Báo cáo khoa học: Presence of melanocortin (MC4) receptor in spiny dogfish suggests an ancient vertebrate origin of central melanocortin system pot

Presence of melanocortin (MC4) receptor in spiny dogfish suggests
an ancient vertebrate origin of central melanocortin system
Aneta Ringholm
1
, Janis Klovins
1
, Robert Fredriksson
1
, Natalia Poliakova
1
, Earl T. Larson
1
,
Jyrki P. Kukkonen
2
, Dan Larhammar
1
and Helgi B. Schio¨th
1
Department of Neuroscience, Division of
1
Pharmacology and Division of
2
Physiology, Uppsala University, Uppsala, Sweden
We report the cloning, expression, pharmacological char-
acterization and tissue distribution of a melanocortin (MC)
receptor gene in a shark, the spiny dogfish (Squalus acanth-
ias) (Sac). Phylogenetic analysis showed that this receptor is
an ortholog of the MC4 subtype, sharing 71% overall amino
acid identity with the human (Hsa) MC4 receptor. When
expressed and characterized by radioligand binding assay for

the natural MSH (melanocyte-stimulating hormone) pep-
tides a-, b-, and c-MSH, the SacMC4 receptor showed
pharmacological properties very similar to the HsaMC4
receptor. Stimulation of SacMC4 receptor transfected cells
with a-MSH caused a dose-dependent increase in intracel-
lular cAMP levels. The SacMC4 receptor has Ala in position
59 where all other cloned MC receptors have Glu. We con-
firmed that this was not due to individual polymorphism and
subsequently mutated the residue ÔbackÕ to Glu but the
mutation did not affect the pharmacological properties of
the receptor. SacMC4 receptor mRNA was detected by RT-
PCR in the optic tectum, hypothalamus, brain stem, telen-
cephalon and olfactory bulb but not in cerebellum or in
peripheral tissues. This study describes the first characteri-
zation of an MC receptor in a cartilaginous fish, the most
distant MC receptor gene cloned to date. Conservation of
gene structure, pharmacological properties and tissue dis-
tribution suggests that this receptor may have similar roles in
sharks as in mammals and that these were established more
than 450 million years ago.
Keywords: GPCR; melanocortin; melanocyte-stimulating
hormone; receptor.
The melanocortin (MC) receptor family consists of five
subtypes, termed MC1-MC5 in mammals. The melanocortin
system is unique because in addition to possessing endo-
genous agonists, it also has an endogenous antagonist, Agrp.
The agonists are the pro-opiomelanocortin (POMC) clea-
vage products a-, b-andc-melanocyte-stimulating hormone
(MSH) and adrenocorticotrophic hormone (ACTH).
POMC has been used extensively as a model for studies of

the evolution of neuropeptides and it is well established that
the sequence of a-MSH is highly conserved between mam-
mals and fishes [1,2]. The centrally expressed MC4 receptor
received great attention by many researchers within the field
of central regulation of food intake after it was Ôknocked-outÕ
in mice [3], causing over-eating and obesity. Centrally
administered MC4 receptor agonists have the ability of
reducing appetite [4,5], while MC4 receptor antagonists are
very effective in increasing food intake in rodents, both in
acute and long-term studies [6–8]. These findings make the
MC4 receptor very interesting for pharmacological research
and drugs against this receptor may become helpful for
people suffering from disorders like obesity and anorexia.
The MC3 receptor has been found exclusively in the brain
and it is involved with regulation of the energy balance [9].
The MC5 receptor is primarily expressed in a wide range of
peripheral tissues and also in the mammalian brain [10]. The
MC2 receptor subtype mediates the function of ACTH but
does not bind a-, b-orc-MSH. This receptor has been found
only in the adrenal gland. The MC1 receptor has a role in
pigmentation and it also mediates the anti-inflammatory
action of MSH [11].
Our understanding of the mechanisms of appetite regu-
lation and metabolism in mammals is increasing rapidly.
Many peptides such as neuropeptide Y, orexins, Agrp
(agouti-related peptide), ghrelin and MSH are involved in
the regulation of energy balance by binding to GPCRs in
the central regions of the brain [12,13]. However, in ÔlowerÕ
vertebrates, the molecular mechanisms for appetite regula-
tion of these peptide binding receptors are poorly known.

