Melanocortin MC₁ receptor in human genetics and model systems

The melanocortin MC(1) receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC(1) receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC(1) receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC(1) receptor and the molecular mechanisms underlying the association between melanocortin MC(1) receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC(1) receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC(1) receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC(1) receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC(1) receptor polymorphism.     

Fish melanocortin system

Melanocortin signalling is mediated by binding to a family of G protein-coupled receptors that positively couple to adenylyl cyclase. Tetrapod species have five melanocortin (MC₁–MC₅) receptors. The number of receptors varies in fish, zebrafish, for example, having six melanocortin receptors, with two copies of the melanocortin MC₅ receptor, while pufferfish have 4 receptors with no melanocortin MC₃ receptor and one copy of melanocortin MC₅ receptor. Fish genomes also exhibit orthologue genes for agouti-signalling protein (ASP) and -related protein (AGRP). AGRP expression is confined to a small area in the hypothalamus but ASP is expressed in the skin. Fish melanocortin MC₂ receptor is specific for ACTH and requires the cooperation of accessory proteins (MRAP) to reach functional expression. The four other melanocortin MC receptors distinctively bind MSHs. The interaction of α-MSH and melanocortin MC₁ receptor plays a key point in the control of the pigmentation and mutations of melanocortin MC₁ receptor are responsible for reduced melanization. Both melanocortin MC₄ and MC₅ receptor are expressed in the hypothalamus, and central melanocortin MC₄ receptor expression is thought to regulate the energy balance through the modulation of feeding behaviour. In addition, the peripheral melanocortin system also regulates lipid metabolism by acting at hepatic melanocortin MC₂ and MC₅ receptors. Both sea bass melanocortin MC₁ and MC₄ receptors are constitutively expressed in vitro and both ASP and AGRP work as inverse agonists but only after inhibition of the phosphodiesterase system. Accordingly, the overexpression of AGRP and ASP transgenes promotes obesity and reduces melanization in zebrafish, respectively.     

Structure, function and regulation of the melanocortin receptors

Melanocortin receptors belong to the seven-transmembrane (TM) domain proteins that are coupled to G-proteins and signaled through intracellular cyclic adenosine monophosphate. Many structural features conserved in other G-protein coupled receptors (GPCRs) are found in the melanocortin receptors. There are five melanocortin receptor subtypes and each of the melanocortin receptor subtypes has a different pattern of tissue expression and has its own profile regarding the relative potency of different melanocortin peptides. α-, β-, and γ-MSH and ACTH are known endogenous agonist ligands for the melanocortin receptors. Agouti and AgRP are the only known naturally occurring antagonists of the melanocortin receptors. We have examined the molecular basis of all five human melanocortin receptors for different ligand binding affinities and potencies using chimeric and mutated receptors. Our studies indicate that human melanocortin MC₁ receptor, human melanocortin MC₃ receptor, human melanocortin MC₄ receptor and human melanocortin MC₅ receptor utilize orthosteric sites for non selective agonists, α-MSH and NDP-?α-MSH, high affinity binding and utilize allosteric sites for selective agonist or antagonist binding. Furthermore, our results indicate that molecular determinants of human melanocortin MC₂ receptor for ACTH binding and signaling are different from that of other melanocortin receptors. Many studies also indicate that agonists can induce different conformation changes of melanocortin receptors, which then lead to the activation of different signaling pathways, even when the expression level of receptor and the strength of stimulus–response coupling are the same. This finding may provide new information for the design of drugs for targeting melanocortin receptors.     

Physiological roles of the melanocortin MC3 receptor

The melanocortin MC₃ receptor remains the most enigmatic of the melanocortin receptors with regard to its physiological functions. The receptor is expressed both in the CNS and in multiple tissues in the periphery. It appears to be an inhibitory autoreceptor on proopiomelanocortin neurons, yet global deletion of the receptor causes an obesity syndrome. Knockout of the receptor increases adipose mass without a readily measurable increase in food intake or decrease in energy expenditure. And finally, no melanocortin MC₃ receptor null humans have been identified and associations between variant alleles of the melanocortin MC₃ receptor and diseases remain controversial, so the physiological role of the receptor in humans remains to be determined.     

