ORIGINAL ARTICLE

Dimerization of the Human Melanocortin 1

Receptor: Functional Consequences and
Dominant-Negative Effects
Berta L. Sánchez-Laorden1, Jesús Sánchez-Más1, Emma Martı́nez-Alonso2, José A. Martı́nez-Menárguez2,
José C. Garcı́a-Borrón1 and Celia Jiménez-Cervantes1

The melanocortin 1 receptor (MC1R), a GS-protein-coupled receptor (GPCR), is a key regulator of proliferation
and differentiation of epidermal melanocytes, and a determinant of human skin phototype and cancer risk.
Homodimerization has been demonstrated for several GPCRs, but little information is available for MC1R. SDS-
PAGE analysis of melanoma cells and heterologous cells expressing epitope-tagged MC1R revealed dimeric and
oligomeric species in detergent-solubilized extracts, confirmed by coimmunoprecipitation of differentially
tagged MC1R forms. Dimerization occurs early during MC1R biosynthesis, and is seen for mutants displaying
intracellular retention. These mutants exerted dominant-negative effects on wild-type (WT) MC1R. Conversely,
partial functional transcomplementation of selected loss-of-function mutants was observed. WT-MC1R lacks
cooperativity in agonist binding, yet coexpression of WT and a C-terminal deletion mutant yielded a form of
different pharmacological properties. The natural diminished function alleles R151C, R160W, and D294H,
associated with red hair, displayed dimerization and heterodimerization with WT. Coexpression of WT and
R151C or R160W reduced the density of binding sites on the plasma membrane of transfected cells, whereas
D294H mediated a dominant-negative effect on functional coupling to adenylyl cyclase. Therefore, subtle
changes of functional properties may be associated with different MC1R haplotypes, contributing to the
complexity of skin phenotype.
Journal of Investigative Dermatology (2006) 126, 172–181. doi:10.1038/sj.jid.5700036

INTRODUCTION pigments (Di Donato and Napolitano, 2003). Tyrosinase

Human hair and skin pigmentation results from the synthesis
of L-tyrosine-derived melanin pigments within epidermal
melanocytes (Rees, 2003). By acting as effective sunscreens
able to absorb harmful ultraviolet radiation, melanin pig-
ments play a major role in photoprotection (Kadekaro et al.,
2003). The synthesis of melanin pigments is governed by
tyrosinase, an enzyme catalyzing the rate-limiting conversion
of L-tyrosine into L-dopaquinone (DQ). In the absence of thiol
compounds, DQ evolves to black eumelanin pigments.
However, in the presence of cysteine or glutathione, addition
of sulfhydryl groups to DQ yields reddish pheomelanin
1
Department of Biochemistry and Molecular Biology, University of Murcia,
Espinardo, Spain and 2Department of Cell Biology, School of Medicine,
University of Murcia, Espinardo, Spain
Correspondence: Dr Celia Jiménez-Cervantes, Department of Biochemistry
and Molecular Biology, School of Medicine, University of Murcia, 30100
Espinardo, Spain. E-mail: celiajim@um.es
Abbreviations: DQ, L-dopaquinone; ER, endoplasmic reticulum; GPCR,
G-protein-coupled receptor; HA, hyaluronate; HEK, human embryonic
kidney; LOF, loss of function; MC1R, melanocortin 1 receptor; aMSH, a
melanocyte-stimulating hormone; NDP-MSH, norleucine4
7
D-phenylalanine -melanocyte-stimulating hormone; PBS, phosphate-buffered
saline; RHC, red hair color; WT, wild type
Received 25 May 2005; revised 22 August 2005; accepted 6 September 2005
activity is controlled by a variety of mechanisms, including
hormonal stimulation by a melanocyte-stimulating hormone
(aMSH) following activation of the melanocortin 1 receptor
(MC1R), a G-protein-coupled receptor (GPCR) positively
coupled to the cAMP pathway (Busca and Ballotti, 2000)
expressed in epidermal melanocytes. In addition, MC1R
activation increases the ratio of black and strongly photo-
protective eumelanins to yellowish and poorly photoprotec-
tive pheomelanins (Abdel-Malek et al., 2000; Barsh et al.,
2000; Kadekaro et al., 2003). Accordingly, MC1R is a key
regulator of mammalian pigmentation.
The human MC1R gene is highly polymorphic. Several
variants are associated with fair skin, red hair color (RHC)
(Box et al., 1997; Smith et al., 1998; Schioth et al., 1999;
Sturm, 2002; Rees, 2003), a high number of freckles
(Bastiaens et al., 2001a), and increased skin cancer risk
(Valverde et al., 1996; Palmer et al., 2000; Bastiaens et al.,
2001b; Sturm, 2002; Sturm et al., 2003). The best character-
ized variants are the RHC alleles R151C, R160W, and
D294H that show diminished coupling to the cAMP signaling
pathway (Schioth et al., 1999; Ringholm et al., 2004). The
RHC alleles are very frequent, and in certain populations
about 40% of the individuals harbor at least one of them
(Rees, 2003). Many other potentially relevant MC1R variants