The spiny dogfish (Squalus acanthias)isasharkanda
member of cartilaginous fishes (also called chondrichth-
yans). Chondrichthyans are characterized by cartilaginous
skeletons, placoid scales and pelvic claspers (in males) [14].
They arose from the Agnatha, the jawless fishes, in the late
Silurian period, approximately 420–430 million years ago.
The spiny dogfish became popular as a research model
among chondrichthyans and it has been extensively studied
due to its peculiar rectal gland, an organ that regulates the
secretion of chloride [15]. Cardiovascular control has also
been widely studied in the spiny dogfish [16]. Several
peptides have been characterized in the dogfish, including
Correspondence to H. B. Schio
¨
th, Department of Neuroscience,
Biomedical Center, Box 593, 75 124 Uppsala, Sweden.
Fax: + 46 18 51 15 40, E-mail:
Abbreviations: ACTH, adrenocorticotrophic hormone; GPCR,
G-protein coupled receptor; MC, melanocortin; MSH, melanocyte-
stimulating hormone; NDP-MSH, [Nle4,
D
-Phe7]a-MSH;
NTS, nucleus of the solitary tract; POMC,
pro-opiomelanocortin.
(Received 18 August 2002, revised 7 November 2002,
accepted 18 November 2002)
Eur. J. Biochem. 270, 213–221 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03371.x
peptide PYY [17] and gastrin/cholecystokinin-like peptides
[18] but very few G-protein-coupled receptors (GPCRs)
have been cloned in this species. Recently, we cloned one

MC4 receptor and two MC5 receptors in a teleost fish, the
zebrafish, Danio rerio [19]. The results showed high conser-
vation in primary structure and pharmacology of these MC
receptors as compared with the mammalian ones. The
teleosts belong to the bony fishes which arose later in the
evolution, after a split from cartilaginous fishes [20–22].
In this paper, we report the cloning, expression, phar-
macological characterization and tissue distribution of a
MC4 receptor in a cartilaginous fish, the spiny dogfish. This
receptor is evolutionarily the most distant MC receptor
from mammals cloned so far.
Materials and methods
Extraction of genomic DNA
Spiny dogfish genomic DNA was extracted from muscle
tissue of four different animals captured in the North Sea
(Hambergs Fisk, Uppsala, Sweden). The muscle tissue
(about 1 g) was homogenized in the lysis buffer [100 m
M
EDTA (TitriplexÒ, Merck, Stockholm, Sweden), 10 m
M
Tris (VWR International, Stockholm, Sweden) and 1%
SDS (Scientific Imaging Systems, Eastman Kodak Com-
pany)] and centrifuged for 5 min at 11 500 g. The superna-
tant was purified first with saturated phenol (BDH
Laboratory Supplies, Poole, UK), then with phenol/chlo-
roform/isoamyl alcohol (1 : 1 : 1) (BDH Laboratory Sup-
plies), and chloroform (KEBO Laboratory AB, Stockholm,
Sweden). The DNA was precipitated with propan-2-ol and
NH
4

Ac (2 : 1) and centrifuged for 30 min at 11 500 g.
Finally the DNA was washed with 70% ethanol and
centrifuged again. The DNA pellet was vacuum-dried and
re-suspended in water.
Cloning
Degenerate primers based on conserved parts of the human,
rat, mouse and chicken MC receptors were used in different
pair-wise combinations. One hundred nanograms of dogfish
genomic DNA was used as template in a low stringency
PCR, using the AmpliTaq DNA polymerase Stoffel Frag-
ment (Perkin Elmer, Roche, Langen, Germany) in a
reaction volume of 20 lL, containing 4 m
M
dNTP,
1 · Stoffel buffer (Perkin Elmer), 60 m
M
MgCl
2
,20pmol
each primer and two units DNA polymerase, Stoffel
Fragment. Touch-down PCR was performed, starting with
an initial 1 min 95 °C denaturation, followed by 22 cycles of
45 s at 94 °C, 45 s at 52 °Cto42°C, 90 s at 72 °C. This was
followedby25cyclesof30sat94 °C, 40 s at 50 °C, 1 min at
72 °C, with a final extension of 5 min at 72 °C. The PCR
gave a product of the expected size, 600 bp. The 5¢ primer’s
sequence was CAY TCN CCN ATG TAY TTY TT and
the 3¢ primer ATN ACI GAR TTR CAC ATD AT. Y
denotes C or T, R denotes A or G, D denotes, A, G or T, I
denotes inosine and N denotes any base. The PCR product

