MC3R and MC4R have also differences in pharmacology: MC3R has a unique preference for c-MSH among different subtypes of MCRs in mammals.. Cloning and expression of receptors Full-length
Trang 1Cloning of two melanocortin (MC) receptors in spiny dogfish
MC3 receptor in cartilaginous fish shows high affinity to ACTH-derived peptides while it has lower preference to c-MSH
Janis Klovins1,2, Tatjana Haitina1, Aneta Ringholm1, Maja Lo¨wgren1, Davids Fridmanis2, Maija Slaidina2, Susanne Stier1and Helgi B Schio¨th1
1 Department of Neuroscience, Unit of Pharmacology, Uppsala University, Sweden; 2 Biomedical Research and Study Centre, University of Latvia, Riga, Latvia
We report the cloning and characterization of two
melano-cortin receptors (MCRs) from the spiny dogfish (Squalus
acanthias) (Sac) Phylogenetic analysis shows that these
shark receptors are orthologues of the MC3R and MC5R
subtypes, sharing 65% and 70% overall amino acid identity
with the human counterparts, respectively The SacMC3R
was expressed and pharmacologically characterized in
HEK293 cells The radioligand binding results show that this
receptor has high affinity for adrenocorticotropic hormone
(ACTH)-derived peptides while it has comparable affinity
for a- and b-melanocyte stimulating hormone (MSH), and
slightly lower affinity for c-MSH when compared with the
human orthologue ACTH(1–24) has high potency in a
second-messenger cAMP assay while a- and c-MSH had
slightly lower potency in cells expressing the SacMC3R We
used receptor–enhanced green fluorescence protein (EGFP)
fusion to show the presence of SacMC3R in plasma
membrane of Chinese hamster ovary and HEK293 cells but the SacMC5R was retained in intracellular compart-ments of these cells hindering pharmacological characteri-zation The anatomical distribution of the receptors were determined using reverse transcription PCR The results showed that the SacMC3R is expressed in the hypo-thalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum of the spiny dogfish while the SacMC5R was found only in the same central regions This report describes the first molecular characterization of a MC3R in fish The study indicates that many of the important elements of the MC system existed before radiation of gnathostomes, early in vertebrate evolution, at least 450 million years ago
Keywords: ACTH; dogfish; melanocortin; MSH; POMC
The melanocortin receptors (MCRs) are G-protein coupled
receptors (GPCRs) that belong to the large rhodopsin
subgroup [1] MCRs respond to the pro-opiomelanocortin
(POMC) cleavage products: a-, b- and c-melanocyte
stimulating hormones (MSH) and adrenocorticotropic
hormone (ACTH), all of which possessing agonistic
prop-erties towards MCRs There are five subtypes of MCRs in
mammals and aves, named MC1R–MC5R (reviewed in
[2,3]) In mammals, MC1R is expressed in melanocytes
and has an important role in determining skin and hair
pigmentation [4] Expression of MC1R is also detected in
other skin cell types and in a number of peripheral tissues,
including leukocytes, where it mediates the broad
anti-inflammatory actions of the melanocortin peptides [5]
MC2R is expressed in adrenal cortex, where it mediates the effects of ACTH on steroid secretion The mammalian MC2R differs pharmacologically from the other MCRs as it
is activated only by ACTH and has no affinity for MSH peptides [6] Some expression of MC2R has been found in human adipose tissue, but its role in this tissue is not clear MC3R and MC4R are expressed in several brain regions, particularly in the hypothalamus These receptors have attracted much attention during recent years due to their involvement in regulation of energy homeostasis MC4R is one of the best-characterized monogenic factors of obesity and a number of mutations in this receptor are linked to obese phenotypes in humans [7–9] Although both receptors are involved in regulation of energy balance, mice deficient
in one of these receptors display separate phenotypes [10,11] Knockout of MC3R causes an 50–60% increase
in adipose mass, but these mice do not become significantly overweight [12]; they do, however, exhibit reduction in locomotory behaviour suggesting reduced energy expendi-ture Mice lacking both MC3R and MC4R become significantly heavier than MC4R knockout mice [11] MC3R and MC4R have also differences in pharmacology: MC3R has a unique preference for c-MSH among different subtypes of MCRs in mammals MC5R is expressed in a number of human peripheral tissues, including adrenal gland, adipocytes and leukocytes [5] The functional prop-erties of MC5R are, however, still not well understood, with
Correspondence to H B Schio¨th, Department of Neuroscience,
Biomedical Center, Box 593, SE-75 124 Uppsala, Sweden.
Fax: +46 18 51 15 40, E-mail: helgis@bmc.uu.se
Abbreviations: MCR, melanocortin receptor; GPCR, G-protein
cou-pled receptor; POMC, pro-opiomelanocortin; MSH, melanocyte
stimulating hormones; ACTH, adrenocorticotropic hormone; EGFP,
enhanced green fluorescence protein; NDP-MSH, (Nle4, D
-Phe7)-a-MSH; EC 50 , 50% effective concentration; TM, transmembrane.
Note: J Klovins and T Haitina contributed equally to this work.