Melanocortin-5 receptor and sebogenesis

The melanocortins (α-MSH, β-MSH, γ-MSH, and ACTH) bind to the melanocortin receptors and signal through increases in cyclic adenosine monophosphate to induce biological effects. The melanocortin MC₅ and MC₁ receptors are expressed in human sebaceous glands, which produce sebum, a lipid mixture of squalene, wax esters, triglycerides, cholesterol esters, and free fatty acids that is secreted onto the skin. Excessive sebum production is one of the major factors in the pathogenesis of acne. The expression of melanocortin MC₅ receptor has been associated with sebocyte differentiation and sebum production. Sebaceous lipids are down-regulated in melanocortin MC₅ receptor-deficient mice, consistent with the observation that α-MSH acts as a sebotropic hormone in rodents. These findings, which suggest that melanocortins stimulate sebaceous lipid production through the MC₅ receptor, led to our search for MC₅ receptor antagonists as potential sebum-suppressive agents. As predicted, an antagonist was shown to inhibit sebocyte differentiation in vitro, and to reduce sebum production in human skin transplanted onto immunodeficient mice. The melanocortin MC₅ receptor antagonists may prove to be clinically useful for the treatment of sebaceous disorders with excessive sebum production, such as acne.     

Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay

The melanocortin MC₄ receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC₄ receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC₄ receptor, we created HEK293 cell lines coexpressing the human melanocortin MC₄ receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z′ = 0.50). A pilot screen run on the Microsource Spectrum compound library (n = 2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β₂AR agonists – the β₂AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.     

Polyclonal antibodies against human melanocortin MC1 receptor: preliminary immunohistochemical localisation of melanocortin MC1 receptor to malignant melanoma cells

Peptides of 11 and 15 residue lengths were synthesised according to the sequence of the N-terminal region of the human MC₁ melanocyte stimulating hormone receptor. The peptides were conjugated to thyroglobulin and used for preparation of antisera in the rabbit. Each of the conjugates raised antisera which showed high titre and specificity for its respective peptide antigen when evaluated in an ELISA test. Both types of antisera immunostained MC₁ receptor expressing COS-7 cells. By contrast, the sera did not stain control COS-7 cells not expressing the MC₁ receptor. Moreover, preimmune sera or antiserum preadsorbed with its respective peptide did not stain the MC₁ receptor expressing cells. The antisera were used to immunostain sections of normal human skin, as well as samples of cutaneous malignant melanoma tumours obtained from a patient. The cells of the melanoma tumours were very strongly immunostained with the MC₁ receptor antisera. By contrast, melanocytes which were present in the normal skin could not be visualised with our antisera.     

Melanocortin MC4 receptor expression sites and local function

The melanocortin MC₄ receptor plays an important role in energy metabolism, but also affects blood pressure, heart rate and erectile function. Localization of the receptors that fulfill these distinct roles is only partially known. Mapping of the melanocortin MC₄ receptor has been stymied by the absence of a functional antibody. Several groups have examined mRNA expression of the melanocortin MC₄ receptor in the rodent brain and transgenic approaches have also been utilized to visualize melanocortin MC₄ receptor expression sites within the brain. Ligand expression and binding studies have provided additional information on the areas of the brain where this elusive receptor is functionally expressed. Finally, microinjection of melanocortin MC₄ receptor ligands in specific nuclei has further served to elucidate the function of melanocortin MC₄ receptors in these nuclei. These combined approaches have helped link the anatomy and function of this receptor, such as the role of paraventricular hypothalamic nucleus melanocortin MC₄ receptor in the regulation of food intake. Intriguingly, however, numerous expression-sites have been identified that have not been linked to a specific receptor function such as those along the optic tract and olfactory tubercle. Further research is needed to clarify the function of the melanocortin MC₄ receptor at these sites.     

Effects of melanocortins on adrenal gland physiology

The melanocortin-2-receptor (MC₂ receptor), also known as the ACTH receptor, is a critical component of the hypothalamic–pituitary–adrenal axis. The importance of MC₂ receptor in adrenal physiology is exemplified by the condition familial glucocorticoid deficiency, a potentially fatal disease characterised by isolated cortisol deficiency. MC₂receptor mutations cause ~ 25% of cases. The discovery of a MC₂ receptor accessory protein MRAP, mutations of which account for ~ 15%–20% of familial glucocorticoid deficiency, has provided insight into MC₂ receptor trafficking and signalling. MRAP is essential for the functional expression of MC₂ receptor. MRAP2, a novel homolog of MRAP, can also facilitate MC₂ receptor cell surface expression and function. Like MRAP, MRAP2 is a small transmembrane domain glycoprotein capable of homodimerising. In addition, MRAP/MRAP2 can heterodimerise. The presence of MRAP2 adrenal expression suggests a possible role for MRAP2 in adrenal physiology, which has yet to be elucidated. Importantly, new data shows that the MRAPs can interact with all the other melanocortin receptors (MC_1,3,4,5 receptor). In contrast to MC₂ receptor, this interaction results in reduced melanocortin receptor surface expression and signalling. MRAP2 is predominantly expressed in brain. Hypothalamic expression has been demonstrated for both MRAP and MRAP2. The ability of MRAPs to modulate different members of the melanocortin receptor family in a bidirectional manner is intriguing. Furthermore, central nervous system expression of MRAPs points to a role beyond MC₂ receptor mediated adrenal steroidogenesis.     