was purified from a 1% agarose gel using Gel Extraction
Kit (Qiagen, Hilden, Germany). Re-amplification was
performed by denaturating for 1 min, followed by 45 s at
95 °C, 45 s at 50 °C, 1 min at 72 °C and 40 cycles with a
final extension of 72 °C for 5 min. An aliquot of the
re-amplified product was cloned into a Topo-vector and
transformed into TOP10 cells (TOPO TA-cloningÒ Kit,
Invitrogen Corporation, Stockholm, Sweden). PCRs were
performed on a GeneAmpÒ PCR System 9700 (Perkin
Elmer).
Screening of a phage genomic library and isolation
of full-length gene
The spiny dogfish phage-DNA library made in kGEM-11
(Promega, Falkenberg, Sweden) with Escherichia coli
KW251-stem (Promega) as a host, was kindly supplied by
Anders Johnsen, Rigshospitalet, Denmark [23]. The inserts
in the phages were about 15–22 kb. Approximately 50 000
phages were plated out on each of 12 different 15-cm Petri
dishes (roughly three genome equivalents) and grown at
37 °C for 8 h. Plaques were lifted over to the nylon transfer
membrane (Amersham Biosciences, Uppsala, Sweden) and
denatured for 1 min in Soak I solution (0.5
M
NaOH + 1.5
M
NaCl), then neutralized for 5 min in Soak II solution [1
M
Tris/HCl (pH 8.0) + 1.5 NaCl] and equilibrated in 2 ·
saline sodium citrate buffer (NaCl/Cit). The filters were
dried and used for hybridization. The MC receptor-like

sequence PCR product was labeled with
32
P using Mega-
prime Labeling System (Amersham Biosciences) and used
as a probe. Hybridization was carried out at 65 °Cin25%
formamide (Merck Eurolab AB, Stockholm, Sweden),
6 · NaCl/Cit, 10% dextran sulfate (Amersham Biosci-
ences), 5 · Denhardt’s solution, and 0.1% SDS overnight.
Thefilterswerewashedfivetimesin0.2· NaCl/
Cit + 0.1% SDS for 1 h at 65 °C. After exposure to
autoradiographic films, one positive signal was selected for
further selection. The procedure of selection and hybridiza-
tion was repeated until a single phage was isolated (three
times). The phage was grown according to the protocol and
the phage DNA was isolated using k-purification kit
(Qiagen, Hilden, Germany). The phage was confirmed to
be true positive and used as template for sequencing to
obtain the full-length receptor. We sequenced about 200–
300 bp both upstream and downstream of the coding region
which did not include any introns.
Sequencing
Sequence determinations were performed using ABI
PRISM Dye Terminator cycle sequencing kits according
to the manufacture’s recommendations (Applied Biosys-
tems, Stockholm, Sweden) and analyzed on an ABI
PRISM-310 Automated Sequencher (Applied Biosystems).
Sequences were compiled and aligned in Sequencher (Gene
Codes). Sequences were compared with National Center for
Biotechnology Information (NCBI) database using BlastX.
Alignments and phylogenetic analysis

The full-length sequence of the SacMC4 receptor was
aligned with other MC receptor sequences using ClustalW
(1.7) software [24] and edited manually after visual inspec-
tion. The sequences (see Figs 1 and 2) were retrieved from
GenBank and have accession codes as follows: Homo
sapiens (Hsa) MC1 (NM_002386), MC2 (NM_000529),
214 A. Ringholm et al. (Eur. J. Biochem. 270) Ó FEBS 2003
MC3 (XM_009545), MC4 (NM_005912), MC5
(XM_008685), Mus musculus (Mmu) MC4 (AF201662),
MC5 (NM_013596), Gallus gallus (Gga) MC4 (AB012211),
MC5 (AB012868), Danio rerio (Dre) MC4 (AY078989),
MC5a (AY078990), MC5b (AY078991) receptors. The
Squalus acanthias (Sac) MC4 receptor gained the accession
number AY169401. Its phylogenetic tree was generated by
using the
MEGA
v.2.1 software [25] applying maximum
parsimony methods. Human cannabinoid 2 receptor
(hCB2) (accession code S36750) was used as an out-group.
A bootstrap consensus tree assessing the robustness of the
nodes was made with 100 replicates.
Cloning into expression vector
The entire coding region of the receptor sequence was
amplified with Pfu Turbo DNA polymerase (Stratagene,
AH Diagnostic, Stockholm, Sweden). Specific primers
containing HindIII and XhoI sites were used under the
following conditions: 60 s at 95 °C for one cycle, then 30 s at
95 °C, 30 s at 53 °Cand70sat72°C for 35 cycles. The
PCR fragments were purified by QIAquick PCR Purifica-
tion Kit (Qiagen) and digested by HindIII and XhoI. The

full-length receptor sequence was re-purified and ligated
into a modified pCEP4 expression vector containing the
CMV promotor [26]. The new construct was sequenced and
found to be identical to the genomic clone.
Site-directed mutagenesis
The A59E mutation was introduced into the SacMC4
receptor coding sequence by PCR. Two complementary
oligonucleotides were designed to contain the required
mutation. The SacMC4-A59E primers were 5¢-CAG CCT
CTT GGA AAA TAT TTT GGT CAT TG and 3¢-GAC
CAA AAT ATT TTC CAA GAG GCT GAA GAT G.
The primers were hybridized to opposite strands of the
receptor gene and the complete 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.
Transfection
HEK 293-EBNA cells were transiently transfected with the
constructs using FuGENE
TM
Transfection Reagent (Boeh-
ringer Mannheim, Roche, Stockholm, Sweden) diluted in
OptiMEM medium (Invitrogen Corporation) according to
the manufacturer’s recommendations. The cells were grown
in DMEM/Nut Mix F-12 with 10% fetal bovine serum
(Invitrogen Corporation) containing 0.2 m
ML
-glutamate

