<scp>MC</scp>1R, the c<scp>AMP</scp> pathway, and the response to solar <scp>UV</scp>: extending the horizon beyond pigmentation

The melanocortins (MCs) α-, β- and γ-melanocortin (melanocyte-stimulating hormone; MSH) and adrenocorticotropic hormone (ACTH) are bioactive peptides derived from the large precursor protein pro-opiomelanocortin (POMC). α-MSH is known to play an important and evolutionarily conserved role in the regulation of integumental pigmentation, inducing rapid color changes in poikilotherms, fish, amphibians and reptiles, and stimulating melanogenesis in mammalian species ( Geschwind, 1966 ; Sawyer et al., 1982 ). Genetic studies conducted on mice provided unequivocal evidence that the receptor for α-MSH is the product of the extension locus, which stimulates the synthesis of eumelanin, and that loss-of-function (LOF) mutation in this locus, recessive yellow (e/e) , results in yellow coat color ( Robbins et al., 1993 ; Tamate and Takeuchi, 1984 ). In the 1960’s, Lerner and McGuire reported that injection of human subjects with α- or β-MSH increased skin darkening ( Lerner and McGuire, 1961 ; Lerner and McGuire, 1964 ). However this increase in pigmentation could not unequivocally be attributed to a direct effect of MCs on melanocytes. Prior to the cloning of the human melanocortin 1 receptor ( MC1R ) gene from cultured human melanocytes ( Chhajlani and Wikberg, 1996 ; Mountjoy et al., 1992 ) and the demonstration that these cells express functional MC1R on their cell surface, there was skepticism about a direct effect of MCs on human melanocytes. That melanocytes are a direct target for MCs and that MCs are synthesized in the skin, mainly by epidermal keratinocytes and melanocytes, strongly suggest that MCs act as physiological paracrine/autocrine regulators of human melanocytes ( Abdel-Malek et al., 1995 ; Fuller and Meyskens, Jr., 1981 ; Halaban et al., 1993 ; Hunt et al., 1994 ; Suzuki et al., 1996 ). Epidemiological studies revealed that the human MC1R is highly polymorphic, with around 200 coding region allelic variants with protein sequence alterations expressed in different human populations ( Box et al., 1997 ; Garcia-Borron et al., 2005 ; Smith et al., 1998 ). A seminal paper by Valverde et al. ( Valverde et al., 1995 ) first reported the association of specific MC1R polymorphisms with the r ed h air c olor (RHC) phenotype which also includes fair and freckled skin, impaired or absent tanning response to UVR and propensity to sunburn. MC1R variants, mainly the RHC alleles, are also associated with increased melanoma and nonmelanoma skin cancer risk ( Davies et al., 2012 ; Dessinioti et al., 2011 ; Scherer and Kumar, 2010 ). These variants are natural models of genotype-phenotype associations and their study provides information on MC1R structure-function relationships, intracellular trafficking and functional regulation. The MC1R RHC alleles result in LOF of the receptor ( Frandberg et al., 1998 ; Herraiz et al., 2009 ; Nakayama et al., 2006 ; Newton et al., 2005 ; Ringholm et al., 2004 ; Roberts et al., 2008 ; Schioth et al., 1999 ; Scott et al., 2002b ). It is now recognized that the MC1R has effects that extend beyond pigmentation, and involve activation of the DNA damage response, including DNA repair pathways in human melanocytes ( Bohm et al., 2005 ; Kadekaro et al., 2005 ; Kadekaro et al., 2010 ; Kadekaro et al., 2012 ; Maresca et al., 2010 ; Song et al., 2009 ). Linking the MC1R not only to the regulation of skin pigmentation, but also to DNA repair pathways, which are pivotal for prevention of photocarcinogenesis, represented a shift in paradigm, and provided an explanation for how MC1R functions as a melanoma predisposition gene, and for why expression of RHC variants increases melanoma risk. This current knowledge about the various effects of MC1R makes it an attractive target for chemoprevention of photocarcinogenesis, including melanoma. Genetic studies describing the penetrance and interactions of common allelic MC1R variants have been recently reviewed ( Beaumont et al., 2011 ), and key aspects of MC1R structure were discussed in a previous review ( Garcia-Borron et al., 2005 ). Interest in understanding the functions of the MC1R, regulation of its expression, its signaling pathways, and