Melanoma-associated melanocortin 1 receptor variants confer redox signaling-dependent protection against oxidative DNA damage.

Cutaneous melanoma, a lethal skin cancer, arises from malignant transformation of melanocytes. Solar ultraviolet radiation (UVR) is a major environmental risk factor for melanoma since its interaction with the skin generates DNA damage, either directly or indirectly via oxidative stress. Pheomelanin pigments exacerbate oxidative stress in melanocytes by UVR-dependent and independent mechanisms. Thus, oxidative stress is considered to contribute to melanomagenesis, particularly in people with pheomelanic pigmentation. The melanocortin 1 receptor gene (MC1R) is a major melanoma susceptibility gene. Frequent MC1R variants (varMC1R) associated with fair skin and red or yellow hair color display hypomorphic signaling to the cAMP pathway and are associated with higher melanoma risk. This association is thought to be due to production of photosensitizing pheomelanins as well as deficient induction of DNA damage repair downstream of varMC1R. However, the data on modulation of oxidative DNA damage repair by MC1R remain scarce. We recently demonstrated that varMC1R accelerates clearance of reactive oxygen species (ROS)-induced DNA strand breaks in an AKT-dependent manner. Here we show that varMC1R also protects against ROS-dependent formation of 8-oxodG, the most frequent oxidative DNA lesion. Since the base excision repair (BER) pathway mediates clearance of these DNA lesions, we analyzed induction of BER enzymes in human melanoma cells of varMC1R genotype. Agonist-mediated activation of both wildtype (wtMC1R) and varMC1R significantly induced OGG and APE-1/Ref1, the rate-limiting BER enzymes responsible for repair of 8-oxodG. Moreover, we found that NADPH oxidase (NOX)-dependent generation of ROS was responsible for AKT activation and oxidative DNA damage repair downstream of varMC1R. These observations provide a better understanding of the functional properties of melanoma-associated MC1R alleles and may be useful for the rational development of strategies to correct defective varMC1R responses for efficient photoprotection and melanoma prevention in fair-skinned individuals.

A Side-by-Side Comparison of Wildtype and Variant Melanocortin 1 Receptor Signaling with Emphasis on Protection against Oxidative Damage to DNA.

Common variants of the MC1R gene coding the α-melanocyte stimulating hormone receptor are associated with light skin, poor tanning, blond or red hair, and increased melanoma risk, due to pigment-dependent and -independent effects. This complex phenotype is usually attributed to impaired activation of cAMP signaling. However, several MC1R variants show significant residual coupling to cAMP and efficiently activate mitogenic extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Yet, residual signaling and the key actions of wildtype and variant MC1R have never been assessed under strictly comparable conditions in melanocytic cells of identical genetic background. We devised a strategy based on CRISPR-Cas9 knockout of endogenous MC1R in a human melanoma cell line wildtype for BRAF, NRAS and NF1, followed by reconstitution with epitope-labeled MC1R constructs, and functional analysis of clones expressing comparable levels of wildtype, R151C or D294H MC1R. The proliferation rate, shape, adhesion, motility and sensitivity to oxidative DNA damage were compared. The R151C and D294H RHC variants displayed impaired cAMP signaling, intracellular stability similar to the wildtype, triggered ERK1/2 activation as effectively as the wildtype, and afforded partial protection against oxidative DNA damage, although less efficiently than the wildtype. Therefore, common melanoma-associated MC1R variants display biased signaling and significant genoprotective activity.

MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker.

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.

Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells.

Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III-IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.

Mahogunin Ring Finger 1 Is Required for Genomic Stability and Modulates the Malignant Phenotype of Melanoma Cells.

