Tài liệu Báo cáo khoa học: Association of feather colour with constitutively active melanocortin 1 receptors in chicken docx

Association of feather colour with constitutively active
melanocortin 1 receptors in chicken
Maria K. Ling
1
, Malin C. Lagerstro¨m
1
, Robert Fredriksson
1
, Ronald Okimoto
2
, Nicholas I. Mundy
3
,
Sakae Takeuchi
4
and Helgi B. Schio¨th
1
1
Department of Neuroscience, Uppsala University, Uppsala, Sweden;
2
Department of Poultry Science, University of Arkansas,
Fayetteville, Arkansas, USA;
3
Department of Biological Anthropology, University of Oxford, Oxford, UK;
4
Department of Biology, Faculty of Science, Okayama University, Okayama, Japan
Seven alleles of the chicken melanocortin (MC) 1 receptor
were cloned into expression vectors, expressed in mam-
malian cells and pharmacologically characterized. Four of
the clones e
+R

,e
+B&D
,e
wh
/e
y
,E
Rfayoumi
gave receptors to
which melanocortin stimulating hormone (a-MSH) and
NDP-MSH bound with similar IC
50
values and responded
to a-MSH by increasing intracellular cAMP levels in a dose-
dependent manner. Three of the cMC1 receptors; e
b
,Eand
E
R
, did not show any specific binding to the radioligand, but
were found to be constitutively active in the cAMP assay.
The E and E
R
alleles are associated with black feather col-
our in chicken while the e
b
allele gives rise to brownish
pigmentation. The three constitutively active receptors share
a mutation of Glu to Lys in position 92. This mutation was
previously found in darkly pigmented sombre mice, but

constitutively active MC receptors have not previously been
shown in any nonmammalian species. We also inserted the
Glu to Lys mutation in the human MC1 and MC4 recep-
tors. In contrast with the chicken clones, the hMC1-E94K
receptor bound to the ligand, but was still constitutively
active independently of ligand concentration. The hMC4-
E100K receptor did not bind to the MSH ligand and was
not constitutively active. The results indicate that the
structural requirements that allow the receptor to adapt an
active conformation without binding to a ligand, as a con-
sequence of this E/K mutation, are not conserved within the
MC receptors. The results are discussed in relationship to
feather colour in chicken, molecular receptor structures and
evolution. We suggest that properties for the ÔE92K switchÕ
mechanism may have evolved in an ancestor common to
chicken and mammals and were maintained over long time
periods through evolutionary pressure, probably on closely
linked structural features.
Keywords: G-protein coupled; MSH; melanocortin receptor;
polymorphism.
Spontaneous or constitutive G-protein coupled receptor
(GPCR) activity was first convincingly described for the
d-opioid receptor [1], and was further established by
the demonstration of constitutive activity in chimeras of
the a
1B
and a
2
receptors (summarized in [2]). Later it was
shown that mutations in the human rhodopsin gene can

constitutively activate transducin in the absence of retinal
and light [3]. It is now known that naturally occurring
constitutively active GPCRs are found to be responsible
for a diverse array of inherited as well as somatic genetic
disorders [4,5].
The melanocortins (a-MSH/ACTH), secreted from a
frog pituitary, were in 1912 found to cause pigmentation.
In higher vertebrates including aves and mammals, these
peptides are expressed throughout the body, and are
involved in a variety of physiological regulatory functions
[6–8]. In the skin, the melanocortins are synthesized locally
(for birds [9], for mammals [10]), and act through a GPCR
named melanocortin (MC) 1 receptor to regulate melano-
genesis. Keratinocytes probably serve as the main physio-
logical source of melanocortins acting on melanocytes in
the epidermis and hair follicle. The MC1 receptor couples
through G proteins to adenylate cyclase to stimulate
tyrosinase, the rate-limiting enzyme in the synthesis of both
classes of melanin pigments, eumelanin and phaeomelanin.
A low level of tyrosinase expression leads to increased
phaeomelanin synthesis, while elevated levels of tyrosinase,
that can result from a-MSH stimulation of melanocytes,
divert the intermediates primarily along the eumelanin
synthetic pathway (for reviews see [8,10–11]).
The extension (E) locus, together with agouti (A) locus,
regulates the relative amount of black pigment (eumelanin)
and red/yellow pigment (phaeomelanin) in mammals [12].
The E locus encodes the MC1 receptor and the A locus
encodes the agouti peptide, an antagonist of the MC1
receptor. Dominant alleles at extension result in dark brown