the mechanisms by which it affects the UVR response has led to important findings that substantiate its central role in regulating human melanocytes and skin cancer (particularly melanoma) predisposition. Therefore, we will focus this review on the regulation of MC1R gene expression and the biosynthesis and intracellular trafficking of the receptor as well as on the various signaling pathways downstream of MC1R and their regulation by specific endogenous ligands or promiscuous GPCR partners such as GPCR kinases (GRKs) and cytosolic β-arrestins (ARRBs). We also present a comprehensive list of mutant alleles corresponding to the natural protein sequence variants described to date. Finally, we will summarize recent insights on the intracellular pathways responsible for the protective role of MC1R against UVR-indu

Compared with most GPCR genes, the structure of MC1R is relatively complex, with occurrence of several splice variants, and a bewildering degree of polymorphism. Although the genes encoding for most GPCRs were for a long time considered intronless, it now appears that approximately 50% of GPCR genes possess at least one intron and can undergo alternative splicing ( Markovic, 2013 ; Markovic and Challiss, 2009 ). The MC1R (MIM# 155555, ID ENSG00000258839) located at 16q24.3 was initially reported as intronless, with a major 2.3 kb transcript and a 951 nucleotides (nt) coding region ( Smith et al., 2001 ). This canonical MC1R transcript (ID ENST00000555147, named MC1R-001) encodes for a 317 amino acid integral transmembrane protein with all the hallmarks of Class A GPCRs ( Katritch et al., 2013 ; Venkatakrishnan et al., 2013 ), namely seven transmembrane fragments, a potentially glycosylated extracellular N-terminus, three extracellular and three intracellular (il) loops and a cytosolic C-terminal extension ( Figure 1 ). The MC1R-001 open reading frame (ORF) predicts that the N-terminal fragment and the extracellular loops are short, compared with most other GPCRs, which may have functional consequences by contributing to a high constitutive activity ( Garcia-Borron et al., 2005 ; Holst and Schwartz, 2003 ). The cytosolic tail of MC1R-001, predicted to extend from His301 to Trp317, is also short. Further studies established that the MC1R gene comprises 4 exons and yields at least one more protein coding transcript. Tan and coworkers first reported an alternative spliced form, designated MC1R-002 (ID ENST00000555427), with a 1149 nt-long ORF ( Tan et al., 1999 ). The resulting 382 amino acids protein is identical to the canonical MC1R up to Ser316, followed by a 66 amino acids C-terminal extension. At least another splice variant has been reported ( Rouzaud et al., 2006 ), which shares with MC1R-001 and MC1R-002 the sequence up to residue Ser316, but the additional C-terminal extension is different from MC1R-002. This structure suggests splicing of the same 5’ exon to a different acceptor site. The occurrence of the new splice variant, named MC1R350, was confirmed in cultured NHMs and skin sections from donors of different ethnic origin ( Rouzaud et al., 2006 ). No specific polymorphisms have been described to date in association with the MC1R splice variants. The 16q24 region is a high density area containing the NULP1, MC1R and Tubulin beta III ( TUBB3 ) genes ( Smith et al., 2001 ), with less than 8 kb separating the coding 3’ end for the upstream NULP1 gene and the MC1R initiation codon, and just 2.5 kb of intergenic DNA between MC1R and its downstream TUBB3 neighbor. In addition, human MC1R has an unusual and inefficient polyadenylation signal ( Dalziel et al., 2007 ). These characteristics are compatible with intergenic splicing, and two intergenic splice products have been reported ( Dalziel et al., 2011 ). One of them contains 2 MC1R exons fused to 3 TUBB3 exons. The resulting 797 amino acids protein is an in-frame fusion containing the first 366 residues of MC1R-002 and most of the TUBB3 sequence ( MC1R - TUBB3 gene, ID ENSG00000198211). The other intergenic chimera arises by out-of-frame fusion of MC1R and exon 2 of TUBB3 , yielding a protein where the first 316 residues correspond to the MC1R sequence and the remaining 116 C-terminal residues share no significant homology with known proteins. The relative