Rate Limiting Factors in Melanocortin 1 Receptor Signalling Throughthe cAMP Pathway

The melanotropic actions of α‐melanocyte‐stimulating hormone (α‐MSH) and other melanocortins are mediated by activation of the melanocortin 1 receptor (MC1R). This G protein‐coupled receptor is positively coupled to Gs and triggers the cyclic adenosine mono‐phosphate (cAMP) pathway. Mutations of the MC1R gene are associated with skin type and pigmentation phenotypes, and with increased risk of skin cancers. Genetic studies have demonstrated an heterozygote carrier effect for these associations, suggesting the importance of variant allele dosage. This could be accounted for, at least partially, if the number of MC1R molecules, rather than the Gs protein or the effector enzyme, adenylyl cyclase, is limiting for the activation of the signalling pathway. However, the nature of the limiting factor(s) in MC1R signalling has not been investigated. We addressed this question by comparing the cAMP output of clones of human melanoma cell lines enriched in MC1R by stable transfection. We also analysed heterologous cell systems widely used for functional studies of MC1R. We show that cAMP production in clones of Chinese hamster ovary cells stably expressing the MC1R is a linear function of receptor number up to high, supraphysiological levels of approximately 50 000 α‐MSH binding sites per cell. Enrichment of human melanoma cell lines with MC1R also results in increased cAMP levels, with a small leftward shift of the agonist dose–response curves. Therefore, at physiological expression levels second‐messenger generation is dependent on receptor density. Within melanoma cells and also likely in normal melanocytes, MC1R appears the limiting factor controlling the output of the cAMP signalling pathway.     

Functional status and relationships of melanocortin 1 receptor signaling to the cAMP and extracellular signal-regulated protein kinases 1 and 2 pathways in human melanoma cells

Melanocortin 1 receptor (MC1R), a major determinant of skin phototype frequently mutated in melanoma, is a Gs protein-coupled receptor that regulates pigment production in melanocytes. MC1R stimulation activates cAMP synthesis and the extracellular signal-regulated (ERK) ERK1 and ERK2. In human melanocytes, ERK activation by MC1R relies on cAMP-independent transactivation of the c-KIT receptor. Thus MC1R functional coupling to the cAMP and ERK pathways may involve different structural requirements giving raise to biased effects of skin cancer-associated mutations. We evaluated the impact of MC1R mutations on ERK activation, cAMP production and agonist binding. We found that MC1R mutations impair cAMP production much more often than ERK activation, suggesting less stringent requirements for functional coupling to the ERK pathway. We examined the crosstalk of the cAMP and ERK pathways in HBL human melanoma cells (wild-type for MC1R, NRAS and BRAF). ERK activation by constitutively active upstream effectors or pharmacological inhibition had little effect on MC1R-stimulated cAMP synthesis. High cAMP levels were compatible with normal ERK activation but, surprisingly, the adenylyl cyclase activator forskolin abolished ERK activation by MC1R, most likely by a cAMP-independent mechanism. These results indicate little crosstalk of the cAMP and ERK pathways in HBL melanoma cells. Finally, we studied cAMP accumulation in a panel of 22 human melanoma cell lines stimulated with MC1R agonists or forskolin. cAMP synthesis was often inhibited, even in cells wild-type for MC1R and NRAS. Therefore, the cAMP pathway is more frequently impaired in melanoma than could be predicted by the MC1R or NRAS genotype.     

Melanocortin 1 receptor mutations impact differentially on signalling to the cAMP and the ERK mitogen-activated protein kinase pathways

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. MC1R activates cAMP and mitogen-activated protein kinase ERK1/ERK2 signalling. When expressed in rat pheochromocytoma cell line cells, the R151C, R160W and D294H MC1R variants associated with melanoma and impaired cAMP signalling mediated ERK activation and ERK-dependent, agonist-induced neurite outgrowth comparable with wild-type. Dose-response curves for ERK activation and cAMP production indicated higher sensitivity of the ERK response. Thus, the melanoma-associated MC1R mutations impact differently on cAMP and ERK signalling, suggesting that cAMP is not responsible for functional coupling of MC1R to the ERK cascade.     