(Invitrogen Corporation) and 250 lg/ml G-418 (Invitrogen
Corporation), 100 U penicillin and 100 lgstrepto-
mycinÆmL
)1
(Invitrogen Corporation).
Radioligand binding
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. Saturation experiments were
performed in a final volume of 100 lL for 3 h at 37 °C
and carried out with serial dilutions of
125
I-labelled [Nle4,
D
-Phe7]a-MSH (NDP-MSH). Non-specific binding was
defined as the amount of radioactivity remaining bound to
the cells after incubation in the presence of 2000 n
M
unlabelled NDP-MSH. Competition experiments were

performed in a final volume of 100 lL. The cells
were incubated in the well plates for 3 h at 37 °Cwith
0.05 ml binding buffer in each well containing a constant
concentration of
125
I-labelled NDP-MSH and appropriate
concentrations of competing unlabelled ligands NDP-, a-,
b-, c-MSH or HS014. The incubations were terminated by
filtration through Filtermat A, glass fiber filters (Wallac
Oy, Turku, Finland), which had been presoaked in 0.3%
polyethylenimine, using a TOMTEC Mach III cell
harvester (Orange, CT, USA). The filters were washed
with 5.0 mL of 50 m
M
Tris/HCl (pH 7.4) at 4 °Cand
dried at 60 °C. The dried filters were then treated with
MeltiLex A (Perkin Elmer) melt-on scintillator sheets and
counted with Wallac 1450 (Wizard automatic Microbeta
counter). The results were analyzed with a software
package suitable for radioligand binding data analysis
(
PRISM
3.0, Graphpad Software, San Diego, CA, USA).
Data were analyzed by fitting to formulas derived from
the law of mass action by the method generally referred to
as computer modeling. The binding assays were per-
formed in duplicate wells and repeated three times.
Nontransfected HEK293-EBNA cells did not show any
specific binding for
125

I-labelled NDP-MSH. NDP-MSH
was radio-iodinated by the chloramine T method and
purified by HPLC. NDP-, a-, b-, c-MSH or HS014 were
purchased from Neosystem, France.
cAMP assay
The experiments were performed essentially as described in
earlier [27]. Briefly, the cells were incubated for 2 h with
5 lCiÆmL
)1
[8-
3
H]adenine (Amersham Biosciences, Upp-
sala, Sweden) and then washed and harvested in a medium
composed of 137 m
M
NaCl, 5 m
M
KCl, 0.44 m
M
KH
2
PO
4
,
4.2 m
M
NaHCO
3
,1.2m
M

MgCl
2
,20m
M
Hepes, 1 m
M
CaCl
2
and 10 m
M
glucose, pH adjusted to 7.4. The pelleted
cells were resuspended in the medium as above containing
0.5 m
M
isobutylmethylxantine (Sigma) and preincubated
for 10 min at 37 °C before adding to appropriate concen-
trations of the stimulant (a-MSH) in 96-well plates (Nunc,
VWR International, Stockholm, Sweden). After an addi-
tional 10 min of incubation in 37 °C with the hormone, the
reactions were stopped by rapid centrifugation at 1000 g
for 1 min, removal of supernatants, and addition of 200 lL
ice-cold 0.33
M
perchloric acid per well. The plates were
frozen down to )20 °C, thawed, and the cell debri were
spun down (1000 g for 10 min). The extent of conversion of
[
3
H]ATP to [
3

H]cAMP was determined by Dowex/alumina
sequential chromatography [28] [
14
C]cAMP (Amersham
Biosciences, Uppsala, Sweden) tracer in 0.75 mL 0.33
perchloric acid (about 1000 cpm) was added to each column
together with the samples. The ATP/ADP and cAMP
fractions were dissolved in an appropriate volume of
scintillation cocktail (Optiphase Hisafe 3, Wallac, Turku,
Finland) and analyzed in a b-counter. The conversion to
[
3
H]cAMP was calculated as a percentage of total eluted
Ó FEBS 2003 Dogfish melanocortin 4 receptor (Eur. J. Biochem. 270) 215
[
3
H]ATP and was normalized to the recovery of [
14
C]cAMP.
The cAMP assay was performed in triplicates and repeated
twice for each receptor.
RT-PCR and Southern analysis
Fresh periphery tissues (muscle, heart, liver, kidney, rectal
gland, spiral valve, eye, colon) and several brain regions
(optic tectum, hypothalamus, brain stem, telencephalon,
cerebellum and olfactory bulb) were collected from spiny
dogfish. The total RNA was isolated according to the
RNeasy Mini Kit (Qiagen) protocol. The RNA prepara-
tions were then DNaseI treated for 20 min at room
temperature using the RNase-Free DNase Set protocol