levels of the various MC1R -derived transcripts may be modified by external signals. The canonical MC1R-001 transcript is induced in α-MSH-treated NHM more strongly than MC1R350 ( Rouzaud et al., 2006 ). Stimulation of melanoma cells with α-MSH or activation of p38 increases the expression of MC1R-TUBB3 fusion chimeras ( Dalziel et al., 2011 ). MC1R-001 and the alternatively spliced MC1R-002 isoform share most of the functionally relevant structural elements, suggesting that the proteins might be pharmacologically similar ( Tan et al., 1999 ). However, expression of the MC1R350 isoform was negatively correlated with total melanin contents in NHM cultures, and forced expression of MC1R350 led to decreased levels of MITF and tyrosinase, suggesting that this splice variant is inactive or even behave as a dominant-negative form ( Rouzaud et al., 2006 ). On the other hand, the properties of MC1R-TUBB3 chimeric proteins are still poorly characterized, but it would appear that whereas the longer in-frame fusion product retains at least part of the signaling potential, the out-of-frame chimera is not functional. Therefore, environmental cues might regulate MC1R signaling by modifications of the relative proportions of different transcripts. However, a detailed comparison of the signaling properties of the resulting proteins is still lacking, and the relative levels of the various transcripts have not yet been determined. Accordingly, the function and physiological relevance of non-canonical MC1R forms rema

After being assembled in the rough endoplasmic reticulum (ER), newly synthesized MC1R undergoes a series of post-translational modifications including oligomerization, glycosylation and, most likely, palmitoylation and phosphorylation. Since the density of MC1R molecules on the cell surface of NHMs is rather low, with around 1000 receptors per cell ( Roberts et al., 2006 ), and most likely a limiting factor for generation of cAMP ( Mas et al., 2003 ), alterations in MC1R processing have a significant impact on the cellular responses to agonists. Many GPCRs exist as dimeric or oligomeric species ( Audet and Bouvier, 2012 ). Oligomerization of newly synthesized monomers is important for anterograde trafficking and may modulate the functional properties of the protein. Concerning the MCR subfamily, dimerization has been demonstrated for MC1R ( Mandrika et al., 2005 ; Sanchez-Laorden et al., 2006a ; Zanna et al., 2008 ), MC2R ( Roy et al., 2010 ; Sebag and Hinkle, 2009 ), MC3R ( Mandrika et al., 2005 ) and MC4R ( Biebermann et al., 2003 ; Piechowski et al., 2013 ). SDS-resistant MC1R dimers and oligomers are found in detergent-solubilized extracts from melanoma cells or heterologous cells overexpressing the MC1R ( Sanchez-Laorden et al., 2006a ). The monomeric units may establish both non-covalent domain swap-type interactions and covalent intermolecular disulphide bonds ( Zanna et al., 2008 ). The minor effect of agonist treatment on the ratio of monomeric and oligomeric species detected by SDS-PAGE suggests that dimerization is constitutive. Dimeric species are found in the ER, showing that oligomerization is an early step in MC1R maturation. Co-immunoprecipitation and functional experiments performed in heterologous cell models show that MC1R variants can heterodimerize with the WT protein, thereby modulating its functional properties ( Sanchez-Laorden et al., 2006a ). This may account for dominant-negative effects detected in genetic studies ( Beaumont et al., 2007 ). Human MC1R contains two potential N-glycosylation sequons, 15 NST 17 and 29 NQT 31 , whose occupancy has been analyzed ( Herraiz et al., 2011b ). Complete removal of oligosaccharide chains with peptide-N4-(acetyl-β-glucosaminyl)-Asparagine amidase (PNGaseF), or simultaneous mutation to Gln of the two potential acceptor residues Asn15 and Asn29, lowers the apparent molecular weight of the protein, determined by SDS-PAGE, from ~35 to ~ 29 kDa. Mutation of each potential acceptor individually causes a smaller change, thus showing that both sites are occupied in the WT protein. The glycan chains bound to each glycosylation site are not functionally equivalent in that cell surface expression is severely compromised for the N29Q mutant, whereas the N15Q mutation has, at best, a