172 Journal of Investigative Dermatology (2006), Volume 126 & 2006 The Society for Investigative Dermatology

have been described (Jimenez-Cervantes et al., 2001a, b;
Sanchez-Más et al., 2002).
Many GPCRs form dimers or oligomers (Milligan, 2001;
Angers et al., 2002; Breitwieser, 2004). Dimerization
can modulate key properties of the receptors, including
ligand binding, coupling efficiency, desensitization, and
intracellular traffic from the endoplasmic reticulum (ER) to
the plasma membrane, and through endocytic pathways. A
recent study suggests that MC1R may dimerize in living
cells, but neither the mechanism nor the functional effect
of dimerization was reported (Mandrika et al., 2005).
Dimer formation has been demonstrated for the related
MC4R and shown to result in a dominant-negative effect
of a mutant associated with severe early-onset obesity
(Biebermann et al., 2003).
We show here that MC1R forms dimers and oligomers
(collectively termed dimers for simplicity) in heterologous
systems and melanoma cells. We also show that dimer
formation may result in dominant-negative effects of mutant
alleles on wild type (WT)-MC1R, as well as some degree of
functional complementation between mutants. Therefore,
MC1R dimerization may underpin the complexity of geno-
type–phenotype associations in human pigmentation.
RESULTS
Functional analysis of epitope-tagged MC1R
Three different epitope-tagged variants of the MC1R were
constructed: (1) a protein with a Flag epitope at the
extracellular N-terminus immediately after the initial Met
residue (Flag-MC1R), (2) a fusion containing one hemagglu-
tinin (HA) epitope after the cytosolic C-terminal W317
residue (MC1R-HA), and (3) a form containing a longer C-
terminal tag with two consecutive HA epitopes followed by a
hexahistidine tail (MC1R-Tag). When expressed in human
embryonic kidney (HEK) 293T cells, Flag-MC1R and MC1R-
HA performed essentially as WT in functional coupling to
adenylyl cyclase, as assessed by determination of agonist-
induced cAMP production. MC1R-Tag did not mediate a
detectable cAMP response (Figure 1a). Cells transfected with
the WT, Flag-MC1R, or MC1R-HA forms bound similar
amounts of a radioactively labeled agonist (Figure 1b), but no
specific binding was demonstrated for MC1R-Tag. For Flag-
MC1R, displacement experiments indicated an affinity for the
melanocortin ligand similar to WT (Figure 1c).
Constitutive oligomerization of MC1R
We used the functional Flag-MC1R and MC1R-HA constructs
in coimmunoprecipitation and Western blot experiments.
Transfected HEK 293T cells were solubilized in the presence
of 10 mM iodoacetamide to prevent spurious disulfide bond
formation. Samples were electrophoresed under reducing or
nonreducing conditions, with increasing concentrations of
SDS (Figure 2a). Samples run in the absence of b-mercap-
toethanol displayed bands of high apparent molecular weight
(Mr) that barely entered the gel, a prominent band of Mr
58 kDa that may correspond to an MC1R dimer, and a minor
band of higher electrophoretic mobility, most likely corre-
sponding to the monomeric species. The oligomeric forms
BL Sánchez-Laorden et al.
Melanocortin 1 Receptor Homodimerization
a MC1R
1.2 Flag-MC1R
pmol cAMP/
g protein
HA-MC1R
0.8 MC1R-TAG
pcDNA3
0.4
0.1
0.0
Control –9 –7
Log[NDP-MSH] (M)
b 30 c 125
c.p.m. bound/dish
100
Bx100/Bmax
(thousands)
20
75
50
10
Flag-MC1R
25
WT-MC1R
0 0
WT FLAG HA TAG pcDNA3 –12 –11 –10 –9 –8 –7 –6 –5
Transfected plasmid
Log[competing peptide] (M)
Figure 1. Functional characterization of epitope-tagged MC1R. HEK 293T
cells transfected with the indicated variants were serum-deprived for 24 hours
before the assays. (a) Functional coupling to cAMP generation. Cells were
treated for 30 minutes with various concentrations of NDP-MSH, or vehicle
(control), lysed, and cAMP was determined by radioimmunoassay. pcDNA3
stands for empty vector. (b) Binding of [125I]NDP-MSH. Cells were incubated
with 10
10 M [125I]NDP-MSH for 1 hour, washed, and the radioactivity
associated was determined. (c) Competition binding analysis of WT- and Flag-
MC1R. Cells were incubated with 10
10 M [125I]NDP-MSH and increasing
concentrations of unlabeled ligand. The specifically bound radioactivity was
measured. Results are given as % maximal binding in the absence of
competitor.
were resistant to SDS, in that increasing its concentration
from 0.5 to 3% did not change the aggregation status. No
immunoreactive bands were detected in cells transfected
with an MC1R construct lacking the Flag epitope.
Under reducing conditions, the majority was a band of
Mr 30 kDa, consistent with dissociation of MC1R oligomers.
A 36-kDa band was also detected (Figure 2a). Upon
deglycosylation with endoglycosidase H, the 36-kDa band
disappeared, but the mobility of the 30-kDa protein did not
change (Figure 2b). This suggests that the 30- and 36-kDa
bands correspond to ‘‘de novo’’ and glycosylated MC1R,
respectively. In spite of the strong effect of mercaptoethanol
on monomer:oligomer ratios, samples still contained bands
consistent with MC1R oligomers. In this case, the 58-kDa
species appeared as a doublet, where only the lower mobility
band was sensitive to endoglycosidase H (Figure 2b).
To demonstrate that the 58 kDa bands were MC1R dimers,
extracts from cells coexpressing Flag-MC1R and MC1R-HA were
immunoprecipitated with an anti-HA antibody and immuno-
blotted with an anti-Flag. Bands consistent with monomeric and
dimeric forms were detected (Figure 2c). These bands were not
seen in extracts from cells expressing either variant alone, or
when these extracts were mixed prior to immunoprecipitation,
excluding dimerization after sample solubilization.
We confirmed MC1R dimerization in a melanocytic
environment, at a lower expression level. We obtained
clones of HBL melanoma cells stably expressing Flag-MC1R,
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Melanocortin 1 Receptor Homodimerization

a SB without -ME SB with -ME b

SDS (% ): 0.5 1.0 2.0 3.0 0.5 1.0 2.0 3.0 WT EndoH

MW (kD a) MW (kDa )