The mouse mahoganoid mutation abrogating Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase expression causes hyperpigmentation, congenital heart defects and neurodegeneration. To study the pathophysiology of MGRN1 loss, we compared Mgrn1-knockout melanocytes with genetically matched controls and melan-md1 (mahoganoid) melanocytes. MGRN1 knockout induced a more differentiated and adherent phenotype, decreased motility, increased the percentage of cells in the S phase of the cell cycle and promoted genomic instability, as shown by stronger γH2AX labelling, increased burden of DNA breaks and higher abundance of aneuploid cells. Lack of MGRN1 expression decreased the ability of melanocytes to cope with DNA breaks generated by oxidizing agents or hydroxyurea-induced replicative stress, suggesting a contribution of genomic instability to the mahoganoid phenotype. MGRN1 knockout in B16-F10 melanoma cells also augmented pigmentation, increased cell adhesion to collagen, impaired 2D and 3D motility and caused genomic instability. Tumors formed by Mgrn1-KO B16-F10 cells had lower mitotic indices, fewer Ki67-positive cells and showed a trend towards smaller size. In short-term lung colonization assays Mgrn1-KO cells showed impaired colonization potential. Moreover, lower expression of MGRN1 is significantly associated with better survival of human melanoma patients. Therefore, MGRN1 might be an important phenotypic determinant of melanoma cells.

Functional characterization of a C-terminal splice variant of the human melanocortin 1 receptor.

The melanocortin 1 receptor (MC1R) is a major determinant of skin pigmentation and sensitivity to ultraviolet radiation. When stimulated by its natural agonists, it promotes the switch from synthesis of poorly photoprotective and lightly colored pheomelanins to production of photoprotective and darker eumelanins. In addition to an unusually high number of single nucleotide polymorphisms, the MC1R is expressed as 3 protein-coding splice variants. Two transcripts display different 5' untranslated sequences but yield the same open reading frame corresponding to the canonical 317 aminoacids protein (termed MC1R). An alternative transcript named MC1R-203 encodes for a 382 amino acids protein of poorly characterized functional properties containing an additional 65 aminoacids C-terminal extension. Given the known roles of the MC1R C-terminal extension in forward trafficking, coupling to intracellular effectors and desensitization, the different structure of this domain in MC1R and MC1R-203 may lead to significant functional alteration(s). We have assessed the functional properties of MC1R-203, as compared with the canonical MC1R form. We show that unstimulated HBL human melanoma cells express the MC1R-203 spliceoform, although at much lower levels than canonical MC1R. When expressed in heterologous HEK293 cells, the presence of the 65 aminoacid-long cytosolic extension immediately after Cys316 in MC1R-203 did not impair the intracellular stability of the protein, but it interfered with functional coupling to the cAMP cascade and with the ubiquitylation of ARRB2 associated with MC1R desensitization. Conversely, MC1R-203 retained full capacity to activate ERK1/2 signaling. Accordingly, MC1R-203 displays biased signaling when expressed in HEK293 cells.

cAMP-independent non-pigmentary actions of variant melanocortin 1 receptor: AKT-mediated activation of protective responses to oxidative DNA damage.

The melanocortin 1 receptor gene (MC1R), a well-established melanoma susceptibility gene, regulates the amount and type of melanin pigments formed within epidermal melanocytes. MC1R variants associated with increased melanoma risk promote the production of photosensitizing pheomelanins as opposed to photoprotective eumelanins. Wild-type (WT) MC1R activates DNA repair and antioxidant defenses in a cAMP-dependent fashion. Since melanoma-associated MC1R variants are hypomorphic in cAMP signaling, these non-pigmentary actions are thought to be defective in MC1R-variant human melanoma cells and epidermal melanocytes, consistent with a higher mutation load in MC1R-variant melanomas. We compared induction of antioxidant enzymes and DNA damage responses in melanocytic cells of defined MC1R genotype. Increased expression of catalase (CAT) and superoxide dismutase (SOD) genes following MC1R activation was cAMP-dependent and required a WT MC1R genotype. Conversely, pretreatment of melanocytic cells with an MC1R agonist before an oxidative challenge with Luperox decreased (i) accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanine, a major product of oxidative DNA damage, (ii) phosphorylation of histone H2AX, a marker of DNA double-strand breaks, and (iii) formation of DNA breaks. These responses were comparable in cells WT for MC1R or harboring hypomorphic MC1R variants without detectable cAMP signaling. In MC1R-variant melanocytic cells, the DNA-protective responses were mediated by AKT. Conversely, in MC1R-WT melanocytic cells, high cAMP production downstream of MC1R blocked AKT activation and was responsible for inducing DNA repair. Accordingly, MC1R activation could promote repair of oxidative DNA damage by a cAMP-dependent pathway downstream of WT receptor, or via AKT in cells of variant MC1R genotype.

