PIGMENT CELL RES 16: 540–547.

2003 Copyright
Blackwell Munksgaard 2003

Printed in UK—all rights reserved ISSN 0893-5785

Rate Limiting Factors in Melanocortin 1 Receptor Signalling Through

the cAMP Pathway

JESÚS SÁNCHEZ MÁS, INEKE GERRITSEN, CHRISTA HAHMANN, CELIA JIMÉNEZ-CERVANTES and
JOSÉ CARLOS GARCÍA-BORRÓN

Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Murcia, Spain
*Address reprint requests to José Carlos Garcı´a-Borrón, Department of Biochemistry and Molecular Biology, School of Medicine, University of
Murcia, Apto 4021. Campus de Espinardo, 30100 Murcia, Spain. E-mail: gborron@um.es
Received 28 April 2003; in final form 4 June 2003

The melanotropic actions of a-melanocyte-stimulating hor-
mone (a-MSH) and other melanocortins are mediated by
activation of the melanocortin 1 receptor (MC1R). This
G protein-coupled receptor is positively coupled to Gs and
triggers the cyclic adenosine mono-phosphate (cAMP) path-
way. Mutations of the MC1R gene are associated with skin
type and pigmentation phenotypes, and with increased risk of
skin cancers. Genetic studies have demonstrated an heterozy-
gote carrier effect for these associations, suggesting the
importance of variant allele dosage. This could be accounted
for, at least partially, if the number of MC1R molecules,
rather than the Gs protein or the effector enzyme, adenylyl
cyclase, is limiting for the activation of the signalling
pathway. However, the nature of the limiting factor(s) in
MC1R signalling has not been investigated. We addressed
this question by comparing the cAMP output of clones of
human melanoma cell lines enriched in MC1R by stable
transfection. We also analysed heterologous cell systems
widely used for functional studies of MC1R. We show that
cAMP production in clones of Chinese hamster ovary cells
stably expressing the MC1R is a linear function of receptor
number up to high, supraphysiological levels of approximately
50 000 a-MSH binding sites per cell. Enrichment of human
melanoma cell lines with MC1R also results in increased
cAMP levels, with a small leftward shift of the agonist dose–
response curves. Therefore, at physiological expression levels
second-messenger generation is dependent on receptor density.
Within melanoma cells and also likely in normal melanocytes,
MC1R appears the limiting factor controlling the output of
the cAMP signalling pathway.
Key words: Melanocortin 1 receptor, Cyclic adenosine mono-
phosphate, Receptor density, Gs, Adenylyl cyclase

INTRODUCTION
Mammalian pigmentation is mainly determined by the
amount and type of the cutaneous melanin pigments. The
rate-limiting enzyme in melanin synthesis is tyrosinase, which
catalyses the conversion of l-tyrosine into l-dopaquinone. In
the absence of thiol compounds such as cysteine or glutathi-
one, l-dopaquinone is converted into black eumelanins, by a
complex pathway including both chemical and enzymatic
reactions. In the presence of thiol compounds, l-dopaqui-
none is the precursor of brown to red pigments, called
pheomelanins (1).
The melanotropic actions of a-melanocyte-stimulating
hormone (a-MSH), which promotes pigmentation and pro-
liferation of human melanocytes (2), are mediated by the
melanocortin 1 receptor (MC1R). Accordingly, the MC1R
gene is a major determinant of human skin and hair
pigmentation characteristics (reviewed in 3 and 4). In the
mouse, activation of MC1R determines the switch from
pheo- to eumelanogenesis (5). MC1R is a G protein-coupled
receptor (GPCR) coupled to adenylyl cyclase via the
a subunit of the heterotrimeric Gs protein, and its activation
triggers the cyclic adenosine mono-phosphate (cAMP)
cascade (6).
After the cloning of the MC1R gene (7, 8), it was realized
that its coding sequence is highly polymorphic and that
naturally occurring variants are associated with specific
pigmentation traits. Notably, the Arg151Cys, Arg160Trp