120
97 120
97
55.4
55.4
36.1

28.9 36.1

Erk- 2 28.9

c IP: -HA; WB: -Flag d HBL Clone 18 WT

MW (kDa)
Flag HA Mix HA+Flag
55.4
113
92

52.3 36.1

35.3 28.9

28.7

Erk- 2

Figure 2. Detection of MC1R dimers and oligomers in solubilized cell extracts. (a) Electrophoretic pattern of Flag-MC1R expressed in HEK 293T cells.
Detergent-solubilized extracts were treated (10 minutes, room temperature) with SDS from 0.5 to 3%, with or without b-mercaptoethanol. Samples were
analyzed by Western blot using an anti-Flag antibody. The mobility of marker proteins is shown on the left. Blots were reprobed with an anti-Erk2 antibody
as a control for comparable loading. (b) Effect of deglycosylation on the electrophoretic pattern of MC1R. Control extracts (WT) or extracts treated with
endoglycosidase H were immunoblotted with an anti-Flag antibody. Endoglycosidase H-sensitive bands in control extracts are highlighted by arrows.
(c) Coimmunoprecipitation of differentially epitope-tagged forms of MC1R. HEK 293T cells were transfected to express either Flag, HA, or a combination
(HA þ Flag) of both epitope-tagged forms of the WT-MC1R, and solubilized. Extracts from cells independently transfected with Flag-MC1R or MC1R-HA were
also mixed and incubated at room temperature for 30 minutes before immunoprecipitation (Mix). Samples were immunoprecipitated with an anti-HA
monoclonal antibody and the precipitates were immunoblotted with an anti-Flag antibody. Arrows highlight the monomer and dimer bands. (d) Detection of
MC1R dimers in human melanoma cells. Extracts from parental HBL, cells stably transfected with Flag-MC1R (clone 18), or HEK 293T cells (WT) transiently
expressing Flag-MC1R were immunoblotted with the anti-Flag antibody. Blots were reprobed with an anti-Erk2 antibody as control. Protein load was 15 (HBL
cells) or 8 mg (HEK cells).

selected on the basis of surface expression of the epitope as
detected by flow-cytometric analysis. A positive clone was
further analyzed, which displayed a moderate six-fold
enrichment in MC1R (Bmax 1.8 vs 0.3 fmol/mg protein for
mock-transfected HBL cells). The electrophoretic pattern was
similar for this clone and transfected HEK cells (Figure 2d).
Oligomerization is an early event taking place in the ER
To locate the subcellular compartment where MC1R dimer-
ization occurs, we first analyzed the electrophoretic pattern
of two MC1R loss-of-function (LOF) mutants, L93R (R93) and
R162P (P162), that show impaired cell surface expression due
to retention in an intracellular compartment (Sánchez-
Laorden et al., 2005). The electrophoretic pattern of these
mutants expressed in HEK 293T cells was similar to WT
(Figure 3a). This suggests that dimerization occurs before
reaching the plasma membrane. Second, we treated Flag-
MC1R-transfected cells with brefeldin A. This drug disrupts
the Golgi apparatus and blocks the post-translational
modifications taking place in this organelle. Brefeldin A
mediated a clear disorganization of the intense, compact, and
perinuclear staining of the cells with a Golgi-specific marker,
confirming that the drug was active (not shown). Western
blotting revealed a similar distribution of MC1R species in the
presence or absence of the drug (Figure 3b), suggesting that
dimerization occurs in a pre-Golgi compartment. Finally, we
took advantage of the observation that the LOF MC1R-Tag
variant is retained in the ER, as shown by immunolabeling
and electron microscopy. This variant was employed rather
than the L93R or R162P alleles, because in our hands the
anti-HA antibody used for its detection with protein A-gold
particles performed better than the anti-Flag. In cells
expressing MC1R-Tag, labeling was concentrated in endo-
membranes with the morphology of ER cisternae, with very
little labeling of plasma membranes (Figure 3c). Extracts of
cells expressing MC1R-Tag showed bands corresponding to
MC1R dimers (Figure 3f). Thus, the MC1R dimer is most likely
formed in the ER.