Implication of the melanocortin-3 receptor in the regulation of food intake

The melanocortin system is well recognized to be involved in the regulation of food intake, body weight, and energy homeostasis. To probe the role of the MC₃ in the regulation of food intake, JRH322-18 a mixed MC₃ partial agonist/antagonist and MC₄ agonist tetrapeptide was examined in wild type (WT) and melanocortin 4 receptor (MC₄) knockout mice and shown to reduce food intake in both models. In the wild type mice, 2.0 nmol of JRH322-18 statistically reduced food intake 4 h post icv treatment into satiated nocturnally feeding wild type mice. The same dose in the MC₄KO mice significantly reduced cumulative food intake 24 h post treatment. Conditioned taste aversion as well as activity studies supports that the decreased food intake was not due to visceral illness. Since these studies resulted in loss-of-function results, the SHU9119 and agouti-related protein (AGRP) melanocortin receptor antagonists were administered to wild type as well as the MC₃ and MC₄ knockout mice in anticipation of gain-of-function results. The SHU9119 ligand produced an increase in food intake in the wild type mice as anticipated, however no effect was observed in the MC₃ and MC₄ knockout mice as compared to the saline control. The AGRP ligand however, produced a significant increase in food intake in the wild type as well as the MC₃ and MC₄ knockout mice and it had a prolonged affect for several days. These data support the hypothesis that the MC₃ plays a subtle role in the regulation of food intake, however the mechanism by which this is occurring remains to be determined.     

Chondroprotective and anti-inflammatory role of melanocortin peptides in TNF-α activated human C-20/A4 chondrocytes

BACKGROUND AND PURPOSE

Melanocortin MC₁ and MC₃ receptors, mediate the anti-inflammatory effects of melanocortin peptides. Targeting these receptors could therefore lead to development of novel anti-inflammatory therapeutic agents. We investigated the expression of MC₁ and MC₃ receptors on chondrocytes and the role of α-melanocyte-stimulating hormone (α-MSH) and the selective MC₃ receptor agonist, [DTRP⁸]-γ-MSH, in modulating production of inflammatory cytokines, tissue-destructive proteins and induction of apoptotic pathway(s) in the human chondrocytic C-20/A4 cells.

EXPERIMENTAL APPROACH

Effects of α-MSH, [DTRP⁸]-γ-MSH alone or in the presence of the MC_3/4 receptor antagonist, SHU9119, on TNF-α induced release of pro-inflammatory cytokines, MMPs, apoptotic pathway(s) and cell death in C-20/A4 chondrocytes were investigated, along with their effect on the release of the anti-inflammatory cytokine IL-10.

KEY RESULTS

C-20/A4 chondrocytes expressed functionally active MC_1,3 receptors. α-MSH and [DTRP⁸]-γ-MSH treatment, for 30 min before TNF-α stimulation, provided a time-and-bell-shaped concentration-dependent decrease in pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) release and increased release of the chondroprotective and anti-inflammatory cytokine, IL-10, whilst decreasing expression of MMP1, MMP3, MMP13 genes.α-MSH and [DTRP⁸]-γ-MSH treatment also inhibited TNF-α-induced caspase-3/7 activation and chondrocyte death. The effects of [DTRP⁸]-γ-MSH, but not α-MSH, were abolished by the MC_3/4 receptor antagonist, SHU9119.

CONCLUSION AND IMPLICATIONS

Activation of MC₁/MC₃ receptors in C-20/A4 chondrocytes down-regulated production of pro-inflammatory cytokines and cartilage-destroying proteinases, inhibited initiation of apoptotic pathways and promoted release of chondroprotective and anti-inflammatory cytokines. Developing small molecule agonists to MC₁/MC₃ receptors could be a viable approach for developing chondroprotective and anti-inflammatory therapies in rheumatoid and osteoarthritis.     

