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

Abstract

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-β-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.

Full text

RESEARCH ARTICLE
Functional Characterization of MC1R-TUBB3
Intergenic Splice Variants of the Human
Melanocortin 1 Receptor
Cecilia Herraiz*, Conchi Olivares, Maria Castejón-Griñán, Marta Abrisqueta,
Celia Jiménez-Cervantes, José Carlos García-Borrón
Department of Biochemistry, Molecular Biology and Immunology, School of Medicine, University of Murcia,
Campus de Espinardo, Murcia and IMIB-Arrixaca, El Palmar, Murcia, Spain
*ceciliahs@um.es
Abstract
The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determi-
nant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melano-
cyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing
intergenic splicing with its downstream neighbour Tubulin-β-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 thanpredicted, and impaired
intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affin-
ity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased sur-
face expression mostly resulted from aberrant forward trafficking, rather than high rates of
endocytosis. Functional coupling of both isoforms tocAMP 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 iso-
forms 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.
Introduction
The melanocortin 1 receptor (MC1R), a major determinant of skin phototype, is a G protein-
coupled receptor (GPCR) that regulates pigment production in melanocytes. When stimulated
by α-melanocyte stimulating hormone or related peptides (the melanocortins, MCs) MC1R
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 1/19
OPEN ACCESS
Citation: Herraiz C, Olivares C, Castejón-Griñán M,
Abrisqueta M, Jiménez-Cervantes C, García-Borrón
JC (2015) Functional Characterization of MC1R-
TUBB3 Intergenic Splice Variants of the Human
Melanocortin 1 Receptor. PLoS ONE 10(12):
e0144757. doi:10.1371/journal.pone.0144757
Editor: Mauro Picardo, San Gallicano Dermatologic
Institute, ITALY
Received: September 10, 2015
Accepted: November 23, 2015
Published: December 11, 2015
Copyright: © 2015 Herraiz et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This work was supported by grant
SAF2012-32134 (to JCGB) from the Comisión
Interministerial de Ciencia y Tecnología, Spain,
FEDER funds (European Community). CH holds a
post-doctoral fellowship from the University of Murcia.
MC is a fellow of the Fundación Seneca (Agencia de
Ciencia y Tecnologia de la Región de Murcia, Spain).
Competing Interests: The authors have declared
that no competing interests exist.

triggers cAMP synthesis leading to activation of the rate-limiting melanogenic enzyme tyrosi-
nase. MC signaling also activates the MAP kinase module leading to ERK1 and ERK2, by a
cAMP-independent mechanism involving transactivation of cKIT [1]. The cAMP and ERK
pathways cooperate to regulate expression, activity and stability of Microphthalmia (MITF)
transcription factor, a key positive modulator of melanocyte differentiation [2] that also regu-
lates expression of the cell cycle regulatory proteins p21 and p27 [3,4]. Increased tyrosinase
activity in response to MC1R activation leads to the synthesis of dark eumelanin pigments as
opposed to reddish pheomelanins, so as to increase the ratio of photoprotective eumelanins to
pro-oxidant pheomelanins [5], thus providing an effective shield against mutagenic ultraviolet
radiation (UVR). Moreover, MC1R orchestrates a complex series of events that coordinately
improve antioxidant defenses and DNA repair mechanisms in UVR-exposed melanocytes
(reviewed in [6]). In addition to direct effects on melanocytes, UVR activates transcription of
the POMC gene in keratinocytes followed by release of MC peptides, thus achieving a paracrine
stimulation of melanocytes [7].
The genes encoding for most GPCRs were thought to be most frequently intronless [8], but
current evidence shows that over 50% of GPCR genes contain more than one intron [9]. The
MC1R (MIM# 155555, ID ENSG00000258839) located at 16q24.3 is in fact quite complex as
it contains 4 exons and yields several transcripts as a result of intra- and intergenic splicing,
with usage of alternative splice donor-acceptor sites, retention of intronic sequences and
skipping of exons and of translation termination and polyadenylation signals. The canonical
2.3 kb MC1R transcript containing a 951 nucleotides (nt) coding region [10] (Ensemble ID
ENST00000555147, named MC1R-001) encodes for a 317 amino acid integral transmembrane
protein with the typical structural characteristics of Class A GPCRs [9,11], and contains exons
2, 3 and 4 with retention of unspliced intervening sequences located between exons 2–3 and
3–4(Fig 1A and 1B). On the other hand, Tan and coworkers [12] reported an alternative
spliced MC1R form designated MC1R-002 (ID ENST00000555427), which contains exons 1–4
resulting in a 1149 nt-long ORF encoding for a 382 amino acids protein. This splice isoform is
identical to MC1R-001 up to Ser316, followed by an additional 65 amino acids C-terminal
extension. Finally, the MC1R-003 transcript (ENST00000539976) lacks a functional open read-
ing frame and is most likely a non-coding defective transcript.
In addition to these intragenic splice isoforms, a number of potentially functional intergenic
splice variants involving the MC1R gene have been described [13]. These intergenic splice vari-
ants would arise as a result of the high gene density in the 16q24 region [10], where less than
8 kb separate the coding 3’end of the next upstream gene and the MC1R initiation codon, and
the intervening DNA fragment located between MC1R and the downstream TUBB3 is only
2.5 kb-long. This dense packing, and the presence of an unusual and inefficient polyadenyla-
tion signal in human MC1R have been reported to promote intergenic splicing to the TUBB3
gene [13,14]. Two intergenic splice products have been described to date [13]. One of them
contains MC1R exons 3 and 4 fused to TUBB3 exons 3, 4 and 5 (Fig 1A). This transcript
(MC1R-TUBB3 gene, ID ENSG00000198211) encodes for a 797 amino acids in-frame fusion
chimera named Iso1, corresponding to the first 366 residues of MC1R-002 and most of the
TUBB3 sequence (Fig 1B). The other intergenic splice variant is an out-of-frame fusion of
MC1R exon 3 and exon 3 of TUBB3 (Fig 1A). The size of the predicted Iso2 protein product is
432 amino acids, with the first 316 amino acids matching the MC1R sequence. The remaining
116 C-terminal residues in this chimera share no homology with known proteins (Fig 1B)[13].
Since both Iso1 and Iso2 proteins virtually conserve all the structural elements in MC1R
known to be important for agonist binding and coupling to downstream signaling pathways
[6], they might retain a significant signaling potential. Interestingly, treatment of cultured
melanocytes with αMSH or activation of p38-MAPK, both key molecules associated with UVR
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 2/19