marginal effect. Occupancy of the 15 NST 17 glycosylation sequon is striking in that is does not adhere to the C-terminal Pro rule stating that a Pro residue immediately C-terminal to a potential glycosylation site abolishes its occupancy ( Bause, 1983 ). Also unexpectedly, mature and active WT MC1R is sensitive to endoglycosidase H (Endo H) digestion ( Herraiz et al., 2011b ; Perez Oliva et al., 2009 ). This enzyme cleaves core high-mannose N-glycan chains and hybrid-type chains present in incompletely processed forms of glycoproteins found in the ER, but not the complex oligosaccharides found in the Golgi network. Thus, resistance to EndoH digestion is often taken as indicative of normal processing and trafficking beyond the ER, but this rule does not hold for MC1R. Natural MC1R variants are differentially glycosylated, at least in terms of the ratios of glycosylated to native protein ( Herraiz et al., 2011b ). Unfortunately, sensitivity to EndoH of WT MC1R excludes this enzyme as a reliable tool to compare trafficking and subcellular compartmentalization of mutant forms. Acylation of specific residues in the C-terminal cytosolic extension is frequent in GPCRs ( Qanbar and Bouvier, 2003 ), but there is no direct evidence that it occurs in the MC1R. Nevertheless, in silico analysis suggests that Cys315 is a high probability palmitoylation site. Mutation of Cys315 to Ala impairs significantly the number of MC binding sites and agonist-mediated stimulation of cAMP synthesis ( Sanchez-Mas et al., 2005b ), and a Cys315Gly mutation causes complete LOF by disrupting the signaling of the receptor but not its binding to agonists ( Frandberg et al., 2001 ). Phosphorylation of specific residues seems to be critical for MC1R trafficking and cell surface expression, and known MC1R mutations might inhibit phosphorylation and thus reduce the number of MC1R per cell. Eight Ser/Thr residues located in intracellular segments of MC1R provide potential phosphorylation targets ( Figure 1 ). Except for Ser71, which lays in il1, these Ser/Thr residues form two clusters. One is located in the short C-terminal tail (residues Ser302, Thr308, Thr314, Ser316) and the other in il2 (Ser145, Ser154 and T

Like most GPCRs, MC1R is coupled to several intracellular signaling cascades. It is established that binding of α-MSH (or ACTH in the case of the human receptor) to MC1R results in its functional coupling to the cAMP pathway, evidenced by sequential activation of the Gs protein and adenylate cyclase (AC), leading to increased cAMP formation ( Figure 2 ) ( Pawelek et al., 1973 ; Suzuki et al., 1996 ; Wong and Pawelek, 1973 ). Increased levels of cAMP lead to the activation of PKA, followed by phosphorylation of members of the cAMP responsive-element binding protein (CREB) family of transcription factors, and CREB-mediated activation of Microphthalmia transcription factor ( MITF ) gene expression ( Bertolotto et al., 1998 ; Price et al., 1998 ). MITF is a key positive regulator of melanocyte differentiation markers including the melanogenic enzymes ( Levy et al., 2006 ). Thus, the second messenger cAMP is critically involved in the regulation of mammalian pigmentation and it also plays a central role in UVR-induced tanning ( Busca and Ballotti, 2000 ; D'Orazio and Fisher, 2011 ; Im et al., 1998 ). Several lines of evidence indicate that cAMP is the main intracellular messenger responsible for the melanogenic actions of α-MSH ( Busca and Ballotti, 2000 ). cAMP-elevating agents such as the natural diterpene forskolin (FSK) stimulate tyrosinase activity, which triggers the switch from pheomelanogenesis to synthesis of eumelanins and mimics many of the effects of α-MSH ( Im et al., 1998 ; Ito and Wakamatsu, 2003 ; Ito and Wakamatsu, 2011 ; Kadekaro et al., 2010 ; Scott et al., 2002a ). Application of FSK onto recessive yellow mice harbouring the LOF mutation MC1R e/e stimulated eumelanogenesis, and rescued the yellow color phenotype by directly activating AC ( D'Orazio et al., 2006 ). Similarly, treatment of NHMs expressing LOF RHC MC1R alleles with FSK markedly increased tyrosinase activity, indicative of stimulation of eumelanin synthesis ( Kadekaro et al., 2010 ; Scott et al., 2002a ). Mouse and human MC1R display significant agonist-independent constitutive