Use of chimeric melanocortin-2 and -4 receptors to identify regions responsible for ligand specificity and dependence on melanocortin 2 receptor accessory protein

The melanocortin 2 (MC₂) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC₂ receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC₂ receptor responsible for these characteristics, we created chimeras between MC₂ and MC₄ receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC₂ but not MC₄ receptors. Replacing the first transmembrane segment of the MC₂ receptor with the corresponding region from the MC₄ receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC₄ receptor with the corresponding regions from the MC₂ receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC₂ receptors with MC₄ sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC₂ receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC₂ receptor as important for MRAP dependent-signal transduction and ligand specificity.     

The early origin of melanocortin receptors, agouti-related peptide, agouti signalling peptide, and melanocortin receptor-accessory proteins, with emphasis on pufferfishes, elephant shark, lampreys, and amphioxus

There are conflicting theories about the evolution of melanocortin MC receptors while only few studies have addressed the evolution of agouti-related peptide (AgRP) and agouti signalling peptide (ASIP), which are antagonists at the melanocortin receptors (MCRs), or the melanocortin MC₂ receptor accessory proteins (MRAP1 and MRAP2). Previously we have cloned melanocortin MC receptors (MC_a and MC_b) genes in river lamprey and here we identify orthologues to these melanocortin MC receptor sequences in the sea lamprey. We investigate the putative presence of the melanocortin MC receptor genes in lancelet (amphioxus; Branchiostoma floridae) but we find it unlikely that such gene exists, due to a sharp drop in sequence similarity beyond sequence clusters of known receptors. We show the presence of AgRP and ASIP in elephant shark, a cartilaginous fish belonging to the subclass of Elasmobranchii. However, we do not find any of these genes in lamprey or lancelet after detailed analysis of both targeted and whole proteome regular expression scans. We found MRAP2, but not MRAP1, to be present in elephant shark and sea lamprey while Fugu (T. rubripes) has both genes. This study shows that the most ancient presence of these melanocortin-related sequences is found in elephant shark and lampreys considering the current available sequence data.     

Recent advances in the development of melanocortin-4 receptor agonists

The melanocortin receptors are an area of intense current research, both in academia and in the pharmaceutical industry. The large body of evidence to support a critical role for the melanocortin-4 receptor (MC₄R) in energy homeostasis (as well as indications of involvement in other interesting physiological processes) has prompted research efforts to investigate its pharmacology. This review will focus on recent advances toward the identification of potential therapeutic agents working via activation of MC₄R, supplementing the more general review on ‘Ligands to the melanocortins’ published in this journal in 2001 [1]. Several patent applications have been filed in the last year on compounds with MC₄R agonist activity for a variety of indications, most notably the treatment of feeding and body weight disorders and sexual dysfunction.     

Discovery of novel melanocortin4 receptor selective MSH analogues

1. We synthesized a novel series of cyclic melanocyte stimulating hormone (MSH) analogues and tested their binding properties on cells transiently expressing the human melanocortin₁ (MC₁), MC₃, MC₄ and MC₅ receptors.
2. We discovered that compounds with 26 membered rings of [Cys⁴,D-Nal⁷,Cys¹¹]α-MSH(4–11) displayed specific MC₄ receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys⁴D-Nal⁷Cys¹¹]α-MSH(4–11) analogues are distinct from all other known MSH analogues, particularly as they bind the MC₄ receptor with high and the MC₁ receptor with low relative affinities.
3. HS964 and HS014 have 12 and 17 fold MC₄/MC₃ receptor selectivity, respectively, which is much higher than for the previously described cyclic lactam and [Cys⁴,Cys¹⁰]α-MSH analogues SHU9119 and HS9510.
4. HS964 is the first substance showing higher affinity for the MC₅ receptor than the MC₁ receptor.
5. HS014, which was the most potent and selective MC₄ receptor ligand (K_i 3.2 nM, which is ∼300 fold higher affinity than for α-MSH), was also demonstrated to antagonize α-MSH stimulation of cyclic AMP in MC₄ receptor transfected cells.
6. We found that a compound with a 29 membered ring of [Cys³,Nle¹⁰,D-Nal⁷,Cys¹¹]α-MSH(3–11) (HS010) had the highest affinity for the MC₃ receptor.
7. This is the first study to describe ligands that are truly MC₄ selective and a ligand having a high affinity for the MC₃ receptor. The novel compounds may be of use in clarifying the physiological roles of the MC₃, MC₄ and MC₅ receptors.