Characterization of MC1R-TUBB3 Intergenic Splice Variants
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responses, shifts expression from MC1R-001 in favor of chimeric MC1R-TUBB3 isoforms
[13]. Accordingly, the intergenic chimera might contribute to fine-tune the complex array of
melanocytic adaptive responses to UVR insults orchestrated by the MC1R. However, their
functional properties remain largely unknown. Here we report a study of the trafficking and
signaling properties of the MC1R chimeric proteins that may shed light on their possible physi-
ological role.
Materials and Methods
Materials
Igepal CA-630, BSA, EDTA, PMSF, iodoacetamide, bicinchoninic acid, anti-FLAG M2–Peroxi-
dase conjugate antibody, anti-HA-peroxidase conjugate and anti-β-Tubulin III antibody were
from Sigma (St. Louis, MO). The anti-pERK1/2 and anti-ERK2 rabbit polyclonal IgGs were
from Santa Cruz Biotechnology (Santa Cruz, CA). The synthetic αMSH analogue [Nle4,
D-Phe7]-αMSH (NDP-MSH) and the protein synthesis inhibitor cycloheximide were from
Calbiochem (Darmstadt, Germany). The radioligand [
125
I]-NDP-MSH, specific activity 2000
Ci/mmol was from Amersham (Little Chalfont, Buckinghamshire, UK). The cAMP immuno-
assay kit was from Arbor Assays (Eisenhower Place, Michigan, USA). Lipofectamine 2000 was
from Invitrogen (Carlsbad, CA). Reagents for SDS-PAGE and Western blot were from Bio-
Rad (Richmond, CA, USA). Other reagents were from Merck (Darmstadt, Germany) or Pro-
labo (Barcelona, Spain).
Cell culture, transfection and functional expression
HEK293T cells, PC12 cells and all human melanoma cells were grown in Dulbecco’s modified
Eagle’s medium enriched with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml
streptomycin sulfate.
All expression constructs were prepared in pcDNA3 (Invitrogen). The following expression
constructs have been described: the Flag-tagged wild-type (WT) MC1R-001 [15], R151C and
D294H [16], V60L and V92M [1]. N-terminal 3xHA-labeled MC1R-001 construct was from
the Missouri University of Science and Technology cDNA Resource Center (Rolla, MO).
MC1R/TUBB3 locus constructs inserted into the pcDNA3.1-His expression vector were
kindly provided by Prof A. Furger (University of Oxford, UK) and have been previously
described [13]. To generate the Flag-tagged constructs, MC1R-TUBB3 chimeric transcripts
were subcloned into pcDNA3 (Invitrogen) using EcoRI and XbaI as restriction enzymes. Site-
directed mutagenesis using the QuickChange kit (Stratagene, La Jolla, CA) was performed to
ablate BamHI restriction site within TUBB3 gene in pIso1 and pIso2 (primer pIso1: 5’-CTACT
TCGTGGAGTGGATTCCCAACAACGTGAA-3’and primer pIso2: 5’-CTACTTCGTGGAG
TGGCTCCCCAACAACGTG-3’). N-terminal Flag epitope Iso1 and Iso2 constructs were
obtained using a Flag-WT MC1R-001 as a template [17] and cleaving with BamHI and XbaI.
Constructs were verified by automated sequencing in both strands.
Fig 1. MC1R transcripts and intergenic splice isoforms of MC1R and TUBB3. (A) Schematic panel showing the exon organization of MC1R splice
variants (MC1R-001 and MC1R-002) and MC1R-TUBB3 chimeric transcripts, Iso1 and Iso2. Exons of all MC1R derived transcripts are represented in
colored boxes and the number of nucleotides in the ORF is shown below. (B) Diagram representing the structural domains of MC1R-001, MC1R-002, β-
tubulin III (TUBB3) and chimeric proteins Iso1 and Iso2. Structural and functional domains are depicted in colored boxes and the number of key residuesin
the proteins is shown. TM indicates transmembrane regions of MC1R. Dashed lines indicate residues of MC1R and TUBB3 linked in fused proteins Iso1 and
Iso2. (C) MC1R-001, Iso1 and Iso2 expression in human melanoma cell lines. Data are shown as relative expression of each isoform (as indicated in each
bar graph) as compared with the levels of the isoform in HBL cells. (D) Expression of Iso1 and Iso2 mRNA as a functionof the levels of the canonical MC1R-
001 transcript in a panel of human melanoma cell lines. Data are represented as mRNA expression of the two intergenic splicing forms relative to MC1R-001
in each cell line.
doi:10.1371/journal.pone.0144757.g001
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 4/19