Abbreviations – DME, Dulbecco’s modified Eagle’s medium; Fsk, forskolin; GPCR, G protein-coupled receptor; a-MSH, a-melanocyte-stimulating
hormone; MC1R, melanocortin 1 receptor; NDP-MSH, [Nle4, D-Phe7] a-melanocyte-stimulating hormone; RHC, red hair colour

540 Pigment Cell Res. 16, 2003

and Asp294His alleles are associated with pigmentary traits
such as red hair, fair skin, poor tanning ability and
propensity to freckle. This human cutaneous phenotype is
termed as RHC phenotype (reviewed in 4), and is also
associated with increased risk of melanoma and non-melan-
oma skin cancers (4, 9–12). MC1R variants also increase the
penetrance of melanoma predisposing alleles of the cyclin-
dependent kinase inhibitor gene CDKN2A, and decrease the
age of onset of the malignancy (13, 14).
Case-control studies have shown a clear heterozygote
carrier effect of MC1R RHC alleles on pigmentation traits
and skin cancer risk, suggesting the importance of MC1R
variant allele dosage (reviewed in 4). The RHC alleles are
loss-of-function variants as shown by functional studies in
heterologous cells (15–17), and in cultured human melano-
cytes (18), although they may display some residual activity
(19). This allele dosage effect could be partially explained if
the density of MC1R receptors in the plasma membrane of
melanocytes is limiting for the melanocortin-induced activa-
tion of second-messenger generation. In this case, cAMP
generation in response to a-MSH within a melanocytic cell
bearing one loss-of-function variant and one wild-type allele
would be decreased when compared with melanocytes
homozygous for wild-type MC1R and expressing the same
number of receptors.
Consistent with this hypothesis, the number of specific
a-MSH binding sites in human melanocytes, when compared
with mouse melanocytes, is low (reviewed in 20). Indeed,
melanoma cells overexpressing the MC1R at the mRNA and
protein levels (7, 21, 22) hardly display more than a few
thousands of binding sites per cell (23, 24). Under these
conditions, it seems likely that MC1R density, instead of the
concentration of other early components of the signalling
pathway, namely the Gs protein and adenylyl cyclase, will
limit the cellular production of cAMP in melanocortin-
stimulated melanocytes. However, this simple hypothesis has
not yet been tested. Moreover, the activation of GPCR-
dependent signalling pathways is often described as a
ÔcatalyticÕ process, where a single ligand-receptor complex
can activate several molecules of the transducing G protein.
Accordingly, a modest number of receptors can, in some
cases, be in excess over other components of the transducing
machinery. For instance, b-adrenergic receptors are ex-
pressed by S49 lymphoma cells at an approximate density
of 1000 sites per cell, and positively coupled to adenylyl
cyclase via Gs, thus constituting a system similar to MC1R in
melanocytes. In S49 cells, the limiting factor for cAMP
generation appears to be adenylyl cyclase, rather than either
the b-adrenergic receptors or Gs, of which more than 100 000
copies per cell are present (25). In other systems, mainly
heterologous cells employed for transient or stable over-
expression of receptor genes, but also some natural systems,
G protein availability seems limiting (reviewed in 26).
However, the number of G proteins activated by a ligand–
receptor complex will certainly be variable for different
receptor types and cell systems, depending on factors such as
the half-life of the complex, its lateral mobility within the
membrane, and the efficiency of G protein activation. Other
factors such as the rate of receptor turnover can also have an
impact on the responsiveness of the system, which further
Pigment Cell Res. 16, 2003
complicates an Ôa prioriÕ prediction of the effects of receptor
density on the level of second-messenger generation.
Therefore, the relationship between the levels of MC1R
expression in the plasma membrane of melanocytes and
melanoma cells on one hand, and the cAMP intracellular
levels in response to the melanocortins, on the other, remains
unclear. Knowledge of this relationship is required to
evaluate properly the basis of the allele dosage effect detected
in genetic studies and the relevance of changes in the number
of a-MSH binding sites in response to physiological regula-
tory stimuli. Using heterologous cells expressing the wild-
type MC1R gene, and human melanoma cells enriched in
MC1R by stable transfection, it is shown that cAMP