(Qiagen). Absence of genomic DNA in all RNA prepara-
tions was confirmed in PCR reaction using 10–100 ng of
total RNA as a template. Messenger RNA was reverse
transcribed using the 1st Strand cDNA Synthesis kit
(Amersham Pharmacia Biotech) with a reverse primer
specific to dogfish MC4 receptor. The produced cDNA was
used as a template for PCR with the specific primers for the
receptor gene. The conditions for PCR were: 1 min initial
denaturation, then 30 s at 95 °C, 40 s at 55 °C, 60 s at 72 °C
for 35 cycles and finished by 5 min at 72 °C, using Taq
polymerase (Invitrogen Corporation). The following prim-
ers were used: 5¢-AGG CAC TTA ACG GCC CCG GA-3¢
and 5¢-AGA GCG AGG CCA TGA GGG CG-3¢ giving
the expected size of the PCR product of around 300 bp. The
PCR products were analyzed on a 1% agarose gel. The
DNA products on the gel were transferred to nylon filters
over night using 0.4
M
NaOH. The filter was hybridized
with a random-primed
32
P-labeled, species and receptor
specific probe (Megaprime kit, Amersham Biosciences) at
65 °C in 25% formamide, 6 · NaCl/Cit, 10% dextran
sulfate, 5 · Denhardt’s solution and 0.1% SDS over night.
Thefilterwasthenwashedfivetimesin0.2· NaCl/
Cit + 0.1% SDS for 1 h at 65 °C and exposed to
autoradiography film (Amersham Biosciences). Due to the
appearance of double bands (not shown), the PCR products
were denatured in 3% formaldehyde, 25% formamide

solution and separated on 1.4% agarose gel using Mops
buffer (20 m
M
Mops, 2 m
M
sodium acetate and 1 m
M
EDTA) resulting in a single band. The original double
band had different relative intensity in the ethidium bromide
staining and the hybridization signal. It is thus likely that the
double band was caused by an extra signal of single-
stranded DNA. As positive control for the Southern blot,
the genomic DNA was used in PCR reaction. Sequence
analysis of this PCR product confirmed the presence of
SacMC4 receptor sequence. Water was used as negative
control. The RT-PCR reactions and Southern blotting were
performed three times.
Results
Several 600-bp PCR products were cloned and about 25
clones were sequenced of which two identical clones showed
high identity to the MC receptors. After library screening, a
single phage was confirmed to contain a MC receptor-like
protein of 331 amino acids. Among the mammalian and
chicken MC receptor subtypes, our clone had highest
identity to the MC4 receptors (69–71%) and was designated
as the SacMC4 receptor. The protein sequence of the spiny
dogfish receptor are shown in Fig. 1, aligned with the
human MC receptors, the GgaMC4 and GgaMC5 recep-
tors, the MmuMC4 receptor and the recently cloned
DreMC4, DreMC5a and DreMC5b receptors [19]. The

SacMC4 receptor has 71% identity to the HsaMC4, 69% to
the GgaMC4 and 75.8% to the DreMC4 receptor.
Phylogenetic analysis was performed using the maximum
parsimony method (MP) (Fig. 2). The MC3, MC4 and
MC5 receptors are more similar to each other in amino acid
sequence comparison, than the MC2 and MC1 receptors
are (see also previous analysis in [19]). The Hsa and
MmuMC4 receptors are most similar to each other,
followed by the Gga- and DreMC4 receptors. As expected
the SacMC4 branched out at basal to the DreMC4 receptor.
As an out-group, we used the human cannabinoid 2 (hCB2)
receptor.
The SacMC4 receptor had an unusual amino acid in a
very conserved region (TM1). All previously cloned MC
receptors regardless of subtype, share the acidic amino acid
Glu in position 59 in transmembrane (TM) region 1,
whereas the new SacMC4 receptor surprisingly had Ala in
this position (see Fig. 1). In order to investigate if this was a
single amino acid polymorphism, we prepared genomic
DNA from three additional individuals, ran PCR to
generate an  490 bp fragment containing position 59,
cloned it and sequenced. All the individuals had identical
sequence in this region including the Ala in position 59.
Subsequently we inserted Glu into position 59 by site-
directed mutagenesis in order to enable pharmacological
characterization (see below).
The coding sequence of the genomic clone SacMC4 and
mutant SacMC4-A59E receptors were transferred to the
expression vector and control sequenced. The constructs
were transiently expressed in mammalian cells and the