HEK293T cells grown to approximately 80% confluence were transfected with 0.6 or 0.3 μg
of plasmid DNA/well for 6 or 12-well plates, respectively, using Opti-MEM to dilute DNA and
Lipofectamine (Invitrogen). HBL cells were transfected with 1 or 0.5 μg of plasmid DNA/well
for 6 or 12-well plates, respectively.
Binding and internalization assays
Radioligand binding assays have been described [17–19]. Cells grown in 12-well plates were
transfected as required. Twenty-four hours after transfection, cells were serum deprived for 3 h
and incubated with
125
I-labelled NDP-MSH (5x10
-11
M) and increasing concentrations of
non-labelled competing NDP-MSH, from 10
−12
to 10
−7
M. To estimate internalization of ago-
nist–receptor complexes, an acid wash procedure was used [20]. Cells were incubated (90 min,
37°C) with [
125
I]-NDP-MSH isotopically diluted to a final concentration of 10
−9
M and 10
5
cpm, washed with cold serum-free DMEM followed by two 2–3 min ice-cold acid washes with
0.5 ml of 50 mM glycine and 150 mM NaCl, pH 3.0. The acid washes were pooled and counted
to determine the amount of non-internalized ligand bound on the cell surface. Cells were tryp-
sin-harvested and counted for internalized receptor. Internalization indexes were defined as
the percentage of internal relative to total ligand bound.
Functional assays
For cAMP assays, HEK293T cells were grown in 12-well plates, transfected with MC1R-001
(WT, V60L, V92M, R151C or D294H), Iso1 or Iso2, serum-deprived for 24 h. They were then
incubated with 10
-7
M NDP-MSH for 30 min. The medium was aspirated and the cells quickly
washed with 800 μl ice-cold phosphate buffered saline (PBS). Stimulated cells were lysed with
preheated 200 μl 0.1 N HCl (70°C) per well and scraped. The mix was freeze dried, washed
with 100 μlH
2
O and freeze dried again twice. cAMP levels were measured by a commercial
ELISA immunoassay, as per instructions. Briefly, samples and standards, a known concentra-
tion of cAMP-peroxidase conjugate, and a sheep antibody to cAMP were added into the wells
of a microtiter plate coated with an antibody to capture sheep IgG. After washes the substrate
was added. The substrate reacts with the bound cAMP-peroxidase conjugate and after a short
incubation the reaction was stopped and the intensity of the generated color was detected at
450 nm in a BioTek™ELx800™microtiter plate reader (BioTek, Bedfordshire, UK). Parallel
dishes were used for protein determination performed with the bicinchoninic acid method.
Immunoprecipitation and Western blotting
Cells grown on 6-well plates were washed with PBS and solubilized at 4°C in 200 μl solubiliza-
tion buffer (50 mM Tris-HCl pH 7.5, 1% Igepal, 1 mM EDTA, 0.1 mM PMSF, 10 mM iodoace-
tamide). Samples were centrifuged (21,000 g, 30 min). Suitable volumes of supernatant were
mixed (2:1 ratio) with sample buffer (180 mM Tris-HCl, pH 6.8, 15% glycerol, 9% SDS, 0.075%
Bromophenol Blue, 7.5% β-mercaptoethanol), electrophoresed and blotted as described [21].
Blots were probed with the required antibodies and stained with a chemiluminescent substrate
(Amersham, Little Chalfont, UK). Comparable loading was ascertained by stripping and
reprobing with an anti-ERK2 antibody. Stripping was performed by washing the membranes
with PBS, followed by treatment with 0.5 N NaOH, 10 min at room temperature, and a final
10-min wash with H
2
O.
For immunoprecipitation, cells were washed, solubilized and centrifuged as above. Superna-
tants were incubated with 20 μl of EZview Red ANTI-FLAG M2 Affinity Gel beads (Sigma) for
1 h at 4°C. The mixture was washed, centrifuged, eluted with pre-heated (95°C) sample buffer,
and electrophoresed and blotted as above.
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 5/19

Confocal microscopy
Cells grown on coverslips were transfected, fixed with 4% paraformaldehyde in PBS and per-
meabilized with 0.05% Triton. Cells were labelled with anti-HA monoclonal (1:5000), followed
by an Alexa 488 secondary antibody for detection of MC1R. For co-localization of HA-MC1R-
001 and Flag-isoforms, cells were incubated simultaneously with anti-HA monoclonal and
anti-Flag rabbit polyclonal, followed by Alexa 488-conjugated anti-mouse and Alexa 568-con-
jugated anti-rabbit secondary antibodies. Samples were mounted with a medium from Dako
(Glostrup, Denmark) and examined with a Leica laser scanning confocal microscope AOBS
(Leica Microsystems GmbH, Wetzlar, Germany). Images were taken in sequential scan mode
between frames, with a HCX PL APO CS 63x objective. Co-localization analysis was performed
in single cells using the line scan analysis of ImageJ.
Flow cytometric analysis
Approximately 10
5
cells were incubated in a final volume of 100 μl with anti-Flag M2 (1:25) for
30 min at 4°C. Cells were washed twice (2% fetal bovine serum, 0.01% NaN
3
in PBS), and fur-
ther incubated with a phycoerythrin-labelled anti-mouse IgG, at a final dilution of 1:50, for 30
min at 4°C. Cells were washed, resuspended in 500 μl PBS, and analyzed in a Becton Dickinson
FACScan system.
Real Time PCR
Several human melanoma cells were serum-deprived at least for 3 h before RNA extraction.
Total RNA was extracted with the commercial kit RNeasy1Mini Kit (QIAGEN, Hilden, Ger-
many) and measured with a NanoDrop 2000c spectophotometer (Thermo Scientific™Wal-
tham, Massachusetts, USA). One μg of RNA was reverse transcribed into cDNA using The
SuperScript1III First-Strand Synthesis System for RT-PCR kit (Invitrogen Life technologies™,
Carlsbad, California, USA) according to the manufacturer’s instructions.
qRT-PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosys-
tem, Foster City, California, USA) on ABI 7500 Fast Real Time PCR System with the following
cycling conditions: 95°C for 30 s and 40 cycles of 95°C for 5 s and 60°C for 60 s. The β-actin
gene was used as the endogenous normalizer. Relative mRNA expression levels were calculated
based on the Ct values using the formula 2
-ΔΔCt
.
Primers for MC1R-001 Iso1, Iso2 and β-actin, used after determination of their efficiencies,
were provided by Biolegio BV (Biolegio BV, Nimega, Netherlands). Their sequences are:
MC1R-001 forward 5’-GCCCTCATCATCTGCAATGC-3’
MC1R-001 reverse 5’-CCCTCTGCCCAGCACACTTA-3’
Iso1 forward 5’-GCTCCTGCAAAAGGAGTTCTG-3’
Iso1 reverse 5’-GCTCGAGGCACGTACTTGTG-3’
Iso2 forward 5’-TGCTGACATGCTCCTGTTCTG-3’
Iso2 reverse 5’-GCTCGAGGCACGTACTTGTG-3’
β-actin forward 5'-GACAGGATGCAGAAGGAGATCA-3’
β-actin reverse 5'-GCTCAGGAGGAGCAATGATCTT-3’
Statistical analysis
Unpaired two-tailed Student’s t-test and one-way ANOVA with Tukey post-test (for multiple
comparisons) were performed using GraphPad Prism (GraphPad Software, San Diego Califor-
nia USA, www.graphpad.com). Data were presented as mean ± standard error mean (SEM).
All p values were calculated using two-sided tests. P values of less than 0.05 were considered
statistically significant. indicates p<0.05,  p<0.01,  p<0.001 and  p<0.0001.
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 6/19