generation in a-MSH-stimulated cells is a linear function of
receptor number over a wide range of densities of binding
sites. These data prove that MC1R is the limiting factor for
the hormonal stimulation of the main signalling pathway
involved in the melanogenic response of melanocytes to
melanocortins.
MATERIALS AND METHODS
Reagents
The radioligand [125I]NDP-MSH, specific activity 2000 Ci/
mmol, a cAMP radioimmunoassay kit and all restriction
endonucleases were from Amersham Biosciences (Little
Chalfont, Buckinghamshire, UK). Igepal CA-630, bovine
serum albumin (BSA), ethylenediaminetetraacetic acid
(EDTA), phenylmethylsulfonyl fluoride (PMSF) and
bicinchoninic acid were from Sigma (St Louis, MO, USA).
a-MSH, the superpotent analogue [Nle4, D-Phe7] a-melano-
cyte-stimulating hormone (NDP-MSH), forskolin (Fsk) and
G418 sulphate were from Calbiochem (Darmstadt, Ger-
many). Reagents for cell culture were obtained from either
Nunc (Hereford, UK) or Gibco (Paisley, UK) and plastic-
ware from TPP (Trasadingen, Switzerland). Taq polymerase
was from Biotools (Madrid, Spain). Other reagents were
from Merck (Darmstadt, Germany) or Prolabo (Barcelona,
Spain), unless otherwise specified.
Cell Culture, Transfection and Selection of Clones
The human melanoma cell lines HBL and LND1, a gift from
Prof. G. Ghanem (Free University of Brussels, Belgium),
were cultured in DME supplemented with 10% fetal calf
serum, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml
streptomycin sulphate. Chinese hamster ovary (CHO) cells
were cultured in HAM-F12 with the same supplements and
1% sodium pyruvate. Transfection was performed with the
Superfect transfection reagent (Qiagen, Hilden, Germany), as
per manufacture’s instructions. In order to obtain clones
stably expressing the MC1R protein, the usual culture
medium was supplemented with G418 sulphate at a final
concentration of 400 lg/ml for CHO cells or 800 lg/ml for
human melanoma cells, immediately after transfection. Cells
were given fresh G418-containing medium every 2 days, until
individual clones resistant to the antibiotic could be isolated.
Single colonies were then expanded, in the continuous
presence of G418. Human embryonic kidney (HEK) 293T
541
cells were cultured and used for transient expression of
MC1R (17).
Radioligand Binding Studies
Two types of assays have been performed. In order to
compare the relative levels of binding sites in different
clones, a simpler assay performed at a single ligand
concentration was employed. Equal numbers of cells from
each clone under study were seeded in triplicate wells of
12 well plates and grown to approximately 80% confluence.
In order to avoid interferences from melanocortin peptides
present in serum, the culture medium was replaced by
serum-free medium 24 h before the assay. Then, the
medium was aspirated and the cells incubated for 1 h at
37C in serum-free medium containing 10)10 M of the
radioactive tracer. Cells were washed twice with complete
medium for 5 min at room temperature, trypsinized and
bound radioactivity was measured in a c counter. Non-
specific binding was estimated in the presence of competing,
unlabelled, 10)6 M NDP-MSH or with cells transfected
with empty vector. Parallel dishes were employed to
determine cell number and total protein.
In order to quantify the number of binding sites in the
plasma membrane of cells, these were incubated (1 h, 37C)
with increasing concentrations of NDP-MSH, ranging from
10)12 to 10)6 M and a fixed amount of [125I]-NDP-MSH,
corresponding to 10)10 M and 0.1 lCi per well, in a final
volume of 0.5 ml of medium, washed and counted as above.
Maximal binding (Bmax) values and dissociation constants
were calculated by non-linear regression, with the Graphpad
software (San Diego, CA, USA).
Measurement of Agonist-induced cAMP Intracellular Levels
Cyclic adenosine mono-phosphate levels were determined
after a 30 min stimulation with NDP-MSH or Fsk, using a
commercial radioinmunoassay (17, 27). Because of the short
incubation time prior to the measurement of the second
messenger levels, the results obtained with the stable NDP-
MSH synthetic melanocortin can very likely be extrapolated
to the more labile natural a-MSH.
RT-PCR Analysis of MC1R Transcript Abundance
Total RNA was extracted with guanidinium thiocyanate
and purified by cesium chloride gradient centrifugation
(21). cDNA was synthesized with the Superscript kit
(Invitrogen, Carlsbad, CA, USA), as per manufacture’s
instructions. Preliminary PCR runs were performed with a