     

The melanocortin MC1 receptor agonist BMS-470539 inhibits leucocyte trafficking in the inflamed vasculature

Background and purpose:

Over three decades of research evaluating the biology of melanocortin (MC) hormones and synthetic peptides, activation of the MC type 1 (MC₁) receptor has been identified as a viable target for the development of novel anti-inflammatory therapeutic agents. Here, we have tested a recently described selective agonist of MC₁ receptors, BMS-470539, on leucocyte/post-capillary venule interactions in murine microvascular beds.

Experimental approach:

Intravital microscopy of two murine microcirculations were utilized, applying two distinct modes of promoting inflammation. The specificity of the effects of BMS-470539 was assessed using mice bearing mutant inactive MC₁ receptors (the recessive yellow e/e colony).

Key results:

BMS-470539, given before an ischaemia–reperfusion protocol, inhibited cell adhesion and emigration with no effect on cell rolling, as assessed 90 min into the reperfusion phase. These properties were paralleled by inhibition of tissue expression of both CXCL1 and CCL2. Confocal investigations of inflamed post-capillary venules revealed immunostaining for MC₁ receptors on adherent and emigrated leucocytes. Congruently, the anti-inflammatory properties of BMS-470539 were lost in mesenteries of mice bearing the inactive mutant MC₁ receptors. Therapeutic administration of BMS-470539 stopped cell emigration, but did not affect cell adhesion in the cremasteric microcirculation inflamed by superfusion with platelet-activating factor.

Conclusions and implications:

Activation of MC₁ receptors inhibited leucocyte adhesion and emigration. Development of new chemical entities directed at MC₁ receptors could be a viable approach in the development of novel anti-inflammatory therapeutic agents with potential application to post-ischaemic conditions.     

Aza-scanning of the potent melanocortin receptor agonist Ac-His-D-Phe-Arg-Trp-NH

The melanocortin pathway is an important participant in the regulation of skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. Melanocortin agonists contain the putative sequence 'His-Phe-Arg-Trp', which has been designated as the 'message' sequence for melanocortin peptides, and this sequence has been hypothesized to adopt a bioactive reverse turn conformation. Exploring the relationship between its structure and biological activity, we report the synthesis and evaluation of seven aza-analogs of the potent melanocortin receptor agonist Ac-His-D-Phe-Arg-Trp-NH2. Aza-amino acids, in which the alpha-carbon was replaced by nitrogen, were inserted along the peptide sequence to probe the importance of local configuration and turn conformation on the biology of this tetrapeptide. Although systematic substitution of aza-amino acids for the D-Phe and Arg residues led to a significant loss of activity relative to the parent peptide for all melanocortin receptor subtypes examined, substitution of aza-amino acids at the C-terminal Trp residue gave analogs equipotent to the parent peptide. In summary, the aza-scan has demonstrated that recognition of this tetrapeptide by the melanocortin receptors is particularly sensitive to modifications of configuration and conformation at the D-Phe and Arg residues versus the Trp amino acid. In light of aza-residues imparting resistance from enzymatic degradation, C-terminal aza-amino acid analogs may be used to design new peptide mimics with enhanced metabolic stability.     

Association between melanism, physiology and behaviour: a role for the melanocortin system

The melanocortin system is implicated in the expression of many phenotypic traits. Activation of the melanocortin MC₁ receptor by melanocortin hormones induces the production of brown/black eumelanic pigments, while activation of the four other melanocortin receptors affects other physiological and behavioural functions including stress response, energy homeostasis, anti-inflammatory and sexual activity, aggressiveness and resistance to oxidative stress. We recently proposed the hypothesis that some melanocortin-physiological and -behavioural traits are correlated within individuals. This hypothesis predicts that the degree of eumelanin production may, in some cases, be associated with the regulation of glucocorticoids, immunity, resistance to oxidative stress, energy homeostasis, sexual activity, and aggressiveness. A review of the zoological literature and detailed experimental studies in a free-living population of barn owls (Tyto alba) showed that indeed melanic coloration is often correlated with the predicted physiological and behavioural traits. Support for predictions of the hypothesis that covariations between coloration and other phenotypic traits stem from pleiotropic effects of the melanocortin system raises a number of theoretical and empirical issues from evolutionary and pharmacological point of views.