Results
Expression of MC1R-TUBBIII splice variants
The Iso1 and Iso2 intergenic splicing chimerae were originally reported in HBL and M14
human melanoma cells [13]. Since gene expression patterns in human melanoma cell lines are
very variable [22], it was of interest to compare the occurrence and abundance of the chimeric
transcripts in a wider panel of melanoma cell lines of known genetic characteristics. This was
approached by qRT-PCR (Fig 1C and 1D). We found detectable expression of the canonical
MC1R-001 transcript in all cell lines, with large differences (~ 40-fold) between the cell lines
with the highest and lowest levels (HBL and C8161 cells, respectively). The patterns of expres-
sion of Iso1 and Iso2 in the various cell lines were similar. However, when compared with
MC1R-001, there where clear differences. For instance, the cell line expressing the highest level
of MC1R-001 (HBL cells) was not the same as the one expressing more Iso1 and Iso2 (HMEL1
cells), and SKMEL28 cells with good expression of MC1R-001 had low levels of the intergenic
spliced variants. Accordingly, the ratio of expression of chimeric mRNA normalized for
expression of MC1R-001 was variable, with an approximately 20-fold difference between the
cells expressing the highest and lowest ratios (HMEL1 and C8161, Fig 1D). Furthermore, simi-
lar analysis in human epidermal melanocytes (HEM) (cDNA kindly provided by Prof. J. Nep-
tuno Rodríguez López, University of Murcia, Spain) revealed that basal expression of chimeric
spliced variants in non-stimulated normal melanocytes was very low, comparable to the mela-
noma cell line C8161 expressing the lowest levels of Iso1 and Iso2 (Fig 1C). In addition, when
intergenic spliced variants expression levels were normalized to MC1R-001 expression, we
found that HEM expressed mainly the canonical MC1R-001 (Fig 1D).
MC1R-TUBB3 chimeric intergenic splice isoforms Iso1 and Iso2 contain the N-terminus
and 7 transmembrane (TM) domains from MC1R (Fig 1A and 1B). Thus, they are potentially
able to perform MC1R functions. In order to compare the signaling properties of the chimeric
proteins and MC1R-001, we cloned Iso1 and Iso2 (with or without an N-terminal fused Flag
epitope) and expressed them in heterologous HEK293T cells, along with the canonical recep-
tor. When expressed in HEK293T cells (Fig 2A), as previously reported [1,16], MC1R
migrated as a doublet with a majority band of apparent Mr ~ 29 kDa and a minority band of ~
34 kDa, corresponding to de novo WT MC1R-001 and an EndoH-sensitive glycoform, respec-
tively. Moreover, Iso1 migrated with the expected apparent molecular weight (Mr ~ 88 kDa)
whereas Iso2 showed a Mr around 38 kDa, lower than predicted. As expected, the Iso1 in-
frame fusion of MC1R and TUBB3 cross-reacted with anti-TUBB3 antibodies (Fig 2A) whereas
the out-of-frame chimera Iso2 did not (blot below). Comparison of band intensities for the
Flag-tagged proteins showed lower steady-state levels of Iso1 and Iso2 compared with WT
MC1R-001, suggesting a lower intracellular stability for the chimeric forms. A higher rate of
intracellular proteolysis would also be consistent with the Mr of Iso2, lower than expected on
the basis of its predicted amino acid sequence, as well as with the finding of more than one dis-
crete immunoreactive band. We further analyzed the electrophoretic pattern of Iso1 and Iso2
intergenic splicing chimeras expressed in the human melanoma cell line HBL (Fig 2B). We
found a similar pattern of migration for Iso1, with an apparent Mr of 88 kDa and cross-reactiv-
ity with TUBB3 antibody. However, Iso2 was detected as a faint band of very high Mr, suggest-
ing protein aggregation and/or ubiquitylation followed by degradation.
Therefore, we tested the possibility of a higher intracellular proteolytic processing by follow-
ing the decay of MC1R-001, Iso1 and Iso2 in HEK293T cells treated with the protein synthesis
inhibitor cycloheximide (Fig 2C and 2D). The decay rate of the proteins was faster for Iso2,
intermediate for Iso1 and slower for MC1R-001, corresponding with half-life values of 1.6, 2.3
and 5.3 h, respectively (Fig 2C and 2D) consistent with the steady state protein levels.
Characterization of MC1R-TUBB3 Intergenic Splice Variants
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Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 8/19