or black coat colour, while animals homozygous for
recessive alleles have yellow or red coats. The opposite is
true for the agouti allele. A dominant mutation at the A
Correspondence to H. B. Schio
¨
th, Department of Neuroscience,
Biomedical Center, Box 593, 75 124 Uppsala, Sweden.
Fax:+4618511540,
E-mail:
Abbreviations: GPCR, G-protein coupled receptor; IC
50
, 50%
inhibitory concentration; EC
50
, 50% effective concentration;
IL, intracellular loops; MC, melanocortin; MSH, melanocortin
stimulating hormone; NDP-MSH, [Nle4,
D
-Phe7]a-MSH;
TM2, transmembrane region 2.
(Received 3 December 2002, revised 26 January 2003,
accepted 6 February 2003)
Eur. J. Biochem. 270, 1441–1449 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03506.x
locus (A
y
/A) in mice causes uniform yellow coat and obesity.
A similar phenotype, but with normal body weight, is found
in recessive yellow mice (e/e), where a loss of function
mutation in the MC1 receptor results in animals producing
phaeomelanin [13]. The dominant extension locus alleles,

sombre (E
SO
and E
SO)3J
)andtobacco (E
tob
), which all have
dominant melanizing effects, result from point mutations
that produce constitutively active receptors. The sombre
alleles produce a fairly uniform black coat, while tobacco
darkening only involves the dorsal portion of the animal.
The tobacco alleles produce a receptor that remains
hormone responsive but produces a greater activation of
adenylyl cyclase than does the wild-type allele. Both sombre
alleles were found to produce constitutively active receptors,
defined as being able to significantly elevate adenylyl cyclase
activity in the absence of ligand, and thereby enhance the
eumelanin production resulting in a dark pigmentation in
mice [13]. Null-mutations of POMC, the precursor for the
melanocortins, causes yellow coat, obesity and adrenal
insufficiency. These mice are somewhat darker, Ôdirty blondÕ
suggesting that the basal MC1 receptor activity in the
absence of ligands may be higher than a nonfunctioning
receptor [14].
Mutations in MC1 receptors, related to hair or skin
colour, have been found in several other mammalian
species, although pharmacological characterization of these
changes has usually not been carried out. Two species,
whose receptors have been pharmacologically investigated,
are fox and sheep. Va

˚
ge cloned and characterized the fox
MC1 receptor and found a mutation that caused constitu-
tive activity in the dark coated Alaska Silver fox [15]. This
dominant mutation was however, also found in foxes with
significant red coat colouration, and it was suggested that
the fox agouti protein counteracted the signalling activity of
a constitutively active fox MC1 receptor. In sheep, two
mutations in the MC1 receptor showed complete cosegre-
gation with dominant black coat colour in a family lineage.
These mutations were transferred into the corresponding
mouse receptor in which they produced constitutive activity
[16]. Loss of function mutations are common in the human
MC1 receptor and these are over-represented in Northern
European redheads and in individuals with pale skin [17,18].
These variants are a risk factor, possibly independent of skin
type, for melanoma [19].
Variants of the MC1 receptor gene were found to be
associated with the extension (E) locus in chickens and it
was proposed that they might be linked to feather
pigmentation [20]. A dominant mutation, identical to
one of the mutations in the sombre mouse, was found in
chicken with black feathers [20]. The very same mutation,
Glu to Lys in transmembrane region 2 (TM2), has also
been shown to be present in melanic individuals of the
bananaquit, a neotropical passerine bird, and absent in all
yellow individuals [21]. The molecular pharmacology of
these mutations has however, not yet been investigated.
In this study, we performed the first expression studies on
avian MC receptor genes. We expressed and pharmacologi-