receptors were tested in radioligand binding assay on intact
cells using radioligand-binding assay. Figure 3 shows
saturation and competition curves for these dogfish recep-
tors. Table 1 shows the K
d
and the K
i
values obtained from
saturation and competition analysis, respectively. Our
results suggest that
125
I-labelled NDP-MSH bound to the
SacMC4 receptor with indistinguishable affinity as com-
pared with the Hsa- and DreMC4 receptors. Both the wild-
type SacMC4 receptor and the SacMC4-A59E mutant
receptor had also the same affinities for the endogenous
peptides a-MSH, and the high potency synthetic ligand
NDP-MSH, as compared to the Hsa- and DreMC4
receptors. c1-MSH had also the same affinity to the Hsa-
and SacMC4 receptors. The SacMC4 receptor had how-
ever, slightly lower affinity for b-MSH, as compared with
the Hsa- and DreMC4 receptors. The synthetic compound
HS014 had the same affinity for the Dre- and SacMC4
receptors, while it had 90-fold lower affinity for the
HsaMC4 receptor. The binding profile for the mutant
A59E receptor was not determined for the low affinity
ligands b-, c1-MSH and HS014.
In order to investigate if the SacMC4 and SacMC4-A59E
receptors were able to influence intracellular cAMP after
stimulation of a-MSH, we tested both receptors in a cAMP

assay. The results are shown in Fig. 4. We found that both
SacMC4 and SacMC4-A59E receptors responded to the
stimulation by a-MSH with the same potency. These results
216 A. Ringholm et al. (Eur. J. Biochem. 270) Ó FEBS 2003
are in line with that which we have observed for the
HsaMC4 receptor in response to a-MSH [11]. Non-
transfected HEK293-EBNA cells, which showed no
response to the a-MSH, were used as controls.
Tissue distribution was determined by RT-PCR. The
results of the RT-PCR are shown in Fig. 5. The SacMC4
receptor was expressed in five brain regions (Fig. 6) but not
in any of the peripheral tissues. There were strong signals in
the brain stem and the hypothalamus and slightly weaker
signals in the optic tectum, olfactory bulb and telencepha-
lon. However, it should be noted that the PCR assay was
not designed for quantification. The experiments were
performed three times and there were no qualitative
differences between the runs. The integrity of the mRNAs
was tested by using zebrafish and fugu based actin primers
for RT-PCRs. We received a distinct product of the
expected length for the cDNA from all the different tissues.
This PCR product was confirmed to be dogfish actin by
sequencing.
Discussion
We describe here the cloning of the first MC receptor in
spiny dogfish. The phylogenetic analysis indicates that the
new receptor is an ortholog of the MC4 receptor and we use
thus the nomenclature SacMC4 receptor and the gene is
entered in the gene database under this name. The SacMC4
receptor appears basal in the MC4 receptor phylogenetic

cluster (see Fig. 2). The spiny dogfish belongs to the
chondrichthyans, the cartilaginous fishes, which diverged
prior to the split leading to ray-finned and lobe finned fishes.
The sharks are thus more distant from mammals than the
bony fishes, including zebrafish which we cloned earlier [19].
The phylogenetic positioning of the SacMC4 receptor is
Fig. 1. Amino acid sequence alignment made using
CLUSTALW
(1.7) software and edited by manual inspection. TheSacMC4receptorservedasa
master for the HsaMC1-5, MmuMC4, GgaMC4, GgaMC5, DreMC4, DreMC5a and DreMC5b sequences. The lines mark putative trans-
membrane (TM) regions (according to [34]). The accession numbers are listed in Material and methods.
Fig. 2. Phylogenetic analysis of the MC-receptor family using the full-
length amino acid sequences. The tree was generated by maximum
parsimony analysis (
PAUP
4.0). The human cannabinoid 2 receptor
(hCB2) sequence was used to root the tree. The numbers above the
nodes indicate percentage of bootstrap replicates. The accession
numbers are listed in Material and methods.
Ó FEBS 2003 Dogfish melanocortin 4 receptor (Eur. J. Biochem. 270) 217
thus in agreement with that of the cartilaginous fishes. This
shows that the MC4 receptor originated before the radiation
of gnathostomes.
It is interesting that despite the fact that cartilaginous
fishes diverged from the lineage leading to mammals
over 450 million years ago, the SacMC4 receptor shows
very similar pharmacological properties as the HsaMC4
receptor. The characteristics of the HsaMC4 receptor, such
as relatively low affinity for c-MSH and a slightly higher
affinity for b-MSH as compared with a-MSH, seem to be