Human melanocortin 1 receptor-mediated ubiquitination of nonvisual arrestins. Role of Mahogunin Ring Finger 1 E3 ligase.

Signaling from the melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) crucial for melanocyte proliferation and differentiation, is regulated by cytosolic ß-arrestins (ARRBs). MC1R signaling is also negatively modulated by the E3-ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1), whose mutation causes hyperpigmentation, congenital heart defects and neurodegeneration in mice. We showed previously that although MC1R interacts stably with human ARRB1 or ARRB2, only ARRB2 mediates receptor desensitization and internalization. We analyzed MC1R-dependent ARRB ubiquitination, and the possible role of MGRN1. ARRB1 expressed in heterologous cells or human melanoma cells migrated in SDS-PAGE as a 55kDa protein whereas ARRB2 migrated as two major bands of apparent molecular weight near 45 and 55kDa, with an intermediate mobility band occasionally detected. These forms were related by post-translational modification rather than by proteolysis. Presence of MC1R favored expression of the 45kDa protein, the form that interacted preferentially with MC1R. MC1R also mediated poly- or multimonoubiquitination of ARRB2. Ubiquitination was agonist-independent, but required a native MC1R conformation and/or normal receptor trafficking to the plasma membrane, as it was not observed for loss-of-function MC1R variants. In a heterologous expression system, MC1R-dependent ARRB ubiquitination was enhanced by overexpression of MGRN1 and was impaired by siRNA-mediated MGRN1 knockdown thus pointing to MGRN1 as the responsible E3-ligase. Co-immunoprecipitation experiments demonstrated interaction of MGRN1 and ARRBs in the presence of MC1R, suggesting a scaffolding role for the GPCR that may determine the selectivity of E3-ubiquitin ligase engagement and the functional outcome of ARRB ubiquitination.

Functional interplay between secreted ligands and receptors in melanoma.

Melanoma, the most aggressive form of skin cancer, results from the malignant transformation of melanocytes located in the basement membrane separating the epidermal and dermal skin compartments. Cutaneous melanoma is often initiated by solar ultraviolet radiation (UVR)-induced mutations. Melanocytes intimately interact with keratinocytes, which provide growth factors and melanocortin peptides acting as paracrine regulators of proliferation and differentiation. Keratinocyte-derived melanocortins activate melanocortin-1 receptor (MC1R) to protect melanocytes from the carcinogenic effect of UVR. Accordingly, MC1R is a major determinant of susceptibility to melanoma. Despite extensive phenotypic heterogeneity and high mutation loads, the molecular basis of melanomagenesis and the molecules mediating the crosstalk between melanoma and stromal cells are relatively well understood. Mutations of intracellular effectors of receptor tyrosine kinase (RTK) signalling, notably NRAS and BRAF, are major driver events more frequent than mutations in RTKs. Nevertheless, melanomas often display aberrant signalling from RTKs such as KIT, ERRB1-4, FGFR, MET and PDGFR, which contribute to disease progression and resistance to targeted therapies. Progress has also been made to unravel the role of the tumour secretome in preparing the metastatic niche. However, key aspects of the melanoma-stroma interplay, such as the molecular determinants of dormancy, remain poorly understood.

MC1R signaling. Intracellular partners and pathophysiological implications.