commercial d-glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)-specific primer set (Clontech, Basel, Switzerland)
to ascertain comparable loadings and that the reactions
were run in the exponential phase. Then, the selected
amounts of cDNA were amplified with MC1R-specific
primers yielding a 1023 bp full length product (21). Suitable
aliquots of the PCR were electrophoresed in 1.5% agarose
gels, ethidium bromide-stained, and band intensities were
quantified in a Gel Doc system (BioRad, Hercules, CA,
USA).
542
Other Procedures
Cell number was estimated by the trypan blue exclusion
method. Total protein was measured by the bicinchoninic
acid method, in extracts prepared by solubilization of
cells in 10 mM phosphate buffer pH 7, 1% Igepal CA-640,
0.1 mM EDTA, 0.1 mM PMSF, using BSA as standard.
RESULTS AND DISCUSSION
cAMP Production in Clones of CHO Cells Stably Expressing
the MC1R
As a first step to study the relationship between the number
of MC1R molecules in the plasma membrane of a given cell
population and the agonist-induced intracellular production
of cAMP, clones of CHO cells were obtained stably
expressing MC1R. CHO cells were chosen because they can
be transfected with relatively high efficiencies and allow for
the stable expression of high levels of a wide variety of
GPCRs, including the members of the melanocortin receptor
family (17, 28, 29). After transfection of CHO cells with a
wild-type MC1R construct, 23 G418-resistant clones were
selected and analysed for their response to a saturating
concentration of NDP-MSH (10)7 M). Approximately, 60%
of the clones responded to the melanocortin with reprodu-
cible and high increases in cAMP (Fig. 1A). The potency of
the response correlated reasonably well with MC1R gene
expression, detected by semiquantitative reverse transcriptase
polymerase chain reaction (RT-PCR) (Fig. 1B). As expected,
MC1R transcripts were not found in cells transfected with
empty vector, whereas mRNA levels were low for poorly
responsive clones such as clone 9, and higher for potently
responsive clones such as 2 and 29.
In order to define the components of the MC1R signalling
pathway that limit second-messenger generation at saturating
concentrations of agonists, the relationship between MC1R
expression and cAMP levels was analysed in melanocortin-
responsive clones challenged with 10)7 M NDP-MSH. The
rationale of these experiments was that a linear relationship
between binding capacity and cAMP levels would indicate
that the limiting factor for cAMP production is the availab-
ility of MC1R. Conversely, should the availability of Gs for
functional coupling, or the adenylyl cyclase activity be the
limiting factor, the shape of the curve would be hyperbolic
rather than linear, as reflecting the titration of these
components of the pathway by activated receptors. As a
first approximation, equal numbers of cells from all respon-
sive clones were seeded in 12 well plates, grown to semicon-
fluence, and incubated with 10)10 M radioactive tracer.
A plot of cAMP maximal production vs. the specific binding
of the clones yielded a strait line (Fig. 1C). Based on these
results, a limited number of clones covering the complete
range of observed binding capacities were selected for further
study, and the density of receptors in their plasma membrane
was measured. The values ranged from 900 to 49 000 binding
sites per cell, and further cAMP production varied linearly
with receptor number (Fig. 1D). Therefore, receptor densities
much higher than those present in human melanocytes and
melanoma cells, and more similar to those of mouse
Pigment Cell Res. 16, 2003
 (A)
(B)
(C) (D)
Fig. 1. Relationship between cyclic adenosine mono-phosphate (cAMP)
production and melanocortin 1 receptor (MC1R) expression in Chinese
hamster ovary (CHO) cells. (A) Maximal agonist-induced stimulation of
cAMP production. Clones of CHO cells transfected with a wild-type
MC1R construct were selected on the basis of their resistance to G418.
Equal numbers of cells were seeded in six well plates, and serum-deprived
for 24 h before determination of cAMP, with (closed bars) or without
(open bars) stimulation with 10)7 M [Nle4, D-Phe7] a-melanocyte-
stimulating hormone (NDP-MSH). Results are given as mean ± SEM
(n ‡ 3). (B) Relative levels of MC1R mRNA in CHO clones with different
responsiveness to NDP-MSH. Equal amounts of cDNA from the
parental cell line (lane labelled CHO) and three NDP-MSH responsive