conserved. The ligand b-MSH, whose physiological roles
are still obscure, also has a higher affinity for the SacMC4
receptor than a-MSH, which is in line with our previous
results for the human and rat MC4 receptors [29,30]. This
Fig. 3. Saturation binding with Scatchard plots (left) and competition curves for the SacMC4 and SacMC4-A59E receptors expressed in intact
transfected cells. The figure shows competition curves (right) for
125
I-labelled NDP-MSH (m), a-MSH (j), c-MSH (h) and HS014 (d)tothe
SacMC4 receptors, and the two first mentioned for the SacMC4-A59E receptor. The binding curves were obtained by using a fixed concentration of
2n
M
125
I-labelled NDP-MSH and varying concentrations of the nonlabeled competing peptide. Lines represent the computer-modeled best fit of
the data assuming that ligands bound to one-site.
Table 1. K
i
and K
d
values (mean±SEM) obtained from competition and saturation curves, respectively, for melanocortin peptides analogs on SacMC4,
SacMC4-A59E, DreMC4, HsaMC4 receptor transfected EBNA cells.
Ligand
SacMC4
(nmolÆL
)1
)
SacMC4-A59E
(nmolÆL
)1
)
DreMC4

a
(nmolÆL
)1
)
HsaMC4
a
(nmolÆL
)1
)
125
I-labelled NDP- MSH 1.21 ± 0.44 1.93 ± 0.30 2.39 ± 0.96 2.35 ± 1.18
NDP-MSH 1.50 ± 0.054 1.41 ± 0.070 3.35 ± 0.31 3.57 ± 0.30
a-MSH 198 ± 42 198 ± 28 243 ± 27 289 ± 29
b-MSH 570 ± 278 ND 163 ± 14 126 ± 15
c1-MSH 1950 ± 70 ND 2200 ± 550 3690 ± 260
HS014 368 ± 110 ND 493 ± 40 5.60 ± 0.22
a
Data taken from Ringholm et al. [19]. ND, not done.
218 A. Ringholm et al. (Eur. J. Biochem. 270) Ó FEBS 2003
feature was also conserved in the zebrafish receptors (see
Table 1) and our new results thus provide additional
evidence for our speculation that b-MSH may have a
specific and also an evolutionarily conserved role for this
receptor subtype. HS014, an antagonistic substance, is a
synthetic cyclic peptide and was developed to be selective for
the human MC4 receptor [31]. This substance is the only
one that has lower affinity for the SacMC4 receptor as
compared with the human ones. It could be speculated that
even though the ability of the receptors to bind the natural
peptides is highly conserved, the 3D binding cavity of the

SacMC4 receptor may not be as well conserved to fit this
synthetic ligand. Moreover, our results also show that the
SacMC4 receptor is a functional receptor and able to
activate the Gs pathway when stimulated with a-MSH, in
agreement with the other MC receptors from mammals and
zebrafish.
The SacMC4 receptor exhibits high sequence identity
with the mammalian orthologs. It also displays several of
the structural characteristics typical for the mammalian MC
receptors such as conserved Cys in the first extra-cellular
loop, lack of Pro in TM5, short extra-cellular and intracel-
lular loops, and short divergent C- and N-terminals (see
Fig. 1). This is in line with what we found for the DreMC
receptors. The SacMC4 receptor sequence had however,
Ala in position 59 in TM1. We found this remarkable as all
the five MC receptor subtypes in all the species cloned so far
have Glu in this position. TM1 is believed to contribute to
the main binding region in MC receptors [32,33]. Some
earlier studies suggested that this acidic and hydrophilic Glu
may play an important role in the ligand binding [34] while
other studies have indicated that this residue may not be
participating in the ligand binding [35] for the mammalian
receptors. It was possible that this was a mutation that was
only found in the genome of the individual we cloned. We
investigated if this residue was conserved in the SacMC4
receptor gene by sequencing additional three individuals.
The results show that the missing Glu was not due to
polymorphism. The putative importance of the unique
amino acid exchange in the SacMC4 receptor was investi-
gatedbymutatingtheAlaÔbackÕ to Glu. We investigated the