cally characterized seven polymorphic variants of the
chicken MC1 receptors derived from different E locus
alleles. We also introduced the Glu to Lys mutation in to the
human MC1 and MC4 receptors to investigate the pharma-
cological impact of these mutations.
Experimental
Receptor clones
Oligonucleotide primers were designed to amplify the entire
coding region of each cMC1 receptor variant. To facilitate
cloning and establishment of orientation of the PCR-
amplified DNA fragments, recognition sites for HindIII and
BamHI were introduced into 5¢ and 3¢ primers, respectively,
by altering original nucleotide sequences. The primer
sequences were 5¢-GGAAGCTTTGTAGGTGCTGCA
GTT-3¢ for the 5¢ primer and 5¢-CATGGATCCTCCTC
CTGTCTGTGCCGC-3¢ for the 3¢ primer, corresponding
to positions )55 to )78 and 1049–1072, respectively, of the
cMC1 receptor gene, where the A of the translation
initiation codon ATG was defined as +1. The amplified
DNA fragmentswere cloned into pGEM3Zf(+), sequenced,
and subsequently subcloned into pCEP4 Turbo expression
vector [22] and resequenced. The human MC1 [23] and
human MC4 receptors [24] were used as templates for the
mutagenesis.
Site-directed mutagenesis
The E94K mutation in hMC1 and E100K mutation in
hMC4, were introduced into the receptor coding sequence
by PCR. Two complementary oligonucleotides were
designed to contain the required mutation. The hMC1-
E94K primers were (5¢ primer) 5¢-CAGGAGGATGA

CGGCCGTCTTCAGCACGTTGCTCCC-3¢ and (3¢ pri-
mer) 5¢-GGGAGCAACGTGCTGAAGACGGCCGTCA
TCCTCCTG-3¢. The hMC4-E100K primers were (5¢
primer) 5¢-TAGGGTGATGATAATGGTTTTTGATCC
ATTTGAAAC-3¢ and (3¢ primer) 5¢-GTTTCAAATG
GATCAAAAACCATTATCATCACCCTA-3¢. The pri-
mers were hybridized to opposite strands of the receptor
gene and the complete MC receptor coding sequence was
amplified. The end-primer was complementary to 3¢ or 5¢
depending on which mutagenesis primer was used (forward
or reverse). The two products were used as templates and
linked together in a second PCR, in which only the end-
primers were used.
Expression
DNA for transfection was prepared using Qiagen Plasmid
Maxi Kit (Merck). HEK 293 EBNA cells 50–70% confluent
on 10-cm plates, were transfected with 15 lgofthe
construct using FuGENE
TM
Transfection Reagent (Boeh-
ringer Mannheim), diluted in Optimem medium (Gibco
BRL). After transfection, cells were grown in Dulbecco’s
MEM/Nut Mix F-12 (Gibco BRL) containing 10% foetal
bovine serum (Biotech Line), 2.4 m
ML
-glutamine,
2.5 mgÆmL
)1
G418, 2.5 lgÆmL
)1

amphotericin, and
100 lgÆmL
)1
kanamycin solution (all from Gibco BRL)
until harvesting, after 48 h.
Binding assays
Intact transfected cells were re-suspended in 25 m
M
Hepes
buffer (pH 7.4) containing 2.5 m
M
CaCl
2
,1m
M
MgCl
2
and 2 gÆL
)1
bacitracin. Competition experiments were
1442 M. K. Ling et al. (Eur. J. Biochem. 270) Ó FEBS 2003
performed in a final volume of 100 lL. The cells were
incubatedin96-wellplatesfor3hat37°Cwithconstant
concentration of [
125
I]NDP-MSH and appropriate concen-
trations of competing unlabelled ligands, [Nle4,
D
-Phe7]
a-MSH (NDP-MSH) or a-MSH. The incubations were