clones (clone number indicated on top of each lane) were amplified with
primers yielding a full-length product (upper gel), or with a commercial
d-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer set (low-
er gel) to ensure comparable loading. The lane on the right shows the
mobility of markers of the indicated size, expressed in base pairs.
(C) Linear relationship between cAMP production and binding capacity.
Receptor density in agonist-responsive clones was estimated by incuba-
ting the cells at a fixed 10)10 M concentration of labelled NDP-MSH and
measurement of specifically bound radioactivity, in triplicate. A plot of
the maximal levels of cAMP (after stimulation with 10)7 M agonist) vs.
specific binding yielded a straight line (r2 ¼ 0.6984). (D) Plot of maximal
cAMP production as a function of receptor number. Five NDP-MSH-
responsive clones with different binding capacity were selected, and their
levels of MC1R expression were determined. There was a linear
relationship between their maximal cAMP response and the density of
NDP-MSH binding sites (r2 ¼ 0.8053).
Pigment Cell Res. 16, 2003
melanocytic cells, apparently fail to saturate the second-
messenger generating machinery, at least in CHO cells.
cAMP Production in Melanoma Cells is Increased by
Overexpression of MC1R
The data obtained for heterologous CHO cells strongly
suggest that MC1R density should also be limiting for cAMP
generation in malignant melanocytes. This was tested by
examining the effect of increased MC1R expression on the
cAMP response of human melanoma cells. The study was
performed with two cell lines, HBL and LND1, homozygous
for the MC1R wild-type form (17), to avoid possible artefacts
due to the presence of mutant alleles. Moreover, the HBL cell
line already displays several thousands of binding sites per
cell (23, 24).
Cells were transfected with wild-type MC1R and G418-
resistant clones were stimulated with a saturating concen-
tration of NDP-MSH. In eight of nine HBL clones, a
30 min stimulation with 10)7 M NDP-MSH yielded cAMP
levels at least two fold-higher than the parental cell line. In
one case (clone HBL20) the maximal response increased as
much as ninefold (Fig. 2A). MC1R mRNA was increased in
responsive clones, and was the highest for clones 17 and 20,
which also yielded the higher fold-increases in cAMP upon
agonist stimulation (Fig. 2B). It should be noted that in the
RT-PCR protocol used in this study, low target cDNA
loads and only 25 amplification cycles were employed in
order to keep the reaction in the exponential phase and
allow for comparison of mRNA levels in clones expressing
high amounts of the messenger. Under these conditions, the
amplification band corresponding to the parental cell line is
barely detectable by ethidium bromide staining of the gels.
This band becomes evident if a higher number of amplifi-
cation cycles are performed. For the LND1 cell line, five of
nine clones also responded with significantly higher cAMP
increases, when compared with the parental cell line
(Fig. 2C).
It is worth noting that the levels of cAMP in melanoma
clones were consistently higher than those observed in CHO
clones expressing the MC1R protein, under identical condi-
tions of stimulation. This might result from higher intracel-
lular concentrations of the Gs protein, or higher adenylyl
cyclase activity as demonstrated by the higher cAMP levels in
Fsk-treated melanoma cells (Table 1). Another interesting
aspect is that there was a trend towards increased basal
cAMP levels, in the absence of any stimulation by exogenous
ligands, in those clones that showed an increased respon-
siveness. This suggests that MC1R might display constitutive
activity, or that an autocrine stimulatory loop could be
operating in these cells, as a result of the expression of MSH-
related peptides by melanoma cells (30). Obviously, both
possibilities are not mutually exclusive. In any case, the
finding of an increased cAMP response in melanoma cells
expressing higher levels of MC1R mRNA points to the
receptor protein as the limiting factor in the transducing
pathway in those cells. As the HBL cell line displays higher
numbers of a-MSH binding sites than most human melan-
oma cell lines (23, 24), and overexpresses the MC1R gene
when compared with normal melanocytes and congenital
543
 (A)
(B)
(C)
Fig. 2. Agonist induced generation of cyclic adenosine mono-phosphate
(cAMP) in melanocortin 1 receptor (MC1R)-enriched melanoma cells.
(A) Equal numbers of cells from clones derived from the HBL cell line
