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,1
* Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA;
Department of Medicinal Chemistry, University of Florida, Gainesville, Florida, USA;
Department of Dermatology and Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, University of Münster, Munster, Germany; and
¶ Department of Dermatology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
1Correspondence: University of Florida Department of Medicinal Chemistry, Rm. P5–26, 1600 SW Archer Rd., Gainesville, FL 32610, USA. frouzaud{at}ufl.edu
UV radiation (UVR) is among the most ubiquitous agents in the environment, and humans are inevitably exposed to it. Constitutive skin pigmentation dramatically affects the incidence of sun-induced skin cancer, and the photoprotective function of melanin in the skin is highly significant. Since epidermal melanocytes respond to UVR by increasing their expression of 
-MSH which up-regulates the expression of MC1R and consequently enhances the response of melanocytes to melanocortins, we studied the expression of MC1R in skin of different ethnic origins and in primary normal human melanocytes (NHM) displaying a large range of pigmentation phenotypes.
PRINCIPAL FINDINGS
1. Detection and characterization of MC1R isoforms expressed by NHM
Western blot was used to detect MC1R expression in 15 NHM cultures differing in their melanin content. Immunodetection by PEP19, a MC1R-specific antibody (Ab), revealed three patterns of expression. Pattern 1 had a single band at 60 kDa whereas pattern 2 revealed two sets of doublet bands: one at 30 and 36 kDa and the other at 
60 and 70 kDa. Pattern 3 showed only one band at 30 kDa and one at 60 kDa, and was the most common pattern. An 125I-MSH labeling study helped determine that all those bands can bind -MSH and that the 60 and 70 kDa bands represent a multi-MC1R complex covalent structure. To characterize at the nucleotide level the isoforms depicted in the Western blots, RACE-polymerase chain reaction (PCR) amplifications were performed using two distinct adapter-ligated cDNA libraries for each NHM culture. 5' RACE-PCR analysis revealed only one 5'untranslated region (5'UTR) of 547 bp identical for all NHM. The 3' RACE-PCR investigations revealed the typical 954 nt monoexon with a 762 nt 3'UTR, as well as a smaller fragment corresponding to a 1053 nt coding region with a 283 nt 3'UTR. The sequence translation indicates that the second transcript encodes a 350 amino acid protein, which differs from the typical 317 amino acid MC1R by the replacement of the last three residues with a new 36 amino acid C terminus sequence. Therefore, we named the first transcript MC1R317 and the second transcript MC1R350. A software-based prediction of the secondary structure of the MC1R isoforms indicated that both isoforms have the typical seven transmembrane domain profile, with the carboxyl tail potentially anchored in the melanocyte membrane via the C315 palmitoylation. However, MC1R350 harbors a total of five cysteines in its longer C-tail, each of them being a potential target for palmitic acid fixation to the membrane.
2. MC1R expression and UV response in skin from different ethnic origins
MC1R350 was detected in paraffin skin sections using PEP20, a new rabbit polyclonal antibody (pAb) we raised against the specific C terminus of that isoform. Immunofluorescence measurements indicated similar low levels of MC1R350 expression in Caucasian and Asian skin (Fig. 1
A, B), and a significant 25% higher level in African American skin. Due to the structural characteristics of the two isoforms, it is not possible to design an Ab that would recognize MC1R317 only. Therefore, any Ab that recognizes MC1R317 will also detect de facto MC1R350, revealing the cumulative expression of the two isoforms. Therefore, PEP20 and another previously reported MC1R Ab (N2–18) were used to monitor respectively MC1R350 and total MC1R expression 7 min, 1 day, and 7 days after irradiation, with 1 minimal erythemal dose (MED) of UVR. At 1 day and 7 days after irradiation, a significant increase was detected by staining with N2–18, which targets the total MC1R corresponding to the sum of MC1R350 and MC1R317 (Fig. 1C
). MC1R350 expression did not significantly vary after UV irradiation in any of the ethnic groups examined (Fig. 1D, E
). Therefore, we attribute the increase detected by N2–18 to MC1R317.
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3. Influence of MSH on MC1R expression in cultured NHM
It has been known for a long time that MSH can mimic the melanogenic effects of UVR. Confocal immunofluorescence and Western blot using SC-N19 (a commercially available MC1R Ab) and PEP19 as well as semiquantitative real-time RT-PCR were used to assess changes in MC1R isoforms expression in NHM on -MSH treatment. Fluorescence index measurements indicated that the increase in expression is much greater for MC1R317 than for MC1R350. Western blot profiles revealed that in most of the NHM tested, MC1R317 levels were increased for both the monomer and the multicomplex forms. At the mRNA level, all NHM cultures tested but one increased their total amount of MC1R and all NHM cultures, but one showed a higher relative amount of MC1R317 when stimulated by -MSH, indicating that MC1R317 is a more suitable answer to an increased melanogenesis.
4. Effects of MC1R isoforms on the melanin biosynthesis pathway
Our real-time RT-PCR study shows a good correlation between the total level of MC1R mRNA and the total melanin content in the 10 different NHM cultures tested, and that correlation is even better if we consider MC1R317 only. However, MC1R350 expression shows an inverse correlation with the total melanin content, suggesting that MC1R317 acts as a positive factor toward the total melanin content synthesis whereas the MC1R350 isoform could have an opposite action. Transient transfections of NHM with MC1R317 or MC1R350 show that while MC1R317 increases expression of MITF and tyrosinase, MC1R350 dramatically reduces them (Fig. 2
). These results show the negative impact of MC1R350 on the intracellular signal transduced to the melanin synthesis pathway.
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CONCLUSIONS AND SIGNIFICANCE
In this study, we characterized for the first time a new isoform of the human melanocortin 1-receptor bearing 350 amino acids, namely MC1R350 instead of the MC1R317 classically described. The full implications of this new isoform as well as its mechanism of action remain to be clarified. However, we have reached a first level of understanding by showing that, unlike MC1R317, relative amounts of MC1R350 mRNA correlate inversely with the total melanin content of the NHM cultures tested. This clearly shows that MC1R350 is not involved in stimulating melanin synthesis but is perhaps a negative regulator. We have further demonstrated the negative impact of MC1R350 expression on MITF and tyrosinase, two key actors of melanogenesis as summarized in Fig. 3
. When primary NHM are treated with -MSH, most of the time they increase their level of MC1R350, but in most cases they also decrease their proportion of this isoform, as large increases in MC1R317 appear to be a more suitable answer to stimulation of the melanogenic pathway in melanocytes. Therefore, one can expect that the mechanism revealed for NHM also applies physiologically in the skin. Since our studies have shown that African American skin has higher absolute levels of MC1R350 than Asian and Caucasian skin, similar to the way that -MSH-treated NHM increase their levels of MC1R350, we expect that darker skins have greater relative amounts of MC1R317. Indeed, UVR exposure does not increase MC1R350 expression but significantly increases that of MC1R317, therefore increasing its relative amount as well as melanin production. In our proposed model, the levels of melanin production appear to be regulated by the balance between the two isoforms of MC1R. The relative amounts of MC1R317 and MC1R350 act like a rheostat to control the melanin production via MITF and tyrosinase.
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It was recently reported that MC1R forms dimeric structures, and further studies are necessary to establish whether MC1R can form heterodimers in an MC1R317-MC1R350 complex. Melanocortin peptide structure-activity relationship studies are providing key insights regarding the design and synthesis of melanocortin selective agonists and/or antagonists, and tremendous effort has recently been put into the design and development of ligands for the melanocortin receptors with properties not present in the endogenous peptides, such as improved potency, receptor selectivity, and bioavailability.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5922fje
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