mutant receptor both regarding binding of NDP-MSH and
a-MSH, and the ability to increase production of adenylate
cyclase when stimulated with a-MSH. The SacMC4 and
the SacMC4-A59E had indistinguishable K
d
, K
i
and
EC
50
-values, suggesting that this exchange is not important
for the pharmacological profile of the receptor. It is difficult
to speculate why this Glu59 has been replaced in the
SacMC4 receptor but cloning of other MC4 receptors from
other distant species as well as other MC receptors from
Fig. 4. Generation of cAMP in response to a-MSH forSacMC4 (h)and
SacMC4-A59E (j) receptors in intact transfected cells. Each point
represents the mean ± SEM. Untransfected HEK293-EBNA (m) cells
showed no adenylate cyclase-activity in response to a-MSH. The cAMP
assaywasperformedintriplicatesandrepeatedtwiceforeachreceptor.
Fig. 5. Expression of SacMC4 receptor mRNA as determined by RT-
PCR on total RNA preparations from spiny dogfish tissues. The tissues
and the controls are denoted at the top of the figure. The figure shows
4-h exposure on an X-ray film after hybridization with the SacMC4
receptor probe. The size (bp) of the ladder is shown on the right. The
PCR reactions and the hybridization were performed three times with
qualitatively similar results.
Fig. 6. Side view of spiny dogfish (Squalus acanthias) brain. Shaded
sections represent regions expressing the SacMC4 receptor. Dashed
lines indicate incisions made to divide brain regions for collecting tis-

sues for RT-PCR. OB, olfactory bulb; Tel, telencephalon; OT, optic
tectum; Hyp, hypothalamus; Cb, cerebellum; BS, brain stem.
Ó FEBS 2003 Dogfish melanocortin 4 receptor (Eur. J. Biochem. 270) 219
dogfish may provide additional information about this
unusual replacement.
The results show that the SacMC4 receptor was expressed
in olfactory bulb, telencephalon, optic tectum, hypothala-
mus, and brain stem but not in cerebellum (Fig. 6). The
presence of MC4 in the hypothalamus is not surprising. This
receptor is expressed in hypothalamus in all species inves-
tigated and is important in the regulation of appetite. In the
telencephalon, especially the limbic system, the MC4
receptor is possibly playing a role in the communication
between the melanocortin system and the serotonergic
system and stress effects on appetite [36]. The presence of
SacMC4 receptor in the olfactory bulb may suggest a role in
chemosensory mechanisms, possibly those associated with
feeding [37,38]. In the brainstem, the SacMC4 receptor
could be involved in visceral afferent signals from the gut via
the nucleus of the solitary tract (NTS). The NTS could be
sending signals to the hypothalamus from the gut to
regulate energy balance [39]. a-MSH immunoreactivity has
been found in the spotted dogfish (Scyliorhinus canicula)
[40], showing that perikarya were largely located in the
hypothalamus with projections running to various places
in the diencephalon. The SacMC4 receptor was found
both in the hypothalamus and diencephalon, indicating that
this receptor is expressed in brain regions of sharks where
a-MSH can be found.
The SacMC4 receptor was expressed only in the brain but

not in peripheral tissues. This is in agreement with the
mammalian MC4 receptors that have only been found in
brain tissue [8,41]. The DreMC4 receptor was also found in
the brain but rather surprisingly also in the eye, gastro-
intestinal tract and ovaries. In chicken, the MC4 receptor is
expressed in a wide variety of peripheral tissues, including
the heart, adrenal glands, ovaries, testes, spleen, adipose
tissues and eye, as well as the brain [42,43]. In our previous
report, we speculated that the expression pattern of the MC4
receptor in mammals had become more confined to central
regions, as compared with zebrafish and chicken [19]. Our
new results from the dogfish indicate rather that the MC4
receptor had a predominant and important function in the
CNS very early in vertebrate evolution and later became
more widely expressed in zebrafish and chicken.
In conclusion, our data show that the MC4 receptor had
already arisen before the radiation of gnathostomes. The
spiny dogfish receptor clone will facilitate cloning in
cyclostomes and amphioxus as well as additional gnathos-
tomes. Our results provide an understanding of the
evolutionary origin of the MC receptor system and enhance
further studies on their many important physiological roles.
The high degree of conservation of MC receptors in teleosts
and cartilaginous fish may indicate that the MC receptors
arose even before the appearance of vertebrates. The
conservation of the pharmacological properties and tissue
distribution pattern could demonstrate an early develop-
ment of a mechanism where a peptide binds to a GPCR for
central regulation of the energy balance.
Acknowledgments

We thank Flemming Cornelius, University of Aarhus, Denmark for
providing dogfish tissues and Anders Johnsen, Rigshospitalet, Den-
mark for providing a dogfish phage library. The studies were supported
by the Swedish Research Council (VR, medicin), the Swedish Society
for Medical Research (SSMF), A
˚
ke Wibergs Stiftelse, Svenska
La
¨
karesa
¨
llskapet, Petrus och Augusta Hedlunds Stiftelse, Go
¨
ran
Gustavsson and Lars Hierta foundations and Melacure Therapeutics
AB, Uppsala, Sweden.
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