were analysed for cAMP production in response to [Nle4, D-Phe7]
a-melanocyte-stimulating hormone (NDP-MSH), as described in Fig. 1.
Open bars correspond to the basal cAMP levels, closed bars to the cAMP
levels in cells stimulated for 30 min with 10)7 M NDP-MSH, and HBL
refers to parental cell line. The statistical significance of the differences
observed was estimated by means of the Student’s t-test (*P < 0.5,
**P < 0.05 and ***P < 0.005). (B) MC1R mRNA levels in control
HBL cells (lane labelled HBL) and MC1R enriched clones (clone number
indicated on top of each lane). The experiment was performed as
described in Fig. 1(B). (C) Responsiveness of the parental LND1 human
melanoma cell line and derived clones, studied as in panel A.
naevus cells (21), this conclusion can probably be extended to
most, if not all, melanocytic cells.
The relationship between expression of binding sites and
cAMP response was analysed in deeper detail for HBL cells.
544
Four clones were selected, covering the full range of cAMP
levels from HBL19, whose responsiveness is slightly higher
than the parental cell line, to the very potently responsive
HBL17 and HBL20 clones. These clones exhibited a wide
range of MC1R densities, from approximately 27 000 NDP-
MSH binding sites per cell (clone 19) to values as high as
242 000 sites per cell (clone 17). For comparison, receptor
density in the parental cell line was 3500 sites per cell, in
reasonable agreement with previous estimates by others
(23, 24). Surprisingly, in spite of very high levels of
expression in several of the clones under study, maximal
generation of cAMP seemed a linear function of receptor
number (Fig. 3). It should be noted, however, that there was
not a perfect correlation between the number of receptors per
cell and the cAMP response, at receptor densities within the
200 000 sites per cell range or higher. This may indicate that,
at these extremely high levels of expression, the transducing
machinery begins to be saturated.
Effect of MC1R Number on Agonist Binding and Functional
Coupling Parameters
It has been pointed out that, theoretically, MC1R agonist
binding parameters should not be affected by the level of
receptor expression, whereas the shape of cAMP dose–
response curves could be altered by receptor density (31).
Low MC1R expression would result in activation of only a
fraction of the Gs and/or adenylyl cyclase, thus shifting the
dose–response curves for agonists to the right (Fig. 4).
Conversely, at very high receptor densities yielding an excess
of receptor molecules over Gs, the maximal response would
be achieved at submaximal receptor occupancies, thus
causing an apparent leftward shift of the agonist potency
curves. In order to analyse these aspects, the affinity of
MC1R for NDP-MSH was estimated from equilibrium
binding curves, using the parental cell lines and one of the
strongly overexpressing clones derived from the HBL line
(HBL20). cAMP generation dose–response curves were also
constructed. The results are shown in Fig. 4, and summarized
in Table 1.
The affinity of MC1R for NDP-MSH was similar in
LND1, HBL and HBL20 cells, irrespective of the Bmax
values, as shown by comparable dissociation constants in the
low nanomolar range. Coupling parameters were much more
variable, both in terms of maximal cAMP response, and
EC50 (agonist dose for half-maximal stimulation). For the
HBL and LND1 lines, the EC50 and Kd values were in good
agreement. Therefore, in these cells activation of the signal-
ling pathway parallels receptor occupancy, again suggesting
that MC1R limits the intracellular generation of the second
messenger. Conversely, for the HBL20 clone, the maximal
levels of cAMP were higher than in the parental cell line
(Fig. 4A), and the dose–response curve was slightly left-
shifted (Fig. 4B). This results is a lower EC50 value for the
MC1R overexpressing clone (Table 1) and confirms the
possibility that, at these high receptor levels, the transducing
system is saturated. For comparison, the EC50 value in HEK
293T cells transiently overexpressing the MC1R is further
reduced relative to the HBL20 clone, and the dose–response
curve is further left-shifted. This proves that, in this system,
Pigment Cell Res. 16, 2003
Table 1. Functional parameters of melano-
cortin 1 receptor (MC1R) in melanoma cells Coupling parameters Binding parameters
and heterologous expression systems
NDP-MSH,
Cell system Forskolinea maximalb EC50 (nM) Bcmax Kd (nM)