Agonist binding and cell surface expression of MC1R-TUBBIII splice
variants
We compared agonist binding parameters for MC1R-001, Iso1 and Iso2 expressed in
HEK293T cells, by equilibrium binding assays using [I
125
]-NDP-MSH as the radioactive tracer
and unlabelled NDP-MSH as competing peptide. The data were plotted as specific binding
normalized for protein to compare maximal levels of bound agonist in cells expressing each
variant (Fig 3A) and as % of maximal binding to each isoform for an easier comparison of the
relative affinity of the variants (Fig 3B). Both Iso1 and Iso2 bound radiolabelled agonist specifi-
cally, but maximal binding was low, with residual Bmax values of about 10% (Table 1). How-
ever, affinity remained high with IC50 and Kd values in the low nM range (Fig 3A and 3B and
Table 1). In fact, the affinity for NDP-MSH was even slightly higher for the chimeric forms, as
shown by left-shifted displacement curves (Fig 3B).
These binding parameters suggested aberrant intracellular trafficking of MC1R-TUBB3 chi-
meric proteins with decreased cell surface expression of the fusion proteins. This was further
tested by flow cytometry (Fig 3C). Non-permeabilized cells were stained with an anti-Flag anti-
body directed against the Flag epitope fused to the extracellular N-terminus of the protein. In
these non-permeabilizing conditions, only receptor molecules inserted on the plasma mem-
brane with the correct orientation should be detected. The intensity of staining was much
lower for the chimeric proteins compared with WT receptor. Lower plasma membrane levels
of Iso1 and Iso2 could in turn result from an inefficient forward movement or from an
increased rate of sequestration away from the cell surface. Forward trafficking was assessed by
confocal microscopy analysis of co-localization with calnexin, an endoplasmic reticulum (ER)-
resident chaperone. Extensive co-localization with calnexin was found for Iso1 and Iso2,
whereas expression of the isoforms on the cell surface was almost undetectable (Fig 3D). Con-
versely, co-localization of calnexin and MC1R-001 was much lower and presence of the recep-
tor on the plasma membrane was easily detected. These results indicated massive intracellular
retention and failure to escape the quality control mechanisms of the secretory pathway for
Iso1 and Iso2 proteins. On the other hand, retrograde transport away from the cell surface was
estimated by an acid-wash procedure that allows distinguishing external (acid-sensitive) bind-
ing sites and internalized (acid-resistant) radioligand-receptor complexes (Fig 3E). Internaliza-
tion of Iso1 was also significantly impaired. Conversely, Iso2 was internalized at slightly higher
rates than MC1R-001, which may contribute to its low cell surface expression.
Functional coupling
Activation of MC1R potently stimulates cAMP synthesis, and triggers ERK signaling through a
cAMP-independent pathway [1,23]. We tested the chimeric proteins for activation of the
cAMP and ERK pathways. For comparison, residual signaling from frequent hypomorphic
MC1R variants associated with a red hair color phenotype with lower (“r”variants V60L and
Fig 2. Electrophoretic analysis and intracellular stability of MC1R-TUBB3 isoforms. (A) Expression of canonical and chimeric MC1R proteins in
heterologous HEK293T cells. HEK293T cells were transiently transfected to express Flag-labelled WT MC1R-001, Iso1 and Iso2. Cells were detergent-
solubilized, electrophoresed and blotted. For MC1R detection, cell lysates were probed with an anti-Flag monoclonal antibody (upper blot). Membranes were
also probed for TUBB3 (middle blot) and ERK2 (lower blot), as loading control (n = 5, representative blots are shown). (B) Electrophoretic pattern of
MC1R-TUBB3 transcripts expressed in HBL human melanoma cells. Representative immunoblots for MC1R, TUBB3 and ERK2 are shown as in panel A
(n = 5, representative blots are shown). (C) Intracellular stability of MC1R-TUBB3 chimeric fusion proteins in HEK293T cells. Flag-labelled MC1R-001, Iso1
and Iso2 were expressed in HEK293T cells. Cells were incubated with the protein synthesis inhibitor cycloheximide (Chx, 0.1 mM) for the times indicated,
lysed and the levels of residual proteins in cell extracts were detected by Western blot. Representative immunoblots probed for MC1R-001, Iso1 or Iso2 with
anti-Flag are shown. (D) Semi-log graph for calculation of half-lives. The intensity of receptor bands in the blots as in panel C was quantitated with ImageJ
and the semi-log of residual signals was plotted against time. Half-life (t½) values correspond to the slope of the resulting lines.
doi:10.1371/journal.pone.0144757.g002
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 9/19

Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 10 / 19

V92M) or higher penetrance (“R”forms R151C and D294H) was also estimated. HEK293T
cells were transfected with the MC1R forms, challenged with a saturating concentration of
NDP-MSH for 30 min, and cAMP contents were determined. Functional coupling of Iso1 and
Iso2 to the cAMP pathway was strongly impaired relative to MC1R-001, as shown by lower
cAMP production upon stimulation with NDP-MSH (Fig 4A). The residual signaling potential
of the chimeric fusions was lower than V60L and V92M, and comparable with R151C and
D294H. On the other hand, MC1R activates the mitogen-activated protein kinases ERK1 and
ERK2 by a cKIT transactivation mechanism independent of cAMP, which is less sensitive to
many natural mutations than activation of the cAMP cascade [24]. We analyzed ERK activa-
tion downstream of Iso1 and Iso2 in PC12 cells transfected to express the canonical or the chi-
meric receptors (Fig 4B). Signaling from the intergenic splice isoforms to ERK activation
showed a decreasing trend compared with MC1R-001, but the differences in the maximal levels
of active ERKs did not reach statistical significance (Fig 4B).
Functional interactions of WT MC1R and intergenic splice variants
We have previously shown that MC1R exists as dimeric species [21,25], and that heterodimeri-
zation of WT and mutant forms gives raise to dominant negative effects [21,26]. Since dimer-
ization apparently proceeds through a domain swap mechanism involving the 7 TM fragments
expressed in Iso1 and Iso2 [25], in vivo formation of MC1R/Iso heteromeric species is likely.
We analyzed the occurrence of heterodimerization by co-immunoprecipitation of differentially
epitope-labelled variants. First, MC1R-001 tagged by in frame fusion of the HA epitope to its
N-terminus, and chimeric proteins (or MC1R-001 as positive control) labelled at the N-termi-
nus with the Flag epitope were expressed alone or in combination in HEK293T cells. The inter-
genic chimeras were immunoprecipitated from detergent-solubilized extracts with anti-Flag
Fig 3. Radioligand binding and intracellular trafficking properties of MC1R-TUBB3 isoforms. (A-B) Competition binding assay of HEK293T cells
transfected with MC1R-001, Iso1 and Iso2. Cells were incubated with
125
I-labelled NDP-MSH (5x10
-11
M) and increasing concentrations of non-labelled
competing NDP-MSH, from 10
−12
to 10
−7
M, extensively washed and counted for radioactivity. Non-specific binding was determined with non-transfected
cells or with transfected cells incubated with the radioactive tracer in the presence of excess (10
−6
M) non-labelled peptide, with the same results. Values are
represented as specifically bound [
125
I]-NDP-MSH (A) and as percentage of residual binding (B) at the different ligand concentrations (n = 3, data are given
as mean ±SEM). (C) Flow cytometric analysis of HEK293T cells expressing MC1R-001 and MC1R-TUBB3 chimeric isoforms. Non-permeabilized cells
expressing the indicated proteins were incubated with an anti-Flag antibody labelled with phycoerythrin. Since the Flag epitope was fused in-frame to the
extracellular N-terminus of the MC1R sequence, only cells expressing the constructs of the plasma membrane should be detected. Histograms represent cell
number (counts) as a function of Flag surface staining, on a logarithmic scale. The gray filled curve refers to cells transfected with an empty pcDNA3 (n=3,
representative histograms are shown). (D) Left panel: Representative confocal images of MC1R-001 or the chimeric isoforms (red) and calnexin (green)
immunostaining in HEK293T cells transiently transfected with Flag-labelled MC1R-001 and MC1R-TUBB3 constructs. Scale bar, 10 μm. Representative line
scan (right panel) from multiple experimental repeats across the cell (location indicated in merged image) shows co-localization of MC1R-TUBB3 transcripts
and calnexin. Line scan, 19 μm for MC1R-001, 17 μm for Iso1 and 18 μm for Iso2. (E) Radioligand internalization assay performed on HEK293T cells
expressing MC1R-001, Iso1 or Iso2 incubated with
125
I-labelled NDP-MSH. The radioactive tracer was isotopically diluted to achieve a final concentration of
5x10
-11
M and 5x10
4
counts/well. Externally bound agonist was separated by an acid wash procedure. Both the externally bound ligand present in the acid
washes and the internalized ligand associated with the cell pellets were counted. The internalization index represents the percentage of ligand internalized
referred to total radioligand bound (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, *p<0.05, **p<0.01).
doi:10.1371/journal.pone.0144757.g003
Table 1. Binding parameters of WT MC1R-001 and chimeric transcripts Iso1 and Iso2.
MC1R transcript Bmax (fmoles/mg protein) Kd (nM)
MC1R-001 3.47 ±0.18 1.99 ±0.49
Iso1 0.27 ±0.03 0.59 ±0.27
Iso2 0.37 ±0.04 1.17 ±0.52
Equilibrium binding parameters of [
125
I]-NDP-MSH to MC1R-001 or the MC1R-TUBB3 isoforms Iso1 and
Iso2 expressed in HEK293T cells (n = 3, data are given as mean ±SEM).
doi:10.1371/journal.pone.0144757.t001
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 11 / 19

agarose beads, and the pellets were analyzed for MC1R-001 by Western blot probed with anti-
HA (Fig 5A). Co-immunoprecipitation of MC1R-001 and the MC1R-TUBB3 chimeric pro-
teins was readily detected, indicating efficient heterodimerization. In addition, to mimic a het-
erocygotic MC1R genetic background highly frequent in northern European population, we
tested the heterodimerization capability of two common hypomorphic variant MC1R alleles
with the WT MC1R-derived chimeric protein Iso1. We selected the frequent V60L and R151C
alleles as representative of the r and R types of RHC alleles, respectively. Flag-labelled versions
of these constructs were overexpressed alone or with intergenic splice variant Iso1 in HEK293T
cells. The amount of chimeric protein Iso1 immunoprecipitated with MC1R was comparable
Fig 4. Effects of intergenic splicing on the functional coupling of MC1R to the cAMP and ERK1/2 pathways. (A) Agonist-induced cAMP production in
HEK293T cells expressing MC1R-001, Iso1, Iso2, or the natural MC1R-001 variant allelesV60L, V92M, R151C and D294H. Cells were incubated with 10
−7
M
NDP-MSH for 30 min and cAMP levels were determined by an immunoassay (n = 6, error bars are ±SEM, two-sided Student´s t test was used to generate p
values, *p<0.05, *** p<0.001). (B-C) Representative immunoblots (B) and quantification (C) of ERK1 and ERK2 phosphorylation in PC12 cells transfected
to express MC1R-001, Iso1 or Iso2 and stimulated with NDP-MSH (10
−7
M) for the times indicated (n = 5, error bars are ±SEM, two-sided Student´s t test was
used to generate p values, *p<0.05, ** p<0.01).
doi:10.1371/journal.pone.0144757.g004
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 12 / 19

Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 13 / 19

for WT and the variant alleles V60L and R151C (Fig 5B). Therefore, in a RHC variant allele
background, heterodimerization between MC1R variant alleles and splice variants Iso1 and
Iso2 may occur.
Moreover, we examined the intracellular localization of MC1R-001 and Iso1 or Iso2 by con-
focal microscopy in HEK293T cells co-expressing MC1R-001 and one of the intergenic splice
forms (Fig 5C). We found a high degree of co-localization of MC1R-001 and Iso1 or Iso2 in
internal compartments, suggesting that heterodimerization impairs forward trafficking com-
pared with MC1R-001 homodimers. Nevertheless, we also detected co-localization of MC1R-
001 and Iso1 or Iso2 at the cell periphery, consistent with higher expression of the chimeric
proteins on the cell surface when co-expressed with MC1R-001 compared with cells expressing
the isoforms alone.
We next estimated agonist-induced cAMP production in HEK293T cells co-expressing WT
or variant alleles V60L or R151C and chimeric proteins Iso1 and Iso2 (Fig 5D). The cAMP
response was similar in cells expressing WT or variant alleles MC1R-001 alone, or MC1R-001
and the chimeric forms. On the other hand, co-expression of canonical and the chimeras
slightly decreased cell surface expression of binding sites, although the differences did not
reach statistical significance (Fig 5E). Conversely, no effects on internalization rates were
detected, with comparable results for cells expressing WT MC1R-001 alone, or in combination
with Iso1 or Iso2 under conditions previously shown to result in efficient heterodimerization
(Fig 5F).
Discussion
Recent progress in the analysis of the architecture of genes encoding for GPCRs has shown that
they often contain several introns and can undergo alternative splicing events. However, inter-
genic splicing giving raise to chimeric molecules is a rare event [27] where transcription pro-
ceeds through the region between two adjacent genes to yield a non-canonical chimeric RNA
which is further spliced to a final fusion product composed of sequences from the two neigh-
boring genes. To the best of our knowledge, this process has been described only for the P2Y
receptor and the SSF1 genes [28] on one hand, and the MC1R and TUBB3 genes on the other
within the large GPCR superfamily [13,14].
We confirmed the occurrence of Iso1 and Iso2 MC1R-TUBB chimeric mRNA species in a
panel of 8 human melanoma cell lines and a human epidermal melanocytic cell line. We were
able to detect the corresponding transcripts in all of them even in the absence of external
Fig 5. Heterodimerization of MC1R and MC1R-TUBB3 chimeric isoforms. (A) Co-immunoprecipitation of MC1R-001 and the MC1R-TUBB3 chimeric
isoforms. HEK293T cells expressing the indicated constructs were lysed and immunoprecipitated for Flag-labelled MC1R-001, Iso1 or Iso2 using an anti-
Flag monoclonal antibody. The pellets were electrophoresed and blotted for HA-labelled MC1R-001 (with a specific anti-HA monoclonal antibody) or for Flag-
labelled MC1R-001, Iso1 or Iso2 (with anti-Flag monoclonal antibody) as a control for efficient immunoprecipitation. Total lysates were also electrophoresed
and blotted as expression controls (n = 3, representative blots are shown). (B) Co-immunoprecipitation of V60L or R151C variant MC1R-001 and WT
MC1R-TUBB3 intergenic splice isoform Iso1. The indicated constructs were expressed in HEK293T cells and immunoprecipitated for Flag-labelled MC1R-
001, V60L or R151C using an anti-Flag monoclonal antibody. Immunoblots for Flag-tagged constructs and TUBB3 are shown for immunoprecipitated and
total lysates. (C) Representative confocal images of MC1R-001 (green) and Iso1 or Iso2 (red) immunostaining in HBL cells transiently transfected with HA-
labelled MC1R-001 and Flag-labelled MC1R-TUBB3 chimeric isoforms. Scale bar, 10 μm. Representative line scan (right panel) from multiple experimental
repeats across the cell (location indicated in merged image) shows co-localization of canonical MC1R-001 and chimeric proteins. Line scan, 20 μm for
MC1R-001+Iso1, and 31 μm for MC1R-001+Iso2. (D) Effect of heterodimerization on functional coupling to cAMP. Intracellular cAMP levels in HEK293T
cells expressing WT, V60L or R151C MC1R-001 alone or in combination with Iso1 and Iso2 upon stimulation with 10
−7
M NDP-MSH for 30 min. Results are
presented as residual cAMP production relative to WT MC1R-001 (for which cAMP levels were 0.096±0.043 and 0.889±0.071 pmol/μg protein in resting and
stimulated conditions respectively) (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values *p<0.05, **p<0.01, ***p<
0.001). (E) Specific binding of [
125
I]-NDP-MSH (5x10
-11
M and 5x10
4
cpm) to HEK293T cells expressing MC1R-001 or the MC1R-TUBB3 isoforms Iso1 and
Iso2, alone or in combination (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, ***p<0.001). (F) Agonist
internalization index in HEK293T cells co-transfected with MC1R-001 and Iso1 or Iso2 upon incubation with [
125
I]-labelled NDP-MSH (5x10
-11
M and 5x10
4
cpm) for 90 min (n = 3, error bars are ±SEM, two-sided one-way ANOVA was used to generate p values, ***p<0.001).
doi:10.1371/journal.pone.0144757.g005
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 14 / 19