174 Journal of Investigative Dermatology (2006), Volume 126


a WT R93 P162 - b WT - B1 B 2
MW (kDa)
MW (kDa)
120.0
97.0 120.0
97.0
55.4
55.4
36.1
28.9 36.1
28.9
Erk-2
Erk-2
c f MC1R-Tag - MW (kDa)
120.0
97.0
55.4
d 36.1
28.9
e GPCR dimerization often involves the swapping of helices
Figure 3. Oligomerization of MC1R is an early event occurring in the ER.
(a) Oligomerization of MC1R LOF variants with impaired plasma membrane
expression. Extracts of cells expressing Flag epitope-tagged WT, L93R and
R162P, or empty vector (lane labeled
) were immunoblotted with anti-Flag.
Protein load was 10 mg/lane, and an anti-Erk2 antibody was used as a control.
(b) Detection of MC1R oligomers in brefeldin A-treated cells. HEK 293T cells
were transfected with WT Flag-MC1R or empty pcDNA (
). The electro-
phoretic pattern of control (WT) and two independent brefeldin A-treated
samples (B1 and B2) was analyzed by immunoblotting. Comparable loading
was ascertained with an anti-Erk2 antibody. (c–e) Intracellular localization of
MC1R-Tag. HEK 293T cells were transfected with MC1R-Tag, labeled with an
anti-HA monoclonal antibody, followed by protein A-gold particles, and
visualized by electron microscopy. In the upper image, arrows highlight a
strong internal labeling, as opposed to the lack of immunoreactivity in the
plasma membrane (pm). The middle image shows intense staining of internal
cisternae with the morphology of ER structures. The lower micrograph
illustrates the residual labeling of the plasma membrane. (f) Oligomerization
of MC1R-Tag. Extracts of HEK 293T cells transiently expressing MC1R-Tag
(lane labeled WT) or transfected with empty vector (lane labeled
) were
immunoblotted with anti-HA monoclonal antiserum. Arrows point to the
monomer and dimer bands. Bar=200 nm.
LOF mutants displaying intracellular retention
a dominant-negative effect
Since dimerization is probably an early event in MC1R
biosynthesis, aberrantly processed LOF mutants might exert a
dominant-negative effect by trapping WT receptor in
intracellular compartments. This was tested by cotransfecting
HEK 293T cells with WT-MC1R and several mutants with
impaired plasma membrane expression. These were the
natural mutants R93 and P162 and an artificial mutant with a
BL Sánchez-Laorden et al.
Melanocortin 1 Receptor Homodimerization
deletion of the five C-terminal amino acids, designated D5
(Sánchez-Laorden et al., 2005; Sánchez-Más et al., 2005b).
HEK 293T cells transfected with these mutants did not show
significant specific binding (Figure 4a) and displayed very
weak cell surface expression of the receptor, as detected by
flow cytometric analysis of nonpermeabilized cells (not
shown), confirming previous results (Sánchez-Laorden
et al., 2005; Sánchez-Más et al., 2005b). Coexpression of
WT-MC1R with increasing amounts of plasmid encoding for
the mutants caused a dose-dependent reduction of
[125I]NDP-MSH binding to about 50% of controls (Figure
4a), consistent with a dominant-negative effect. Cotransfec-
tion of WT-MC1R and an unrelated thromboxane receptor
had no effect on [125I]NDP-MSH-specific binding, demon-
strating the specificity of the dominant-negative effect.
Coimmunoprecipitation of differentially epitope-tagged WT
and variants confirmed heterodimerization of the mutants
and WT (Figure 4b). This suggests that dimerization of WT-
MC1R and aberrantly processed variants may lead to
intracellular retention of the heterodimer, and further
demonstrate MC1R dimerization in living cells.
Functional characterization of oligomers
from each monomer within the dimer, so that two defective
monomers can form a correctly folded and partially
functional unit, provided that the mutations are located in
different domains (Breitwieser, 2004). Conversely, if the
mutations fall in the same protein domain, dimerization
should not restore function. We tested functional comple-
mentation for the R93, P162, and D5 LOF forms. Although no
heterozygotes for these variants have been found, these forms
provide excellent models for functional transcomplementa-
tion because the mutations are located in different protein
regions (transmembrane fragments 2 and 4 for R93 and P162,
respectively, and C-terminus for D5), and the mutants are
complete LOF forms, thus yielding no background for
functional analysis. Two other deletion mutants missing the
last 12 and 14 C-terminal amino acids, termed D12 and D14
(Sánchez-Más et al., 2005b), were also analyzed. For some
combinations, substantial binding and coupling activities
were rescued (Figure 5). However, coexpression of the two
forms with the inactivating mutation in the same region, the
C-terminal cytosolic tail, failed to restore ligand binding or
coupling to adenylyl cyclase.
We analyzed other functional effects of MC1R dimeriza-
tion. Equilibrium binding data were analyzed for cooperativ-
ity with the Hill equation. The slope of Hill plots was not
significantly different from 1 (Hill number 1.0370.06, n ¼ 4),
exert indicative of lack of allosteric effects on agonist binding.
Moreover, neither agonist stimulation nor activation of
adenylyl cyclase with forskolin modulated the monomer-
to-dimer ratio as estimated by Western blot (Figure 6a),
suggesting that dimerization is constitutive. However, we
found evidence for a dimerization-dependent modulation of
MC1R pharmacology. Cotransfection of WT and the D5 LOF
variant yielded a form of higher affinity for NDP-MSH than
WT, as assessed from left-shifted competition binding curves
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 BL Sánchez-Laorden et al.
Melanocortin 1 Receptor Homodimerization

IP: -HA; WB: -Flag

a WT/variant(1:1, 1:2, 1:3) b
WT-HA: + + + + +
70 Flag-variant: WT R93 – P162 ∆5
c.p.m. bound/dish (thousands)

60

50

40

30

0
W T R93 P162 ∆5 WT WT+pcDNA W T+R93 W T+P162 W T+∆5
+
TXR

Figure 4. LOF variants with compromised cell surface expression exert a dominant-negative effect on WT-MC1R. (a) Reduction of binding sites in cells
coexpressing variant and WT-MC1R. HEK 293T were transfected with variant and WT-MC1R, alone or in combination, as indicated, and binding of 10
10 M
[125I]NDP-MSH was measured. For expression of a single MC1R form, 0.6 mg of plasmid DNA/well was employed. For cotransfections, the concentration of
WT-MC1R plasmid DNA was kept constant at 0.15 mg/well, and three variant plasmid concentrations were assayed (0.15, 0.3, and 0.45 mg/well), corresponding
to WT-to-variant ratios of 1:1, 1:2, and 1:3, respectively. Total concentration of plasmid DNA was kept constant at 0.6 mg/well with empty pcDNA. As a control
for specificity, WT-MC1R (0.15 mg/well) was cotransfected with the unrelated thromboxane a receptor (TxR) (0.45 mg/well), or with empty pcDNA.
(b) Heterodimerization of variant and WT-MC1R. HEK 293T cells were transfected to express WT-MC1R-HA and one of the following Flag-epitope-tagged
forms: WT, R93, P162, or D5, as indicated on the top of each lane. As a negative control, empty plasmid was used (lane labeled
). Detergent-solubilized
extracts were immunoprecipitated with an anti-HA monoclonal antibody and immunoblotted with anti-Flag.