terminated by filtration through Glass Fibre Filters, Filter-
mat A (Wallac Oy, Turku, Finland), which had been
presoaked in 0.3% poly(ethylenimine), using a TOMTEC
Mach III cell harvester (Orange, CT, USA). The filters were
washed with 5.0 mL 50 m
M
Tris pH 7.4 at 4 °C and dried
at 60 °C. The dried filters were then treated with MeltiLex A
(Perkin Elmer) melt-on scintillator sheets and counted in a
Wallac 1450 (Wizard automatic Microbeta counter). The
results were analysed with a software package suitable for
radioligand binding data analysis (
PRISM
3.0, Graphpad,
San Diego, CA, USA). The binding assays were performed
in duplicate and repeated three times. Nontransfected
HEK293-EBNA cells did not show any specific binding to
[
125
I]NDP-MSH. NDP-MSH was radio-iodinated by the
chloramine T method and purified by HPLC. NDP-MSH
and a-MSH were purchased from Neosystem (France).
cAMP assay
Semi-stable cells, expressing the receptors of interest, were
harvested in growth media containing 3-isobutyl-1-methyl-
xanthine from Sigma. Twohundred microlitres of the cells
were added to tubes containing appropriate concentra-
tions of a-MSH and incubated for 30 min at 37 °C. After
stimulation the cells were lysed and cAMP extracted using
4.4

M
perchloric acid. The cAMP extract was then
neutralized with 5
M
KOH and centrifuged. The intracel-
lular cAMP produced was measured in 50 lLofthe
supernatant after addition of [
3
H]cAMP and bovine
adrenal binding protein and incubation for 2 h at 4 °C.
Standards containing nonlabeled cAMP were assayed in
the same manner. The incubates were then harvested by
adding activated carbon. After centrifugation, the super-
natant was removed into scintillation tubes and counted
in a Tri-carb Liquid scintillation analyser after addition of
3 mL scint solution (Ready Safe
TM
; Beckman Coulter).
The cAMP assay was performed in duplicate and
repeated three times. The results were analysed using the
PRISM
3.0 software package (Graphpad, San Diego, CA,
USA). The protein concentrations were measured using
Bio-Rad Protein Assay (Bio-Rad, Solna, Sweden) with
BSA as standard.
Results
The amino acid sequences for the seven polymorphic cMC1
receptors derived from different E locus alleles are shown in
Table 1. Four clones; e
+R

,e
+B&D
,e
wh
/e
y
,E
Rfayoumi
resulted
in competition curves for a-MSH and NDP-MSH using
iodinated [
125
I]NDP-MSH as radioligand. The binding
curves are shown in Fig. 1. The IC
50
for the MSH ligands
are shown in Table 2. Three clones, e
b
, E and E
R
, did not
induce any specific binding on the transfected cells. We also
measured the intracellular cAMP in response to varying
concentrations of a-MSH. The cells transfected with the
four cMC1 receptors, that showed competition curves in the
binding assays, also responded to a-MSH by accumulation
of intracellular cAMP in a dose-dependent manner. The
cAMP curves are shown in Fig. 2, and the EC
50
values are

showninTable2.
The three cMC1 receptors; e
b
,Eand E
R
, did not show
any specific binding to the radioligand or respond to
a-MSH by accumulation of intracellular cAMP. The results
are shown in Fig. 3. The basal levels were, however,
significantly increased for the cells transfected with the
three clones in all experimental points, as compared with
nontransfected HEK-293 EBNA cells, that were used as a
control. The cells transfected with the receptors (e
+R
,
e
+B&D
, e
wh
/e
y
, E
Rfayoumi
) also served as controls, as at the
initial level and at the lowest concentrations of a-MSH, the
cAMP levels were lower than that observed for the cells
transfected with e
b
,Eand E
R

. All experiments were
performed with a similar number of cells; the amount of
protein per well was determined and the cAMP values were
normalized accordingly. The cAMP levels for these three
clones were significantly higher for all experimental points,
also when corrected for the cell number. The experiments
were repeated three times and qualitatively the results were
the same each time. The activation level did, however, not
reach the maximum level of the other clones. e
b
,Eand E
R
cMC1 receptors were partially activated to  20–60% of the
maximal activation of a ÔnormallyÕ functioning cMC1
receptor (e
wh
/e
y
). The cAMP levels did, however, vary
between the repeats and the internal order between the three
clones was not always the same. Therefore, we do not draw
the conclusion that the cAMP levels for the e
b
,Eand E
R
cMC1 receptors transfected cells differed from each other.
The Glu92 residue in TM2 (see Table 1) is conserved
within the family of MC receptor subtypes. The e
b
,Eand