Signaling from the human melanocortin 1 receptor to ERK1 and ERK2 mitogen-activated protein kinases involves transactivation of cKIT

Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. Upon stimulation by αMSH, MC1R triggers the cAMP and ERK1/ERK2 MAPK pathways. In mouse melanocytes, ERK activation by αMSH binding to Mc1r depends on cAMP, and melanocytes are considered a paradigm for cAMP-dependent ERK activation. However, human MC1R variants associated with red hair, fair skin [red hair color (RHC) phenotype], and increased skin cancer risk display reduced cAMP signaling but activate ERKs as efficiently as wild type in heterologous cells, suggesting independent signaling to ERKs and cAMP in human melanocytes. We show that MC1R signaling activated the ERK pathway in normal human melanocytes and melanoma cells expressing physiological levels of endogenous RHC variants. ERK activation was comparable for wild-type and mutant MC1R and was independent on cAMP because it was neither triggered by stimulation of cAMP synthesis with forskolin nor blocked by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine. Stimulation of MC1R with αMSH did not lead to protein kinase C activation and ERK activation was unaffected by protein kinase C inhibitors. Conversely, pharmacological interference, small interfering RNA studies, expression profiles, and functional reconstitution experiments showed that αMSH-induced ERK activation resulted from Src tyrosine kinase-mediated transactivation of the stem cell factor receptor, a receptor tyrosine kinase essential for proliferation, differentiation, and survival of melanocyte precursors, thus demonstrating a functional link between the stem cell factor receptor and MC1R. Moreover, this transactivation phenomenon is unique because it is unaffected by natural mutations impairing canonical MC1R signaling through the cAMP pathway.     

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.     

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.     

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.     

ACTH1-17 is a more potent agonist at the human MC1 receptor than alpha-MSH.

The melanocortin receptor MC1 is expressed on melanocytes and is an important control point for melanogenesis and other responses. Alpha-MSH, which is considered to be the major ligand at the human melanocortin (MC)1 receptor (hMC1R), is produced from proopiomelanocortin (POMC) in the pituitary and in the skin by melanocytes and keratinocytes. Other POMC peptides are also produced in the skin and their concentrations exceed those of alpha-MSH by several fold. One of the most abundant is ACTH1-17. We have shown that adrenocorticotrophic hormone (ACTH)1-17 is more potent than alpha-MSH in stimulating melanogenesis in human melanocytes and unlike alpha-MSH produces a biphasic dose response curve. In this study we have examined the ability of ACTH1-17 to function as a ligand at the hMC1R. Competitive binding assays with [125I]Nle4 DPhe7 alpha-MSH as labelled ligand were carried out in HEK 293 cells transfected with the hMC1R. ACTH1-17 showed high affinity for the hMC1R with a Ki value of 0.21 +/- 0.03 nM which was slightly higher than that of 0.13 +/- 0.005 nM for alpha-MSH. ACTH1-17 was, however, more potent than alpha-MSH in increasing cAMP and IP3 production in the transfected cells. Our results demonstrate that ACTH1-17 is a potent agonist at the hMC1R. It is therefore possible that ACTH1-17, which is found in the skin in greater concentrations than alpha-MSH, has an important role in the regulation of human melanocytes and other cell types that express the hMC1R.     

MC1R,the cAMP pathway,and the response to solar UV: extending the horizon beyond pigmentation

The melanocortin 1 receptor (MC1R) is a G protein‐coupled receptor crucial for the regulation of melanocyte proliferation and function. Upon binding melanocortins, MC1R activates several signaling cascades, notably the cAMP pathway leading to synthesis of photoprotective eumelanin. Polymorphisms in the MC1R gene are a major source of normal variation of human hair color and skin pigmentation, response to ultraviolet radiation (UVR), and skin cancer susceptibility. The identification of a surprisingly high number of MC1R natural variants strongly associated with pigmentary phenotypes and increased skin cancer risk has prompted research on the functional properties of the wild‐type receptor and frequent mutant alleles. We summarize current knowledge on MC1R structural and functional properties, as well as on its intracellular trafficking and signaling. We also review the current knowledge about the function of MC1R as a skin cancer, particularly melanoma, susceptibility gene and how it modulates the response of melanocytes to UVR.     

Homologous regulation of melanocortin-1 receptor (MC1R) expression in melanoma tumor cells in vivo

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist alpha-melanocyte-stimulating hormone (alpha-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B 16F1 cells, a single injection of 50 to 500 microg alpha-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16-24 h. By continuous administration of alpha-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.     

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

Alpha-melanocyte-stimulating hormone (alpha-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected HEK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to alpha-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent.     