coupling to the cAMP pathway in heterologous cells overexpressing the WT proteins ( Sanchez-Mas et al., 2004 ). For human MC1R overexpressed in HEK cells, agonist-independent cAMP production can reach 40% maximal values obtained after agonist stimulation, within the range of the constitutively active E92K natural MC1R Eso-3J ( somber ) allele associated with hypermelanized coats, or other artificial constitutively active mutants ( Lu et al., 1998 ; Sanchez et al., 2002 ). Constitutive coupling of MC1R to cAMP has been recently confirmed ( Benned-Jensen et al., 2011 ), and may account for certain direct effects of the inhibitory ligand ASIP, which behaves as an inverse agonist rather than a competitive antagonist ( Chluba-de et al., 1996 ; Graham et al., 1997 ; Hida et al., 2009 ; Le Pape et al., 2009 ; Ollmann et al., 1998 ; Sakai et al., 1997 ; Siegrist et al., 1997 ; Suzuki et al., 1997 ). Interestingly, POMC -null mice show significant eumelanin pigmentation, suggesting that constitutive signaling from MC1R is high enough to support eumelanogenesis ( Slominski et al., 2005 ). Alternatively, other paracrine factor might compensate for loss of POMC. The situation for human melanocytes is less clear-cut. Constitutive levels of cAMP in melanocytes expressing functional or non-functional MC1R were found to be similar ( Kadekaro et al., 2010 ). These conflicting results might be explained by the level of expression of MC1R, which is low in human melanocytes, with around 1000 receptors per cell ( Roberts et al., 2006 ), and is expected to be much higher in MC1R transfected cells. Whereas expression of MC1R rescues the pigmentation phenotype of MC1R e/e mice bearing a completely inactive truncated mutant ( Healy et al., 2001 ), patients with mutations in the POMC gene abolishing translation, or interfering with the production or activity of MCs have red hair ( Krude et al., 1998 ; Samuels et al., 2013 ). However, these studies did not report the MC1R genotype of the patients, thus complicating the interpretation of the phenotype. The cAMP pathway is surprisingly complex, given that there are 10 different AC isoenzymes ( Cooper, 2003 ), 4 functionally non-redundant PKA regulatory subunits, and 3 catalytic PKA ( Taylor et al., 2012 ). The cAMP-degrading phosphodiesterases (PDEs) also form a relatively large family of proteins with different catalytic and regulatory properties, encoded by 11 different genes ( Conti and Beavo, 2007 ; Houslay, 2010 ). A further layer of complexity and specificity is provided by the prevalence of scaffold proteins that mediate formation of signaling complexes to regulate not only the substrates of PKA but also the spatiotemporal organization of specific hubs of cAMP signalling ( Houslay, 2010 ; Taylor et al., 2012 ). Despite the significance of the cAMP pathway in melanocyte biology, the specific components and organization of this s

The observation that some NHMs in culture failed to respond to α-MSH suggested that this might be due to a LOF mutation in the MC1R , and led to the first investigation of SNPs in the MC1R gene in British and Irish individuals ( Valverde et al., 1995 ). This was followed by a large twin study in Australia that solidified the association of three MC1R variants, R151C, R160W, and D294H, with red hair color ( Box et al., 1997 ). Other studies ensued from different countries, which established the association of the above variants with RHC phenotype and also with increased melanoma risk ( Ichii-Jones et al., 1998 ; Kennedy et al., 2001 ; Palmer et al., 2000 ). Subsequent studies have shown that the MC1R is extraordinarily polymorphic ( Figure 1 ) ( Gerstenblith et al., 2007 ; Guan et al., 2013 ; Kanetsky et al., 2006 ; Savage et al., 2008 ), with around 200 coding region variants altering the protein sequence. In addition, 50 synonymous changes and another 50 polymorphisms in the 5´- and 3´’ UTRs have been described to date. Nonsynonymous coding region polymorphisms and the corresponding nucleotide changes are listed in Supplemental Table 1 . This unusual nucleotide diversity has been attributed to a high CpG content which increases mutation rates, in combination with some degree of positive selection for mutant alleles associated with lighter skin pigmentation in regions