E
R
cMC1 receptors have Lys in this position. We inserted a
Table 1. Amino acid positions in the seven different cMC1 receptors. Dominance: E >E
R
>e
wh
>e
+
>e
b
>e
y
.
Allele 71 92 133 143 213 215 Line
e
+
Met Glu Leu Thr Arg His Richardson’s RJF (e
+R
)
e
+
– – – – Cys – B & D RJF (e
+B&D
)
E Thr Lys – – Cys – Black Australorp (E)
e
b
Thr Lys – – Cys Pro Smyth Brown line (e
b

)
E
R
– Lys – – – – ADOL line 0 (E
R
)
E
R
– – Gln – – – Fayoumi (E
Rfayoumi
)
e
wh
/e
y
– – – Ala – – NHR, RIR, Buff Min(e
wh
)
– Indicates that the amino acid in this position is the same as the one as described in the allele at the top. Note that e
wh
and e
y
are identical in
amino acid sequence.
Ó FEBS 2003 Constitutively active MC1 receptors in chicken (Eur. J. Biochem. 270) 1443
Lys in the corresponding position in the human MC1 and
MC4 receptors by site-directed mutagenesis and generated
clones termed hMC1-E94K and hMC4-E100K. The clones
were expressed and assayed pharmacologically in the same
manner as the clones mentioned above. The binding results

are shown in Fig. 4. The cells transfected with hMC1-E94K,
in contrast with the chicken receptors with Lys in position
92, did bind NPD-MSH. The IC
50
value was about 18-fold
lower than that of the wild-type human MC1 receptor. The
cells transfected with hMC4-E100K did not, however, show
any specific binding. The cAMP results are shown in Fig. 5.
The cells transfected with hMC1-E94K showed significantly
higher cAMP values at all experimental points, independent
of concentration of a-MSH, as compared with the non-
transfected cells. The cells transfected with hMC4-E100K,
in contrast with the chicken receptors with Lys in position
92, showed the same low levels as nontransfected cells.
Discussion
Like the extension locus in mammals, the extended black
(E) locus of the chicken controls the relative amount of
eumelanin and phaeomelanin in melanocytes. The locus was
originally localized on chromosome 1, but recent genetic
and FISH analyses revealed that it is located on a
microchromosome [25,26]. Several alleles exhibiting differ-
ent pigmentation have been described and there is an
intricate hierarchy among them. But in general, alleles that
make more eumelanin are dominant over those that make
less eumelanin. Unlike the mammalian extension locus, the
phenotype of each allele is expressed mainly in chicks and
adult females. In fact, the phenotypes of adult males with
different nonmelanic alleles, including e
+
, e

wh
, e
y
,ande
b
,are
similar, having black-breasted red feather pattern. The only
phenotypic difference observed among them is the under-
colour, the fluff of the feathers next to the skin; it is white or
cream for the e
wh
and e
y
malesandgreyforthee
+
and e
b
.In
melanic alleles (E, E
R
,andE
Rfayoumi
), adult males are black
in all areas and the flight feathers are also black for E, while
the other melanic alleles (E
R
and E
Rfayoumi
)givehalf-red
half-black feathers. Fig. 6 shows the adult E locus colour

patterns on a wild-type background for all other feather
colour genes.
Our results indicate that the polymorphic e
b
, E and E
R
chicken MC1 receptors are constitutively active. These three
receptors all share a mutation of Glu to Lys in position 92
(see Table 1). Previously it has been shown that constitutive
activation of the MC1 receptor in darkly pigmented sombre
mice results from the very same mutation in position 92 [13].
L98P mutation in the MC1 receptor in mouse, D119N in
Fig. 1. Competition curves of [Nle4,
D
-Phe7]a-MSH (u) and a-MSH (m) obtained with transfected HEK-293 (EBNA) cells using a fixed concen-
tration of 0.2 n
M
[
125
I][Nle
4
,
D
-Phe
7
]a-MSH for four cMC1 receptors, e
+R
,e
+B&D
,e

wh
/e
y
,E
Rfayoumi
. Data points represent means of duplicates and
error bars indicate standard error of the mean (SEM).
Table 2. Pharmacological characterization of chicken MC1 receptors
after expression in HEK cells. The IC
50
values were obtained from
competition using [
125
I] [Nle4, D-Phe7] a-MSH as radioligand and
a-MSH and [Nle4, D-Phe7] a-MSH as competitors. The EC
50
values
are obtained in intracellular cAMP assay using a-MSH as stimulator.
Receptor
a-MSH (IC
50
)
(nmolÆL
)1
)
NDP-MSH (IC
50
)
(nmolÆL
)1