a 3000
R93 P162 ∆5 ∆12

2000
c.p.m. bound

1000

0
V P162 ∆5 ∆12 ∆14 V R93 ∆5 ∆12 ∆14 V R93 P162 ∆12 ∆14 V R93 P162 ∆5 ∆14

b 0.10 0.10 0.10

pmoles cAMP/
g protein

pmol cAMP/
g protein

pmol cAMP/
g protein

R93/pcDNA P162/pcDNA ∆5/pcDNA

R93/P162 P162/R93 ∆5/R93
R93/∆5 P162/∆5 ∆5/P162
0.05 R93/∆12 0.05
P162/∆12 ∆5/∆12
0.05
R93/∆14 P162/∆14 ∆5/∆14

0.00 0.00 0. 00
Control NDP-MSH Control NDP-MSH Control NDP-MSH

Figure 5. Functional transcomplementation of MC1R mutants. (a) The LOF MC1R mutants indicated on the top of each group of bars were cotransfected with
an identical amount of empty vector (V) or the mutants shown. Expression of plasma membrane-binding sites was estimated by incubation with 10
10 M
[125I]NDP-MSH. Combinations that do not yield a partial recovery of binding are highlighted by arrows. (b) The L93R (left), R162P (middle), and D5 (right) forms
were cotransfected with empty pcDNA3 or the indicated variants. Functional coupling was estimated by measuring cAMP in cells challenged with vehicle
(control) or 10
7 M NDP-MSH.

(Figure 6b) and from the calculation of dissociation constants Functional behavior and dimerization of the RHC alleles
(1.8070.18 nM for WT vs 0.7370.08 nM in cotransfected The RHC alleles R151C, R160W, and D294H are frequent
cells, P ¼ 0.0017). Interestingly, maximal cAMP levels (Rees, 2003) and display reduced functional responses
achieved upon agonist stimulation were lower in cotrans- (Schioth et al., 1999; Ringholm et al., 2004). Therefore, it
fected samples than in cells transfected with WT alone was of interest to investigate whether these mutants form
(Figure 6c). homodimers and heterodimerize with WT-MC1R. We first

176 Journal of Investigative Dermatology (2006), Volume 126

 BL Sánchez-Laorden et al.
Melanocortin 1 Receptor Homodimerization

a MSH Fsk b 100 a SB with -ME SB without -ME

C 30´ 60´ 30´ WT C151 W160 H294 C151W160H294

Bx100/Bmax
75
MW (KDa)
120 50 120
97 97
25 WT
55.4 ∆5+WT 55.4
0
–11 –10 –9 –8 –7
36.1 Log[competing peptide] (M) 36.1
28.9
28.9

Erk-2
Erk-2

c 1.25 W T/pcDNA
pmol cAMP/
g protein

W T/∆ 5 b 2.25 Control

1.00

pmol cAMP/
g protein

*** 2.00 10-11
0.75 10-7
1.75
0.50

0.25 1.0

0.00 ***
Control NDP-MSH 0.5

Figure 6. Functional characterization of MC1R dimers. (a) Lack of effect of 0.0

WT-F R151C R160W D294H
agonist and forskolin on WT-MC1R dimerization. HEK 293T cells expressing
Variant
WT-MC1R were challenged with 10
7 M NDP-MSH (30 or 60 minutes),
forskolin (10
5 M, 30 minutes), or vehicle (lane labeled C). Extracts were c 60
WT
immunoblotted with anti-Flag, and an anti-Erk2 antibody was used as a
c.p.m. bound/dish 50 R151C
loading control. (b) Competition binding analysis of HEK cells coexpressing
(thousands) R160W
WT-MC1R and D5. Cells were transfected with WT-MC1R and pcDNA3 (’) 40
D294H
or WT-MC1R and D5-MC1R (m) and incubated with 10
10 M [125I]NDP-MSH 30
and increasing concentrations of unlabeled ligand. Results are expressed as %
20
maximal binding in the absence of competing ligand.
(c) Decreased stimulation of adenylyl cyclase in cells coexpressing WT and 10
D5. Cells cotransfected with WT-MC1R and pcDNA3 (empty bars) or D5
0
(closed bars) were challenged with 10
7 M NDP-MSH or vehicle (control). -10 -9 -8 -7 -6 -5
Their cAMP content was determined by radioimmunoassay (***Pp0.001). Log[competing peptide] (M)

d
70
analyzed the functional behavior of the RHC forms expressed
individually in HEK 293T cells. Western blot demonstrated 60

similar oligomerization characteristics and expression levels 50

Counts

for the three RHC forms and WT (Figure 7a). In functional 40

coupling experiments, the RHC variants showed a dramatic
reduction of agonist-independent constitutive activity
(Sanchez-Mas et al., 2004). R151C and R160W displayed
significant agonist-induced signaling, and only the D294H
receptor was a nearly complete LOF protein (Figure 7b),
consistent with reports by others (Schioth et al., 1999;
Ringholm et al., 2004). Competition binding curves indicated
a lower binding capacity in cells expressing the R151C and
R160W receptors (Figure 7c) compared with WT, but the
affinity of these forms for NDP-MSH was not impaired.
Conversely, transfection with D294H yielded a higher
density of binding sites (Bmax 6.270.7 fmol/mg protein for
D294H and 3.870.6 fmol/mg protein for WT). The affinity of
D294H for NDP-MSH was nevertheless lower than WT, as
demonstrated by a right-shifted displacement curve.
Therefore, the density of binding sites was lower for
R151C and R160W, intermediate for WT, and higher for
D294H. Since previous reports described very similar binding
properties for RHC variants and WT (Schioth et al., 1999), we
wished to confirm this trend. Flow-cytometric analysis of
nonpermeabilized cells indicated higher plasma membrane
D294H
30
WT
20
R151C
10 R160W
0
100 101 102 103 104
Figure 7. Dimerization of RHC variants. (a) Extracts of HEK 293T cells
expressing Flag-epitope-tagged WT-MC1R or RHC variants were electro-
phoresed (8 mg/lane) with or without b-mercaptoethanol, and immunoblotted
with anti-Flag. Comparable loading was checked by staining for Erk2.
(b) Functional coupling of the RHC variants. HEK 293T cells transfected with
WT-MC1R or the indicated variants (0.45 mg/dish) were challenged with
10
11 or 10
7 M NDP-MSH, or vehicle (control). cAMP was determined by
radioimmunoassay. (c) Displacement curves for binding of [125I]NDP-MSH to
HEK 293T cells expressing the RHC variants. Cells transfected with the
indicated variants were incubated with 10
10 M [125I]NDP-MSH and
increasing concentrations of unlabeled ligand, from 10
10 to 10
6 M. The
radioactivity associated was measured. Note the two-fold maximal binding
obtained for WT or D294H compared with R151C and R160W. (d) Expression
of WT-MC1R and RHC variants on the plasma membrane of HEK 293T cells.
Nonpermeabilized cells were stained for the Flag epitope and analyzed by
flow cytometry. A representative overlay histogram is shown. Blank (filled
curve) refers to cells transfected with WT-MC1R lacking the Flag epitope,
treated in parallel to the samples.