of lower UVR incidence ( Jablonski and Chaplin, 2010 ; Martinez-Cadenas et al., 2013 ). Indeed, the frequencies of specific alleles show significant variations in different populations. The consensus sequence for the MC1R coding region is more common in African populations, whereas nonsynonymous variants are at higher frequency in Eurasian individuals ( Harding et al., 2000 ; Martinez-Cadenas et al., 2013 ; Rana et al., 1999 ). Following the discovery of the association of variant alleles with the RHC phenotype ( Valverde et al., 1995 ) and melanoma risk ( Valverde et al., 1996 ), MC1R variants have been reported to be associated with other cutaneous phenotypes such as oculocutaneous albinism ( Saleha et al., 2013 ), congenital melanocytic nevi ( Kinsler et al., 2012 ), vitiligo ( Szell et al., 2008 ), piebaldism ( Oiso et al., 2009 ) or severe photoaging of facial skin ( Elfakir et al., 2010 ). Moreover, the increase in the density of melanocytes in human skin exposed to UVR is significantly reduced in carriers of MC1R variants ( Hacker et al., 2013 ). Associations with unrelated traits or diseases have also been described, including glucocorticoid deficiency ( Turan et al., 2012 ), follicular lymphoma ( Kane et al., 2010 ), depression and antidepressant response ( Wu et al., 2011 ), radiotherapy side effects ( Fogarty et al., 2010 ), anesthetic requirements and recovery time after surgery ( Myles et al., 2012 ), acute pain and pain from inflammatory origin ( Delaney et al., 2010 ; Oertel and Lotsch, 2008 ), multiple sclerosis ( Strange et al., 2010 ) and Parkinson’s disease risk ( Gao et al., 2009 ). MC1R variants whose frequency is high enough to allow for statistically significant association studies have been classified into groups according to their penetrance for the RHC phenotype: strong and penetrant “R” alleles, and the weaker “r” forms, and the pseudoalleles that do not have any noticeable phenotype ( Duffy et al., 2004 ). Extensive evidence shows that all the R alleles yield hypomorphic proteins with decreased or even absent ability to activate the cAMP pathway, when assayed in heterologous expression systems, human melanoma cells or NHMs of defined genotype ( Frandberg et al., 1998 ; Herraiz et al., 2009 ; Kadekaro et al., 2010 ; Nakayama et al., 2006 ; Newton et al., 2005 ; Ringholm et al., 2004 ; Roberts et al., 2008 ; Schioth et al., 1999 ; Scott et al., 2002b ). For the R alleles overexpressed in heterologous systems, both basal and agonist-dependent coupling to cAMP synthesis is impaired, with varying degrees of residual activity ranging from an almost complete loss of functional coupling for D84E and D294H, to 25–50% residual activity for other forms, particularly R160W. Decreased cell surface expression seems the primary cause of LOF for the major RHC alleles D84E, R151C, I155T and R160W ( Beaumont et al., 2005 ; Herraiz et al., 2012 ; Schioth et al., 1999 ). Evidence for extensive intracellular retention with preferential association of R151C with the ER and R160W with the Golgi apparatus has also been provided ( Sanchez-Laorden et al., 2009 ). However, plasma membrane density of R142H is normal and D294H is expressed at even higher cell surface levels than WT. Moreover, both variants bind MC agonists with high affinity, thus suggesting that for these R alleles the molecular defect lies in the inability to undergo the agonist-induced transition to an active conformation. The best characterized r alleles are V60L, V92M and R163Q. The V60L variant is found at frequencies as high as 20% in some European populations, but is mu

Since the early 1990’s, there has been great advancement in our knowledge about MC1R, the MCs, and the significance of their physiological role, particularly in prevention of photocarcinogenesis. It is now established that human MC1R is a main determinant of the diversity of human pigmentation. That human melanocytes express functional MC1R, and that the activated MC1R enhances the repair of UVR-induced DNA damage in addition to its classic role in regulating eumelanin synthesis, strongly suggests that MC1R can be directly targeted in a skin cancer, including melanoma chemoprevention strategy. For many years, there has been interest in developing potent α-MSH analogs for safer sunless