to be the natural antagonist and agonist respectively of MC3R and MC4R. This central melanocortin system is thought to play a fundamental role in the control of feeding and body weight. Knock-out mice models and genetic studies have pointed to the importance of the melanocortins in complex human pathways such as pigmentation, lipolysis, food intake, thermogenesis, sexual behaviour, memory and inflammatory response. Recently the melanocortins and their receptors have been the target for drug-based treatment of human physiological processes. MC3R and MC4R are likely targets for controlling body weight; MCIR may be used in the treatment of inflammation and MC2R for the treatment of glucocortical deficiency. A role for MCSR still remains unclear, but the evidence suggests an exocrine gland function.

Solution structures of cyclic melanocortin agonists and antagonists by NMR.

The melanocortin receptors are involved in many physiological functions, including pigmentation, sexual function, feeding behavior, and energy homeostasis, making them potential targets for drugs to treat obesity, sexual dysfunction, etc. Understanding the conformational basis of the receptor-ligand interactions is crucial to the design of potent and selective ligands for these receptors. The solution structures of the cyclic melanocortin agonists, partial agonist, and antagonists MTII, VJH085, SHU9119, MK5, and MK9 were determined by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy at pH 4.5 and 25 degrees C in water (90% H(2)O/10% D(2)O). The overall backbone structures of these cyclic alpha-melanocyte-stimulating hormone (alpha-MSH) analogues around the message sequence (His(6)-D-Phe(7)/D-Nal(2')(7)-Arg(8)-Trp(9)) were similar and reasonably well defined. beta-Turns spanning His(6) and D-Phe(7)/D-Nal(2')(7) were identified in all analogues, and an amphiphilic molecular surface was obtained for the message sequence residues in most structures within the NMR ensembles. The beta-turn, which most closely resembles a type II beta-turn, leads to stacking between the aromatic rings of His(6) and D-Phe(7) in MTII and VJH085. However, no aromatic stacking between His(6) and D-Nal(2')(7) was found in structures of the D-Nal(2')(7)-containing analogues. The difference in the side-chain dispositions of His(6) and D-Nal(2')(7) may be responsible for the reduced potency or antagonist activity of the D-Nal(2')(7)-containing analogues. In addition, our results suggest that the side-chain orientations may also modulate the receptor selectivity. The information found in this study will be useful for the further design of ligands for melanocortin receptors.

The catabolic action of insulin in the brain is mediated by melanocortins.

Like leptin, the pancreatic hormone insulin is an important adiposity signal to the brain. We report that the hypothalamic melanocortin system is an important target of the actions of insulin to regulate food intake and body weight. Hypothalamic neurons expressing insulin receptors were found to coexpress the melanocortin precursor molecule pro-opiomelanocortin (POMC), and administration of insulin into the third cerebral ventricle of fasted rats increased expression of POMC mRNA. Finally, a subthreshold dose of the melanocortin antagonist SHU-9119 prevented the reduction in food intake caused by third-ventricular insulin administration. These data suggest that the hypothalamic melanocortin system mediates the anorexic effects of central insulin, as well as of leptin.

Functional properties of an agouti signaling protein variant and characteristics of its cognate radioligand.

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.

The melanocortin-1 receptor: red hair and beyond.

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.

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein.

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.

In situ localization of agouti signal protein in murine skin using immunohistochemistry with an ASP-specific antibody.

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.