)
a-MSH (EC
50
)
(nmolÆL
)1
)
e
+(R)
1350 ± 680 9.76 ± 2.51 449 ± 69
e
wh
/e
y
827 ± 167 13.7 ± 8.8 20.4 ± 3.5
e
+(B & D)
601 ± 117 6.00 ± 0.40 21.9 ± 5.3
E
Rfayoumi
1080 ± 137 4.79 ± 0.20 36.2 ± 0.5
1444 M. K. Ling et al. (Eur. J. Biochem. 270) Ó FEBS 2003
sheep and C125R in Alaska silver fox that cause dark
pigmentation have also been shown to be constitutively
active [15,27]. The pharmacology of constitutively active
MC receptors has not been previously shown in any
nonmammalian species, and these results show that the
function of this mutation seems to be similar in chicken and
mice. The results add further support to the hypothesis that
the same point mutation (E92K) in the bananaquit, Coereba

flaveola associated with the melanic plumage morph [21] is
constitutively active.
The clear association between the mainly black feather
colour of chickens possessing E and E
R
and the presence
of constitutively active MC1 receptor is in line with the
previous observation of the E92K mutation in other
species. Males possessing the e
b
allele are described above.
Among the females, the allele gives rise to brownish
pigmentation, although according to the results from the
cAMP assay for this receptor, perhaps a darker pheno-
type would have been expected, as for the other two
constitutively active cMC1 receptors. Likewise, E
Rfayoumi
and e
wh
/e
y
alleles exhibiting phenotypes similar to E
R
allele and yellow-red pigmentation, respectively, were
found to encode normally functioning cMC1 receptors,
which bind agonist and couple to G-protein in a ‘normal’
manner. Thus, our pharmacological results cannot com-
pletely explain the association of cMC1 variants with the
corresponding phenotypes. It is possible that the expres-
sion levels of cMC1 receptor vary with different alleles,

which results in phenotypic difference in alleles encoding
cMC1 receptors with similar pharmacological character-
istics. Alternatively, specific amino acid substitutions
observed in each allele alter the interaction of cMC1
receptor with unidentified factors expressed specifically in
melanocytes. Further analyses are thus required to clarify
molecular mechanisms for all the different feather pig-
mentation patterns in chicken.
Considering the other polymorphic residues in the
chicken clones, the results indicate that the changes of
Fig. 2. Generation of cAMP in HEK-293 (EBNA) cells transfected with four cMC1 receptors: e
+R
,e
+B&D
,e
wh
/e
y
,E
Rfayoumi
in response to a-MSH.
The cAMP levels were normalized to the amount of protein. Data points represent means of duplicates and error bars indicate standard error of
the mean (SEM).
Fig. 3. Cells transfected with the three cMC1 receptors e
b
(n), E (s),
E
R
(m) show elevated cAMP levels and independence of the concentration
of a-MSH in comparison to basal cAMP levels in HEK-293 (EBNA)

cells (j). The cAMP levels were normalized to the amount of protein.
e
wh
/e
y
(d) (from Fig. 2) is shown for comparison. Data points repre-
sent means of duplicates and error bars indicate standard error of the
mean (SEM).
Ó FEBS 2003 Constitutively active MC1 receptors in chicken (Eur. J. Biochem. 270) 1445
Met71 to Thr, Leu133 to Gln, Thr143 to Ala, Arg213 to
Cys, or His215 to Pro do not influence the pharmacological
function of the receptors. Met71, Leu133 and His215 are
conserved through all the MC receptors cloned so far. These
residues are believed to be in the first, second and third
intracellular loops (IL), respectively. Thr143 alternates
between Thr and Ser, and is found in IL2, while Arg213
alternates between Arg and Cys, and is found in IL3, within
the entire MC receptor family. It is interesting that mutation
of Cys215 to Gly in the human MC1 receptor (correspond-
ing to Arg213) resulted in failure to generate cAMP signal in
response to the agonists a-MSH [28]. It seems therefore that
the function of the MC receptor requires a polar residue in
this position, while a hydrophobic nonpolar residue causes
disconnection of the signal transduction. As the key residues
within the MC receptor family are highly conserved, our
new results provide additional information for generating
molecular models of the binding and activation process of
the MC receptors that is an important part of rational drug
discovery [8,29].
In order to shed further light on the structural require-