tanning. The best known α-MSH analog is the tridecapeptide [Nle4, D-Phe7]-α-MSH (NDP-MSH; melanotan or afamelanotide). A clinical trial with systemic application of melanotan showed that in addition to the desired stimulation of pigmentation in the absence of any sun exposure, the analog had several unexpected effects, including nausea and loss of appetite (Levine et al., 1991). These side effects were due to the ability of the analog to bind and activate other MCRs. Nonetheless, NDP-MSH is currently being used on patients with polymorphic light eruption, and tested for treatment of vitiligo ( Fabrikant et al., 2013 ). To alleviate the systemic effects of non-selective α-MSH analogs, novel analogs that are highly selective and specific to MC1R, small in size and lipophilic need to be developed to allow for topical delivery to the skin, and for primarily targeting the melanocytes. Towards this goal, potent tetrapeptide analogs of α-MSH, n-capped-His-D-Phe-Arg-Trp-NH 2 have been developed and tested on NHMs in vitro . Two of these tetrapeptides, 4-phenylbutyryl- and n-pentadecanoyl-His-D-Phe-Arg-Trp-NH 2 , proved to be 100 fold more potent than the full-length physiological hormone α-MSH in activating the MC1R, based on activating cAMP formation, and stimulation of tyrosinase activity, and surpassed α-MSH in enhancing the repair of DNA photoproducts and reducing ROS generation and apoptosis in UVR-irradiated melanocytes. These effects were mediated by activation of the MC1R, and were not evident in melanocytes expressing LOF MC1R ( Abdel-Malek et al., 2006 ). Further testing of n-pentadecanoyl-His-D-Phe-Arg-Trp-NH 2 showed that it is efficacious in stimulating pigmentation, and repairing UVR-induced CPDs in a 3D-skin model. This analog also proved to be highly selective to MC1R, and to have the ability to diffuse through human skin, providing confidence that it can be employed in a strategy to protect from the mutagenic and carcinogenic effects of UVR. Tripeptide α-MSH analogs have also been developed, and found to retain full melanotropic activity, being only 10 fold less potent that α-MSH ( Abdel-Malek et al., 2009 ). This can be compensated for by the low molecular weight of these peptides that should allow for using then at a high concentration in the micromolar range. The significance of targeting the MC1R on melanocytes by selective MC analogs lies in their efficacy in protecting individuals with high risk to skin cancer, including melanoma, from the carcinogenic effects of UVR. Such individuals include those heterozygous for a RHC allele, or harboring mutations in known melanoma-associated genes, such as p16. The identification of MC1R alleles strongly associated with the RHC phenotype and with increased skin cancer risk has fuelled research on the functional properties of WT and variant MC1R, as well as on downstream targets involved in the regulation of pigmentary and non-pigmentary mechanisms to deal with UVR-induced damage. These studies are urgent given the importance of MC1R signaling in prevention of UV-induced skin cancers, mainly melanoma. The signaling properties of the various intra- and intergenic splice forms of the MC1R gene need further analysis to fully understand the physiological output of changes in their relative levels of expression. Our current knowledge of the MC1R life cycle still has important gaps, notably concerning the site and the molecular machinery of its proteolytic degradation. Given that recent research underlined important differences in the behavior of mouse MC1R compared with human MC1R, several current paradigms call for reevaluation. In this respect the functional coupling to the ERK pathway needs further research, since the molecular event(s) leading to transactivation of the cKIT receptor, the differential regulation of signaling to ERKs or cAMP, and the physiological consequences of ERK activation downstream of MC1R remain poorly understood. Efforts will also be made to fully understand the molecular basis of the different penetrance of r versus R alleles, and to find functional criteria to classify rare alleles in one of these two categories. An immediate task will be to define the possible role of MC1R as the hub of a signaling complex undergoing dynamic changes. For instance, r