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 BL Sánchez-Laorden et al.
Melanocortin 1 Receptor Homodimerization

−Igepal + Igepal a WT/variant(1:3)

50

40 *** ***

c.p.m. bound/dish
(thousands)
30
WT
20

10

0
pCDNA R151C R160W D294H

b 2.0 WT
D294H
1.5 ***

pmol cAMP/
g protein

WT + D294H
R151C 1.0
0.50

***
0.25 ***
***
***
0.00
Control 10–11 10–7

Figure 9. Functional analysis of coexpressed WT-MC1R and RHC variants.

(a) Expression of [125I]NDP-MSH binding sites. Cells were cotransfected with
R160W
WT (0.15 mg/dish) and empty pcDNA or the indicated variants (0.45 mg/dish)
and analyzed for binding of the iodinated ligand. (b) Inhibition of functional
coupling. Cells were transfected with WT-MC1R (0.15 mg/dish), D294H
(0.45 mg/dish), or cotransfected with both forms at a ratio of 1:3 (0.15 mg/dish
WT plus 0.45 mg/dish D294H). At 24 hours after transfection, cells were
challenged for 30 minutes with vehicle (control) or with NDP-MSH at a final
concentration of 10
11 or 10
7 M, and their cAMP contents were determined
(***Pp0.001).

D294H
Figure 8. Decreased expression of the R151C and R160W MC1R variants on
the plasma membrane. HEK 293T cells grown on coverslips were transfected
with the indicated variants or with WT-MC1R. Staining with an anti-Flag
monoclonal antibody followed by an Alexa 568-conjugated secondary
antibody was performed without permeabilization, for visualization of
receptor molecules located on the plasma membrane (left column, labeled

Igepal), or after permeabilization with 0.5% Igepal, 15 minutes at room
temperature (right column, labeled þ Igepal).
expression for D294H, and lower staining for R151C and
R160W (Figure 7d). Moreover, fluorescence microscopy
showed a fainter labeling of nonpermeabilized cells trans-
fected with R151C and R160W, as compared with cells
expressing WT or D294H (Figure 8). Conversely, staining of
permeabilized cells was intense for cells expressing R151C or
R160W, suggesting that the decreased membrane staining
was not due to lower expression levels but, rather, due to
partial intracellular retention (Figure 8).
Coimmunoprecipitation of differentially epitope-tagged
variants and WT followed by Western blot demonstrated
that the RHC mutants heterodimerized with WT-MC1R (not
shown). Therefore, we analyzed the functional effects of
cotransfection with the WT and variant forms. Coexpression
of WT and R151C or R160W caused a modest but significant
decrease in binding, but D294H had no effect (Figure 9a).
This is consistent with the behavior of the variants expressed
individually. For analysis of functional coupling to adenylyl
cyclase, we only used the nearly complete LOF D294H form,
as it provides a simpler model system. Cotransfection of WT
and D294H abolished completely the constitutive activity of
MC1R, as well as the response to low concentrations of the
agonist (1011 M). However, when cAMP accumulation was
measured following a challenge with a saturating concentra-
tion of NDP-MSH (10
7 M), no statistically significant
differences were observed for cells expressing WT alone, or
WT and the D294H form (Figure 9b).
DISCUSSION
Human MC1R is a highly polymorphic gene accounting for
much of the normal variation of pigmentation traits (Rees,
2003). By determining skin phototype and sun sensitivity,
MC1R behaves as a modifier of skin cancer risk (Sturm,
2002). The MC1R gene encodes for the aMSH receptor, a
member of the GPCR superfamily. Many members of this
family function as dimers or higher order oligomers (Milligan,
2001; Angers et al., 2002; Breitwieser, 2004). Dimerization