ments needed for generating a constitutively active MC
receptor, we introduced the corresponding E92K mutation
into the human MC1 and MC4 receptors. It was surprising
that the mutant human MC1 receptor showed differences in
pharmacology as compared with the mutant chicken MC1
receptor. It is intriguing that in contrast with the chicken
receptor with Lys, hMC1-E94K bound the ligand but was
still constitutively active, independently of the ligand. This
pharmacology is remarkable, as this receptor seems to have
the ability to bind the ÔagonistÕ peptide ligand with high
(albeit slightly lower) affinity, and undergo the conforma-
tional changes that this binding interaction is proposed to
have, without influencing the interaction with the G-protein.
Even though inactivating mutations associated with red hair
are common in the hMC1 receptors, no constitutive
activating MC1 receptor has yet been found in humans. It
seems clear that the structural properties of the hMC1
receptor are different from those of chicken, mouse and fox,
where the constitutive E/K mutation leads to loss of
binding. It is possible that the evolutionary pressure on the
hMC1 receptor is altered due to changes in the physiological
importance of body hair colour and therefore the structural
pharmacology is evolving differently as compared to MC1
receptors of species in which colour has a more defined
function. It would be interesting to see if this property is
unique to humans or if it has evolved earlier in primates.
In order to find out if these pharmacological properties
are shared beyond the MC1 receptors, we also introduced
the E92K mutation into the hMC4 receptor. This receptor is
mainly expressed in the central regions of the brain,

including the hypothalamus where it is an important
regulator of food intake. The MC4 receptor shares 60%
of the amino acids with the MC1 receptor, but has a
completely different physiological role with no known
overlap in function. The MC1 and MC4 receptors do,
however, share the unique property that they both have
natural antagonists, the agouti and the agouti-related
peptide, respectively, in addition to their natural agonist,
a-MSH. The hMC4-E100K, in contrast with the hMC1
receptor mutation, did not bind the MSH ligand and was
not constitutively active. No naturally occurring activation
mutants have been found for MC receptors other than
MC1, but several inactivation mutants of the MC4 recep-
tors are related to obesity [30,31]. The results indicate that
the structural ability to form constitutively active receptors
with a single amino acid mutation of the conserved Glu in
TM2 has either not been maintained, or was never present
in the MC4 receptors. Unlike constitutively activating
mutations in many GPCRs that give increased agonist
efficacy or affinity, these MC1 receptor mutations have the
opposite effect. The molecular mechanism of constitutive
activation of the MC1 receptor has been studied by inserting
the mutations into the mouse MC1 receptor [16]. These
authors proposed a ligand-mimetic model which explains
Fig. 4. Competition curve of [Nle4,
D
-Phe7]a-MSH obtained with
transfected HEK-293 (EBNA) cells using a fixed concentration of 0.2 n
M
[

125
I][Nle
4
,
D
-Phe
7
]a-MSH for the human wild-type MC1 (u), human
MC1 (E94K) (m), and wild-type MC4 (j), human MC4 (E100K) (r)
receptors. Data points represent means of duplicates and error bars
indicate standard error of the mean (SEM).
Fig. 5. cAMP levels of normal cAMP in HEK-293 (EBNA) cells (j)
and cells transfected with human MC1 (E94K) (d), human MC4
(E100K) (u) receptors, showing cAMP levels independent of the con-
centration a-MSH. The cAMP levels were adjusted to the amount of
protein. e
wh
/e
y
(.) (from Fig. 2) is shown for comparison. Data points
represent means of duplications and error bars indicate standard
deviations.
1446 M. K. Ling et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the lower (read nonexistent) ligand affinity and efficacy. It
was proposed that the mutations transformed the receptor
into its active form, not by disrupting the internal constraint
as proposed by the early rhodopsin studies and the ternary
allosteric model, but by indirectly mimicking ligand-bind-
ing. The amino acid residues that were mutated in the
mouse MC1 receptor are all conserved within the human