HBL (parental) 15.5 ± 2.4 0.69 ± 0.20 3.90 ± 1.02 3500 3.91 ± 1.55
HBL (clone 20) nd 7.84 ± 0.80 0.25 ± 0.09 214 000 7.33 ± 2.89
LND1 (parental) 9.3 ± 2.5 1.13 ± 0.21 2.56 ± 1.32 nd 4.68 ± 1.21
HEK 293Td 2.16 ± 0.13 0.30 ± 0.05 0.10 ± 0.04 490 000 1.54 ± 0.52

Data are given as mean ± SEM (n ‡ 3). nd ¼ not determined.

a
Expressed as pmoles cyclic adenosine mono-phosphate (cAMP)/lg protein after a 30 min challenge
with 10)5 M forskolin.
b
Expressed as pmoles cAMP/lg protein after a 30 min challenge with 10)7 M [Nle4, D-Phe7]
a-melanocyte-stimulating hormone (NDP-MSH).
c
Expressed as NDP-MSH binding sites per cell, mean of three independent determinations with
deviations lower than 25%.
d
In untransfected human embryonic kidney (HEK) 293T cells, the cAMP levels in control cells were
0.0043 ± 0.0003 pmoles/lg protein, and addition of NDP-MSH failed to cause a statistically
significant increase of cAMP. Untransfected cells did not display detectable specific binding of
radiolabelled NDP-MSH.