178 Journal of Investigative Dermatology (2006), Volume 126


has been shown to modulate pharmacological properties
such as agonist-binding affinity and coupling efficacy. For
some GPCRs, dimerization may be a necessary processing
step required for efficient cell surface expression (Salahpour
et al., 2004). Moreover, dimerization seems to account for
the disease-related dominant-negative effects of certain
GPCR mutants, including MC4R, a member of the melano-
cortin receptor subfamily with high sequence similarity with
MC1R (Biebermann et al., 2003).
Our results prove that MC1R undergoes constitutive
dimerization in heterologous cells overexpressing the recep-
tor. This was shown by Western blot and coimmunoprecipi-
tation, and by the finding of dominant-negative effects and of
a modulation of the pharmacological properties of the
receptor in cells coexpressing different variants. The resis-
tance to SDS of oligomeric species, with similar monomer:-
dimer ratios for SDS concentrations ranging from 0.5 to 3.0%,
as well as the strong dissociating effect of the reducing agent
2-mercaptoethanol, suggests a role of disulfide bonding in
dimerization. Concerns have been raised about the use of
heterologous systems as a model to study GPCR dimeriza-
tion, as a strong overexpression may lead to irrelevant
oligomerization. Thus, we also demonstrated dimerization in
melanoma cells stably expressing near-physiological MC1R
levels. Therefore, dimerization of MC1R is not an artifact of
protein overexpression or cell solubilization, and most likely
reflects the situation in melanocytes.
MC1R dimerization occurs early in receptor ontogeny,
probably in the ER. This is shown by (i) the finding of dimers
of the deglycosylated ‘‘de novo’’ form of MC1R, (ii) the
dimerization behavior of LOF mutants displaying intracellu-
lar retention, (iii) presence of dimers in cells with a disrupted
Golgi apparatus following brefeldin A treatment, and (iv)
efficient dimerization of an MC1R artificial variant retained in
the ER. Therefore, MC1R dimerization may be required for
export to the plasma membrane, as shown for other GPCRs
(Angers et al., 2002; Salahpour et al., 2004). Moreover, the
dominant-negative effect exerted by LOF mutants with
intracellular retention is best explained by trapping WT units
in internal compartments. A similar mechanism has been
postulated for dominant-negative mutants of other GPCRs
(Angers et al., 2002; Salahpour et al., 2004). Interestingly, a
heterozygous human melanoma cell line bearing the L93R
allele does not respond to agonists by activating cAMP
production, in spite of expressing an active adenylyl cyclase
(Sanchez-Más et al., 2002), consistent with a dominant-
negative effect of L93R.
We considered the possible functional consequences of
MC1R dimerization. Although no cooperativity in agonist
binding was demonstrated by Hill analysis, cotransfection of
WT and the D5 mutant yielded a form of different affinity and
lower efficacy in activating cAMP synthesis as compared with
WT. This demonstrates that MC1R dimerization can yield
functional units with slightly different pharmacological
properties. This was further shown by studies with the
frequent RHC forms. The D294H variant abolished constitu-
tive signaling as well as signaling at low agonist concentra-
tions in HEK cells coexpressing WT and variant MC1R. These
BL Sánchez-Laorden et al.
Melanocortin 1 Receptor Homodimerization
inhibitory effects were not due to reduced MC1R plasma
membrane expression, as shown by binding studies, and
therefore most likely reflect changes in the coupling efficacy
of the heterodimer.
In summary, we have shown that MC1R dimerization
takes place in living cells and has a wide range of functional
consequences, including partial functional complementation
of inactive forms, dominant-negative effects decreasing WT
expression on the cell surface, and, for certain heterodimeric
combinations, a modulation of pharmacological properties,
such as affinity for agonists and coupling efficacy. Changes in
other functional properties, such as desensitization (Sánchez-
Más et al., 2005a) or internalization, are also possible and are
being investigated. Therefore, the presence of mutant MC1R
alleles may have consequences beyond those based on
dosage effects and haploinsufficiency, thus yielding a
complex spectrum of graded responses to the melanocortins.
Given the high number of MC1R natural variants and the high
frequency of variant alleles in some populations, our results
highlight an unexpected complexity of the MC1R–melano-
cortin system. This complexity is fully consistent with the
diversity of hair color shades, skin phototypes, and sun
sensitivities (Rees, 2003; Bohm et al., 2005), and may extend
to other aspects of skin biology, since MC1R has been shown
to regulate specific functions of skin cells other than
melanocytes (Bohm et al., 2004; Elliott et al., 2004).
MATERIALS AND METHODS
Expression constructs
All expression constructs were prepared with the pcDNA3 vector
(Invitrogen, Carlsbad, CA). The following constructs have been
described: WT-MC1R (Jimenez-Cervantes et al., 2001b), MC1R-Tag
(Sanchez-Más et al., 2002), Flag-MC1R, the C-terminus-deleted
mutants D5-, D12-, and D14-MC1R (Sánchez-Más et al., 2005), and
Flag epitope-labeled L93R and R162P variants (Sánchez-Laorden
et al., 2005). The RHC variants R151C, R160W, and D294H were
obtained by mutagenesis, using the QuickChange kit (Stratagene, La
Jolla, CA) and WT Flag-MC1R as template. An MC1R-HA construct
bearing a single copy of the hemagglutinin (HA) epitope immedi-
ately after the C-terminal W317 residue was obtained by PCR
amplification of WT-MC1R, using the same forward primer as for
cloning of the WT gene and a reverse primer containing the HA
epitope sequence. Sequences of mutagenic primers are available on
request. All constructs were verified by automated sequencing.
Cell culture and transfection
HEK 293T cells were cultured in RPMI 1640 with 10% fetal calf
serum (FCS), 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells
grown to approximately 50% confluence were transfected with
Lipofectamine 2000 (Invitrogen). HBL human melanoma cells were
grown in DMEM supplemented with antibiotics and 10% FCS. Stable
transfectants were obtained with Lipofectamine. Clones were
selected with geneticin (Mas et al., 2003).
Functional assays
All experiments were performed in independent duplicate or
triplicate dishes of 12-well plates and repeated at least twice, so
that each experimental point is the mean7standard error of mean for
www.jidonline.org 179
 BL Sánchez-Laorden et al.
Melanocortin 1 Receptor Homodimerization
nX4. Radioligand-binding assays were carried out with [125I]NDP-
MSH, specific activity 2000 Ci/mmol (Amersham Pharmacia Bio-
tech, Little Chalfont, Buckinghamshire, UK), and unlabeled NDP-
MSH up to 10
6 M when required. The experimental procedure has
been described (Sanchez-Más et al., 2002). Functional coupling was
analyzed in cells serum-deprived for 24 hours prior to a 30-minute
stimulation with 10
13–10
7 M NDP-MSH. Cells were lysed in
preheated 0.1 N HCl (701C) and cAMP was measured with a
radioimmunoassay kit (Amersham) as described (Jimenez-Cervantes
et al., 2001a; Sanchez-Más et al., 2002).
Immunoprecipitation and Western blot
HEK 293T cells were transfected with MC1R-HA, alone or in
combination with Flag epitope-labeled MC1R variants. Approxi-
mately 2  106 cells were washed twice with phosphate-buffered
saline and solubilized in 200 ml of iodoacetamide-containing
solubilization buffer (50 mM Tris-HCl, pH 8, 1% Igepal CA-640,
1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 10 mM iodo-
acetamide). The sample was centrifuged (105,000  g, 30 minutes)
and the supernatant was precleared with 20 ml of protein G-agarose
slurry, for 1 hour, followed by centrifugation. Monoclonal anti-HA
antibody (2 mg) (clone HA-7, from Sigma, St Louis, MO) and protein
G-agarose slurry (20 ml) were added. After a 3-hour incubation, the
sample was centrifuged and the pellet was washed five times
with 10 mM Tris-HCl, pH 7.5, 500 mM KCl, 300 mM NaCl, and
0.05% Triton X-100. Elution was performed in 20 ml of 2% SDS for
30 minutes at room temperature. Eluted supernatants were
mixed in a ratio of 2:1 with an electrophoresis sample buffer
(180 mM Tris-HCl, pH 6.8, 15% glycerol, 9% SDS, 0.075%
bromophenol blue, and 7.5% b-mercaptoethanol). Electrophoresis
and Western blotting were performed as described (Sanchez-Más
et al., 2002; Sánchez-Laorden et al., 2005). Direct Western blot was
performed with extracts of cells expressing Flag-MC1R variants.
Blots were probed with anti-Flag M2 monoclonal antibody-
peroxidase conjugate (Sigma) and stained with a chemiluminescent
substrate (Amersham).
Flow-cytometric analysis
Approximately 250,000 HEK 293T cells expressing the relevant
constructs were incubated for 30 minutes in a volume of 100 ml with
anti-Flag M2 monoclonal antibody (Sigma) at a dilution of 1:25.
Cells were washed twice with 2% FCS and 0.01% NaN3 in
phosphate-buffered saline, and incubated with a phycoerythrin-
labeled anti-mouse IgG (1:50) for 30 minutes at 41C. Cells were
washed, resuspended in 500 ml of 0.4% paraformaldehyde in
phosphate-buffered saline, and analyzed in a Becton Dickinson
FACScan.
Immunofluorescence microscopy
Cells grown on coverslips were fixed with paraformaldehyde (4%) in
phosphate-buffered saline. To assess the effects of brefeldin A,
control and brefeldin A-treated (1 mg/ml, 30 minutes) cells were
permeabilized (0.1% saponine) and immunostained for the Golgi
matrix protein GM130 with a monoclonal antibody and an Alexa
488-conjugated secondary antibody (Martinez-Alonso et al.,
2005). For detection of WT and variant MC1R, cells expressing
the relevant forms were labeled with the anti-Flag M2 mono-
clonal antibody (Sigma), followed by an Alexa 568-conjugated
180 Journal of Investigative Dermatology (2006), Volume 126
secondary antibody. Samples were examined with a Zeiss Axiophot
fluorescence microscope.
Immunoelectron microscopy
HEK 293T cells expressing MC1R-Tag were fixed with 2%
paraformaldehyde and 0.2% glutaraldehyde in 0.1 M sodium
phosphate buffer, pH 7.4, washed, embedded in 10% gelatin,
cooled on ice, and cut into 1 mm3 blocks. Blocks were infused with
2.3 M sucrose at 41C overnight, frozen in liquid nitrogen, and stored
until cryoultramicrotomy. Sections (B50 nm thick) were cut at