MC1 and further studies are thus needed to explain the
pharmacological differences between the mouse and human
MC1 receptors.
The reptilian ancestors of chickens diverged about 300–
350 million years ago from the lineage leading to mammals
[32]. It is remarkable that the same single amino acid
mutation forming a constitutive active receptor is found in
so evolutionary distant species. Recent studies show
remarkable evolutionary conservation within the primary
sequence and pharmacology of MC receptors (including the
E92) for at least 450 million years [33]. Our mutations of the
hMC1 and hMC4 receptors indicate that the structural
requirements that allows the receptors to fall into a
constitutively active state without binding to a ligand are
not automatically conserved within the MC receptors or
within the MC1 receptors. Therefore, we find it unlikely that
it is simply the ability to bind MSH peptides, the main
pharmacological property shared by the MC1 and MC4
receptors, that is the structural feature that automatically
allows the ÔE92K switchÕ mechanism to work. We find it
also unlikely that the complex structural requirements for
single amino acid activation switch were independently
developed in chicken and mammals. It is thus tempting to
speculate that some structural properties evolved once
before the divergence of the avian and mammalian lineages.
The intriguing question is by which mechanism this
property could have been conserved over such long time
period. One theoretical possibility is that the specific
mutation was ancestral and that an E/K92 heterozygote
had a selective advantage over the two homozygotes,

thereby preserving both the alleles and also the structural
constraints. This is, however, very unlikely due to the long
evolutionary times and the divergent lineages involved.
Fig. 6. Cartoon representation of the adult E locus colour patterns on a wild-type background for the feather colour genes. E (dominant extended black)
birdsareblackinallareasinbothsexes.Malesthataree
+
,e
b
, e
wh
or e
y
(wild-type, partridge or brown, dominant wheaten or recessive wheaten,
respectively) all have the black-breasted red feather pattern. The only difference is that the wheaten males have a white or cream feather under-
colour and the e
+
and e
b
males have a grey under-colour. E
R
(birchin) males are different in the wings. The flight feathers are all black instead of the
half-red half-black feathers of the recessive alleles. Birchin females have black bodies and gold hackle feathers. Both male and female birchins have
black melanin in the epidermis of their shanks. The brown (e
b
) females have brown stippled backs and wings and brown stippled breasts, where the
wild-type (e
+
) females have brown stippled backs and wings and salmon breasts. Both females have a grey under-colour. Dominant wheaten (e
wh
)

and recessive wheaten (e
y
) females have the same phenotype, having the salmon colour of the wild-type breast extended into the plumage of the back
and wings. They have a white or cream under-colour and black is diluted in the female plumage.
Ó FEBS 2003 Constitutively active MC1 receptors in chicken (Eur. J. Biochem. 270) 1447
There are only a few examples of the maintenance of specific
alleles by heterozygote advantage (e.g. MHC class II alleles,
haemoglobin S) and in these cases the maintenance has been
for only thousands to at most a few million years [34].
Moreover, it is notable that the E92K mutation in
bananaquit (mentioned above) is believed to have occurred
recently in that lineage [21]. We believe therefore that it is
most likely that the structural properties for the ÔE92K
switchÕ mechanism were maintained through evolutionary
pressure on closely linked structural features. These pro-
perties were subsequently retained in certain lineages, like
mouse and birds, and lost in others, like humans. Further
structural characterization of the protein structure and
studies on the MC receptors in more ancient tetrapod
groups, such as reptiles, may shed further light into the
mechanism on how structural properties for a single amino
acid switch mechanism may have survived such a long
evolutionary distance.
Acknowledgements
We thank Prof D. Larhammar, Uppsala University for valuable
criticism. The studies were supported by the Swedish Medical Research
council (MRC), the Swedish Society for Medical Research (SSMF),
Svenska La
¨
karesa

¨
llskapet, A
˚
ke Wibergs Stiftelse and Melacure
Therapeutics AB, Uppsala, Sweden to H.S., the Japanese Society for
Promotion of Science (Grant-in Aid for Scientific Research) to S.T.
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