stimuli, thus showing that expression of Iso1 and Iso2 is a general phenomenon in human mel-
anocytic cells. We also observed that the ratio of Iso1 and Iso2 transcripts relative to canonical
MC1R-001 transcript was variable in different cell lines. This suggests that Iso1/2 may not
merely result from an unregulated error of the transcriptional machinery occurring at a con-
stant rate within the context of the crowded genomic region where MC1R is located. Should
this be the case, a linear relationship between MC1R-001 and Iso1/2 mRNA would be expected.
In keeping with these data, Dalziel and co-workers reported that Iso1/2 expression is a regu-
lated process, activated by αMSH and by the p38 stress activated kinase [13].
When expressed in HEK293T heterologous cells or in human melanocytic cells, the in-
frame Iso1 intergenic fusion protein exhibited the expected Mr and cross-reacted with anti-
TUBB3 antibodies. Therefore, chimeric mRNA was adequately processed and the resulting
protein accumulated at sufficient levels within transfected cells, although its intracellular stabil-
ity was lower than the one of MC1R-001. On the other hand, Iso2 results from an out-of-frame
fusion with TUBB3 exon 3, skipping exon 1a in the MC1R 3’-UTR. Hence, Iso2 did not cross-
react with anti-TUBB3 antibodies. Moreover, its intracellular levels and half-life were low. The
shorter half-life of the chimeric proteins was consistent with impaired forward trafficking dem-
onstrated by reduced cell surface expression and high co-localization with the ER marker cal-
nexin. Thus, the ER-resident protein quality control system most likely recognized the
chimeric proteins as aberrant, causing their ubiquitylation, extrusion to the cytosol and proteo-
lytic degradation [29].
Both chimeras exhibited very poor ability to activate the cAMP pathway. This might be at
least partially due to decreased cell surface expression of the chimeric receptor compared to
WT MC1R. Residual cAMP levels after stimulation with a saturating concentration of the
superpotent NDP-MSH analog of αMSH were lower than those obtained in cells expressing
known red-hair color-associated MC1R variants with low (V60L and V92M) or high (R151C
and D294H) penetrance [24]. Conceivably, chimeric proteins arising from variant MC1R may
be even more severely reduced in signaling capacity, but we did not test directly this likely
hypothesis. Functional impairment was nevertheless less evident for activation of the ERK cas-
cade. Accordingly, signaling from the MC1R-TUBB3 intergenic splicing isoforms seemed
biased in favor of the ERK pathway. A similar behavior has been shown for many natural
MC1R variant alleles associated with the red hair color phenotype, with increased skin cancer
risk [24], higher mean survival of patients [30,31] and the anatomic site presentation of mela-
nomas [32]. Therefore, when expressed following exposure to UVR, the chimeric isoforms
might prevent excessive stimulation of the cAMP pathway without a parallel decrease in mela-
nocortin-dependent ERK activation. This effect might be enhanced by the ability of the iso-
forms to heterodimerize with MC1R-001, which seemed to impair forward trafficking of the
canonical form and lead to its partial intracellular retention. This was suggested by high co-
localization of the canonical protein and the chimeric isoforms in intracellular locations as well
as a by an apparently decreased number of αMSH binding sites on the cell surface of cells over-
expressing both proteins simultaneously. Although in this case the observed decrease did not
reach statistical significance, it appears likely that under normal expression conditions it may
become a significant factor.
The observation that expression of the intergenic splicing isoforms is a general feature in
human melanoma cell lines (this manuscript) as well as a regulated process [13] strongly sug-
gests that Iso1 and Iso2 might fulfil still uncharacterized specific actions within melanocytes.
Given that both isoforms are hypomorphic in terms of functional coupling to the cAMP path-
way, these actions might likely be related with dampening MC1R signaling under specific phys-
iological conditions. UVR-mediated upregulation of MC1R molecules on the cell surface of
melanocytes has been shown to occur at least partially by transcriptional activation of the
Characterization of MC1R-TUBB3 Intergenic Splice Variants
PLOS ONE | DOI:10.1371/journal.pone.0144757 December 11, 2015 15 / 19

MC1R gene. Direct UVR-mediated upregulation of MC1R expression has been shown in
mouse and human melanocytic cells [33,34] as well as in human epidermis in vivo [35]. In
addition, MC1R gene expression is upregulated, apparently via MITF, in human and mouse
melanocytes stimulated with αMSH or the cAMP inducer forskolin [36–38]. UVR-induced
DNA damage in keratinocytes has been shown to stabilize the p53 tumor suppressor, which
activates transcription of POMC gene encoding for the precursor of αMSH. αMSH is then
released from keratinocytes to activate MC1R in melanocytes [39]. Accordingly, UVR would
increase MC1R expression by at least two types of processes, namely a direct stimulation of
MC1R gene expression in irradiated melanocytes on one hand, and, on the other, an indirect
pathway whereby release of POMC-derived MC1R agonists by keratinocytes in UVR-exposed
skin would activate MC1R signaling in melanocytes, followed by increased cAMP signaling
and induction of MC1R transcription. This would further increase the responsiveness of mela-
nocytes to the paracrine signals resulting in a positive feedback loop that may favor a potent
tanning response, but that may also threaten melanocyte viability owing to the inherent cyto-
toxicity of the melanogenic pathway [40,41]. It has been shown that Iso1/2 expression
increases following stimulation of melanocytes with αMSH or activation of the p38 kinase, in
an isoform switch that favors their expression relative to the canonical transcript [13]. Thus,
the same signaling cascades implicated in induction of MC1R gene expression have also been
shown to promote Iso1 and Iso2 transcripts. The diversion of a fraction of the new transcrip-
tional events towards formation of inactive, and maybe even dominant-negative intergenic
splice isoforms might endeavor the melanocytes with a mechanism to dampen and fine-tune
this potentially dangerous positive feedback loop.
Acknowledgments
We thank Prof. A. Furger (University of Oxford, UK) for gift of Iso1 and Iso2 constructs, Dr P.
Zanna (University of Bari, Italy) for HMEL1 melanoma cells, Prof. G. Ghanem (Université
Libre de Bruxelles, Belgium) for other human melanoma cell lines and Prof. J. Neptuno Rodrí-
guez López for cDNA from normal human epidermal melanocytes (University of Murcia,
Spain).
Author Contributions
Conceived and designed the experiments: JCGB CJC CH. Performed the experiments: CH CO
MCG MA. Analyzed the data: CH JCGB CJC. Wrote the paper: JCGB CH CJC CO.
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