1201C in a Leica Ultracut T/FCS. Ultrathin sections were picked
up in a mix of 1.8% methylcellulose and 2.3 M sucrose (1:1).
Cryosections were collected on carbon- and formvar-coated copper
grids and incubated with an anti-HA monoclonal antibody,
followed by a rabbit anti-mouse antiserum and 10 nm protein
A-gold (Martinez-Alonso et al., 2005). Sections were treated with 1%
glutaraldehyde, counterstained with uranyl acetate, pH 7.0, and
embedded in methyl cellulose-uranyl acetate, pH 4.0 (9:1). Grids
were examined with a Philips Tecnai 12 electron microscope.
Other procedures
For data normalization, cells were solubilized in 10 mM phosphate
buffer, pH 7, and 1% Igepal CA-640, containing 0.1 mM EDTA and
0.1 mM phenylmethylsulfonyl fluoride. The extract was centrifuged
and the supernatant used for protein determination by the
bicinchoninic acid method. For deglycosylation studies, extracts
were incubated at 371C for 4 hours with endoglycosidase H (5 U) in
50 mM phosphate buffer, pH 7.0, 10 mM EDTA, and 0.1% SDS, as
described (Olivares et al., 2003).
Differences between experimental data were assessed for
significance using the Student’s t-test. Curve fitting and statistical
analysis were performed with the GraphPad Prism package
(GraphPad Software, San Diego, CA).
The medical ethical committee of the University of Murcia
approved all described studies.
CONFLICT OF INTEREST
The author states no conflict of interest.
ACKNOWLEDGMENTS
J.S.M. and B.L.S.-L. contributed equally to this work. This work was supported
by Grant SAF2003-03411 (to J.C.G.B.) from the CICYT, Spain, and FEDER
funds (European Community). J.S.M. is a fellow of the Fundación Séneca
(Comunidad Autónoma de la Región de Murcia). B.L.S.-L. and E.M.-A. hold
fellowships from the Ministry of Education (Spain). Work by E.M.-A. and
J.A.M.-M. was supported by Grant BFU2004-05568 (CICYT, Spain). We thank
Professor G. Ghanem (Free University of Brussels, Belgium) for HBL cells and
Dr C. Martı́nez (Centro de Hemodonación, Hospital Reina Sofı́a, Murcia,
Spain) for thromboxane a receptor cDNA.
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