saturation of the signalling pathway is achieved at lower
concentrations of receptor-agonist complexes, and therefore,
that MC1R is not the limiting component of the signalling
pathway, in this case. Moreover, the maximal cAMP levels
achieved after stimulation of HEK 293T cells with Fsk are
much higher than those obtained at a saturating concentra-
tion of the agonist. Thus, it appears that in HEK cells
overexpressing the MC1R, the activity of adenylyl cyclase is
not fully stimulated at saturating hormone concentrations.
This points to the Gs protein as the component of the
cascade that is in defect with respect to the others, and is
therefore rate-limiting for cAMP generation.
In summary, the density of MC1R molecules expressed in
human melanoma cells determines the extent of the response
to agonists, over a wide range that covers most, if not all,
biologically relevant circumstances. Changes in receptor
number, even minor, will therefore be relevant in terms of
the amplitude of the cAMP response, although they may
10.0
pmoles cAMP/µg protein
have little impact on the shape of the dose–response curves.
Indeed, the HBL20 clone expressing a very high number of
receptors per cell, only displays a moderate shift in EC50. It is
more likely that comparable MC1R levels are never achieved
in vivo, in response to the known regulatory mechanisms of
MC1R expression (24). Moreover, overexpression of MC1R
in human melanoma cells when compared with normal
melanocytes may lead to higher cAMP levels in malignant
melanocytes. Together with the occurrence of an autocrine
stimulatory loop (30), this could account, at least partially,
for the well known lack of requirement of cAMP elevating
agents for melanoma cell proliferation when compared with
the stringent requirement observed in normal melanocytes
(32). However, a preliminary analysis of phenotypic effects of
MC1R overexpression, particularly on basal pigmentation or
on the levels of the melanogenic enzymatic activities, did not
yield significant results. For instance, although certain HBL-
derived clones appeared to pigment slightly more than the
parental cell line, this was not always the case, and may result
from normal stochastic variations in the melanogenic poten-
tial of the cells, rather than from differences in MC1R
expression. In particular, HBL20 cells, which express high
cAMP levels even under basal, unstimulated conditions, did

7.5 not seem more pigmented than the parental cells upon visual
inspection, nor did they display significantly increased levels
of tyrosine hydroxylase activity in the absence of added
melanocortins. Possibly, the effects of MC1R overexpression
5.0
may be more evident after exogenous hormonal stimulation
than under basal conditions.
The link between MC1R allelic variants, RHC phenotype
2.5 and skin cancer risk is firmly established. Recent works
provided insights into new, refined and sensitive pheomelanin
markers (33, 34), whose relationship with the MC1R geno-
type has been pointed out (4). The results presented here also
0 50 100 150 200 250 suggest a possible relationship between these markers and the
Number of receptors/cell level of MC1R expression. It will be interesting to see
(thousands) whether low expression of wild-type MC1R correlates with a
chemically defined pheomelanogenic state, thus yielding a
Fig. 3. Maximal levels of cyclic adenosine mono-phosphate (cAMP) in more complete picture of what has been termed the
[Nle4, D-Phe7] a-melanocyte-stimulating hormone (NDP-MSH) stimula- relationship between skin genophototype and chemophoto-
ted HBL clones, as a function of receptor number. A plot of the cAMP
response to a saturating dose of NDP-MSH (10)7 M) versus receptor type (4, 35). Within this context, studies of a possible
number yields a straight line (r2 ¼ 0.7003). association between polymorphisms at the MC1R promoter

Pigment Cell Res. 16, 2003 545

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cAMP (pmoles/µg protein)

7.5 Melanoma Res 2002;12:1–12

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Percentage maximal response

100
75
50
25
0 15.
–12 –11 –10 –9 –8
Log (NDP-MSH)
Fig. 4. Dose–response curves for cyclic adenosine mono-phosphate
(cAMP) accumulation in the parental and MC1R-enriched HBL20
clone. (A) Absolute levels of cAMP in control HBL (squares) or HBL20
(triangles) cells as a function of [Nle4, D-Phe7] a-melanocyte-stimulating
hormone (NDP-MSH) concentration. (B) For easier comparison of curve
shape, the data presented in panel A are normalized as percentage of the
maximal response. Symbols as in panel A. Circles refer to transiently
transfected human embryonic kidney (HEK) 293T cells. Results given as
mean ± SEM (n ‡ 3).
region (36), levels of MC1R density in the plasma membrane
and skin phenotype, may provide valuable information.
Acknowledgements – This work has been supported by grant PM1999-
0138, from the DGI, Ministry of Science and Technology, Spain. Jesús
Sánchez Más is recipient of a fellowship from the Fundación Séneca,
Comunidad Autónoma de la Región de Murcia, Spain.
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