Peripheral effect of alpha-melanocyte-stimulating hormone on fatty acid oxidation in skeletal muscle

To study the peripheral effects of melanocortin on fuel homeostasis in skeletal muscle, we assessed palmitate oxidation and AMP kinase activity in alpha-melanocyte-stimulating hormone (alpha-MSH)-treated muscle cells. After alpha-MSH treatment, carnitine palmitoyltransferase-1 and fatty acid oxidation (FAO) increased in a dose-dependent manner. A strong melanocortin agonist, NDP-MSH, also stimulated FAO in primary culture muscle cells and C2C12 cells. However, [Glu6]alpha-MSH-ND, which has ample MC4R and MC3R agonistic activity, stimulated FAO only at high concentrations (10(-5) M). JKC-363, a selective MC4R antagonist, did not suppress alpha-MSH-induced FAO. Meanwhile, SHU9119, which has both antagonistic activity on MC3R and MC4R and agonistic activity on both MC1R and MC5R, increased the effect of alpha-MSH on FAO in both C2C12 and primary muscle cells. Small interference RNA against MC5R suppressed the alpha-MSH-induced FAO effectively. cAMP analogues mimicked the effect of alpha-MSH on FAO, and the effects of both alpha-MSH and cAMP analogue-mediated FAO were antagonized by a protein kinase A inhibitor (H89) and a cAMP antagonist ((Rp)-cAMP). Acetyl-CoA carboxylase activity was suppressed by alpha-MSH and cAMP analogues by phosphorylation through AMP-activated protein kinase activation in C2C12 cells. Taken together, these results suggest that alpha-MSH increases FAO in skeletal muscle, in which MC5R may play a major role. Furthermore, these results suggest that alpha-MSH-induced FAO involves cAMP-protein kinase A-mediated AMP-activated protein kinase activation.     

Association of feather colour with constitutively active melanocortin 1 receptors in chicken.

Seven alleles of the chicken melanocortin (MC) 1 receptor were cloned into expression vectors, expressed in mammalian cells and pharmacologically characterized. Four of the clones e(+R), e(+B&D), e(wh)/e(y), E(Rfayoumi) gave receptors to which melanocortin stimulating hormone (alpha-MSH) and NDP-MSH bound with similar IC50 values and responded to alpha-MSH by increasing intracellular cAMP levels in a dose-dependent manner. Three of the cMC1 receptors; e(b), E and E(R), did not show any specific binding to the radioligand, but were found to be constitutively active in the cAMP assay. The E and E(R) alleles are associated with black feather colour in chicken while the eb allele gives rise to brownish pigmentation. The three constitutively active receptors share a mutation of Glu to Lys in position 92. This mutation was previously found in darkly pigmented sombre mice, but constitutively active MC receptors have not previously been shown in any nonmammalian species. We also inserted the Glu to Lys mutation in the human MC1 and MC4 receptors. In contrast with the chicken clones, the hMC1-E94K receptor bound to the ligand, but was still constitutively active independently of ligand concentration. The hMC4-E100K receptor did not bind to the MSH ligand and was not constitutively active. The results indicate that the structural requirements that allow the receptor to adapt an active conformation without binding to a ligand, as a consequence of this E/K mutation, are not conserved within the MC receptors. The results are discussed in relationship to feather colour in chicken, molecular receptor structures and evolution. We suggest that properties for the 'E92K switch' mechanism may have evolved in an ancestor common to chicken and mammals and were maintained over long time periods through evolutionary pressure, probably on closely linked structural features.     

Identification and functional characterization of natural human melanocortin 1 receptor mutant alleles in Pakistani population

Melanocortin 1 receptor (MC1R), a Gs protein‐coupled receptor of the melanocyte's plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α‐melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole‐exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in‐frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist‐induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild‐type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss‐of‐function (LOF) allele, while p.Val174del displayed a partial LOF attribute.     

Thyrotropin releasing hormone (TRH) selectively binds and activates the melanocortin 1 receptor.

We tested the endogenous tripeptide TRH (thyrotropin releasing hormone) ability to bind to MC (melanocortin) receptor subtypes. We discovered that TRH binds to the human MCI receptor expressed in COS cells and to murine B16 melanoma cells with 5790+/-1010 nM and 6370+/-1260 nM Ki's, respectively. TRH did not bind to the human MC3, MC4 or MC5 receptor subtypes. Moreover, TRH also stimulated cAMP production in murine B16 melanoma cells reaching the same maximum level of cAMP as found for alpha-MSH. However, several analogues of TRH, including TRH-OH, TRH-Gly-NH2 and other analogues, where each of the three amino acid residues in TRH had been exchanged by a related residue, did not bind to any of the MC receptors tested in this study. C(alpha) atoms of molecular models of TRH and the core of a MSH peptide were aligned with r.m.s. of 0.01 A. Moreover, TRH could be docked into a binding pocket of a molecular model of the MC1 receptor at only a little higher energy than a short cyclic MSH peptide. The data indicate a similarity in the mode of TRH and MSH activation of the MCI receptor.     

Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor function.

The alpha-melanocyte-stimulating hormone (alphaMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of alphaMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent alphaMSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships.     

ACTH, alpha-MSH, and control of cortisol release: cloning, sequencing, and functional expression of the melanocortin-2 and melanocortin-5 receptor in Cyprinus carpio

Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.     

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.