The melanocortin-1 receptor (MC1R) preferentially expressed in melanocytes is best known as a key regulator of the synthesis of epidermal melanin pigments. Its paracrine stimulation by keratinocyte-derived melanocortins also activates DNA repair pathways and antioxidant defenses to build a complex, multifaceted photoprotective response. Many MC1R actions rely on cAMP-dependent activation of two transcription factors, MITF and PGC1α, but pleiotropic MC1R signaling also involves activation of mitogen-activated kinases and AKT. MC1R partners such as ß-arrestins, PTEN and the E3 ubiquitin ligase MGRN1 differentially regulate these pathways. The MC1R gene is complex and polymorphic, with frequent variants associated with skin phenotypes and increased cancer risk. We review current knowledge of signaling from canonical MC1R, its splice isoforms and natural polymorphic variants. Recently discovered intracellular targets and partners are also discussed, to highlight the diversity of mechanisms that may contribute to normal and pathological variation of pigmentation and sensitivity to solar radiation-induced damage. This article is part of a Special Issue entitled: Melanocortin Receptors - edited by Ya-Xiong Tao.

Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor.

The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-ß-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters.

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.

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.

MC1R is a potent regulator of PTEN after UV exposure in melanocytes.

The individuals carrying melanocortin-1 receptor (MC1R) variants, especially those associated with red hair color, fair skin, and poor tanning ability (RHC trait), are more prone to melanoma; however, the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers phosphatase and tensin homolog (PTEN) interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAF(V600E), MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes' response to UVB exposure and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis.

Differential and competitive regulation of human melanocortin 1 receptor signaling by ß-arrestin isoforms.

The melanocortin 1 receptor (MC1R) is a G-protein-coupled receptor (GPCR) crucial for the regulation of melanocyte proliferation and differentiation. MC1R activation by melanocortin hormones triggers the cAMP pathway and stimulates the extracellular-signal-regulated protein kinases ERK1 and ERK2 to promote synthesis of photoprotective eumelanin pigments, among other effects. Signaling from most GPCRs is regulated by the ß-arrestin (ARRB) family of cytosolic multifunctional adaptor proteins, which mediate signal termination and endocytosis of GPCR-agonist complexes. The ubiquitously expressed non-visual ß-arrestin1 (ARRB1) and ß-arrestin2 (ARRB2) are highly similar but not functionally equivalent. Their role in the regulation of MC1R is unknown. Using a combination of co-immunoprecipitation, gel filtration chromatography, confocal microscopy, siRNA-mediated knockdown and functional assays, we demonstrated agonist-independent competitive interactions of ARRB1 and ARRB2 with MC1R, which might also be independent of phosphorylation of Ser/Thr residues in the C-terminus of the MC1R. The effects of ARRBs were isoform specific; ARRB2 inhibited MC1R agonist-dependent cAMP production but not ERK activation, stimulated internalization and showed prolonged co-localization with the receptor in endocytic vesicles. By contrast, ARRB1 had no effect on internalization or functional coupling, but competed with ARRB2 for binding MC1R, which might increase signaling by displacement of inhibitory ARRB2. These data suggest a new mechanism of MC1R functional regulation based on the relative expression of ARRB isoforms, with possible activatory ARRB1-dependent effects arising from partial relief of inhibitory ARRB2-MC1R interactions. Thus, competitive displacement of inhibitory ARRBs by functionally neutral ARRB isoforms might exert a paradigm-shifting signal-promoting effect to fine-tune signaling downstream of certain GPCRs.

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.

N-glycosylation of the human melanocortin 1 receptor: occupancy of glycosylation sequons and functional role.

The melanocortin 1 receptor (MC1R), a major determinant of skin pigmentation and phototype, mediates the actions of α-melanocyte-stimulating hormone on melanocytes and is critical for melanocyte proliferation and differentiation. MC1R has two putative N-glycosylation targets, Asn15 and Asn29. It has been shown that MC1R is a glycoprotein with an unusual sensitivity to endoglycosidase H digestion. However, the occupancy and functional importance of each specific glycosylation sequon remains unknown. We demonstrate that MC1R is N-glycosylated at Asn15 and Asn29, with structurally and functionally different glycan chains. N-glycosylation is not necessary for high affinity agonist binding or functional coupling but has a strong effect on the availability of MC1R molecules on the plasma membrane, most likely by a combination of improved forward trafficking and decreased internalization. Finally, we found that MC1R variants exhibit different degrees of glycosylation which do not show a simple correlation with their functional status or intracellular trafficking.

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.

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.