(Received 25 May 2004, revised 27 August 2004,
accepted 22 September 2004)
Trang 2the exception of its participation in exocrine function,
regulating sebaceous gland secretion in mice [13])
Understanding of how the energy balance is regulated in
vertebrates other than mammals is very important, both
from a global ecological and economic perspective
consid-ering the importance of growth rate of, for example, fish
We have shown recently that MCRs exist in teleost fish
[14–16] and that MC4R plays an important role in
regulation of food intake in teleosts [17,18] Previously, we
also showed that the MC4Rs is likely to regulate food intake
in birds [19] All five MCR subtypes can be found in teleosts,
such as zebrafish, but there are still important differences in
the repertoire in teleosts as compared with that in mammals
The zebrafish has six MCRs due to an additional copy of
the MC5R that, we have suggested, were created through a
lineage-specific tetraploidization within teleosts Another
interesting difference is that the teleost Fugu has only four
MCRs as it is lacking an MC3R [15] Moreover, there are
some interesting differences in the pharmacology between
teleosts and mammals ACTH seems to play a more
prominent role for the MCRs in Fugu as compared with
a-MSH, which has the highest affinity to the four MSH
binding MCRs in mammals
The spiny dogfish (Squalus acanthias) is a shark that has
become the most popular research object among
cartilagin-ous fishes (also called chondrichthyans) They arose from
the Agnatha, the jawless fishes, in the late Silurian period,
approximately 450 million years ago The lineage leading
to cartilaginous fishes split from the lineage leading to
mammals prior the split of teleosts, and can thus be
considered in evolutionary term as older species than the
teleosts There are also other differences The generation
time in spiny dogfish is fairly long among fish; 18 years as
compared with 1 year in zebrafish Another difference is
that the chondrichthyans produce a relatively small number
of offspring [20] According to genome duplication theory,
the first tetraploidization occurred before the divergence of
agnathans, but the second genome duplication took place in
the lineage leading to gnathostomes [21] The teleost fishes
however, are believed to have undergone another
tetra-ploidization [22] followed by subsequent large genome
reorganizations The teleosts have thus gained and lost
many gene copies, which may reflect the different numbers
of MCR genes in the different teleost species POMC
peptides are well studied in dogfish [23,24] and the POMC
gene structure is more conservative than in any ray-finned
fishes One of the major features of POMC in dogfish is that
it contains a c-MSH peptide, like in mammals Moreover,
two copies of this gene are found in several ray-finned fish
species [25,26] Notable is that the c-MSH region in
ray-finned fishes is quite variable and degenerate, either missing
the c-MSH core motif [27,28] or even the complete c-MSH
sequence [29], and at the same time displaying a high level of
conservation in the a-, b-MSH and ACTH sequences The
modern sharks and rays (Chondrichthyes) also contain an
additional MSH sequence (d-MSH), which may be the
result of internal domain duplication of b-MSH sequence
All of this together makes cartilaginous fishes particularly
interesting species, not only to understand the evolution of
the melanocortin system in other vertebrates, but also to
study functional features of MCRs We have recently cloned
and characterized the MC4R gene from dogfish The results
showed high conservation in sequence and pharmacology of this receptor to its human counterpart [30] but it was not known if cartilaginous fishes have other subtypes of the MCRs
We now report the cloning, expression, pharmacological characterization, intracellular localization and tissue distri-bution of MC3R and MC5R in the spiny dogfish These receptors represent in evolutionary terms the most distant receptor subtypes cloned so far Moreover, the dogfish MC3R is the first MC3R that has been pharmacologically characterized in any lower vertebrate
Materials and methods
Cloning and sequencing Genomic DNA was extracted from muscle tissue of spiny dogfish captured in the North Sea (Hambergs Fisk, Uppsala, Sweden) About 1 g of the muscle tissue was homogenized in lysis buffer [100 mMEDTA, 10 mMTris, 1% (w/v) SDS] and centrifuged for 5 min at 11 500 g The supernatant was extracted with saturated phenol, phenol/ chloroform/isoamyl alcohol (25 : 24 : 1, v/v/v) and chloro-form The DNA was precipitated with 1 vol isopropanol in the presence of 1M NH4Ac and washed with 70% (v/v) ethanol Degenerate primers based on conserved parts of the human, rat, mouse and chicken MC3, MC4 and MC5R were used in a low stringency PCR with 100 ng of dogfish genomic DNA PCR was performed using the Taq DNA polymerase (Invitrogen) in a reaction volume of 20 lL, containing 200 lMdNTP, 20 mMTris/HCl pH 8.4, 50 mM
KCl, 2 mMMgCl2and 20 pmol each primer Touch-down PCR was performed, starting with an initial 1 min 95C denaturation, followed by 22 cycles of 45 s at 94C, 45 s
at 52C to 42 C, 90 s at 72 C This was followed by 25 cycles of 30 s at 94C, 40 s at 50 C, 1 min at 72 C, with a final extension of 5 min at 72C The primer sequences were 5¢-CAYTCNCCNATGTAYTTYTT-3¢ and 5¢-AT NACIGARTTRCACATDAT-3¢ The degenerated posi-tions were designed considering the conservation of the MC3, MC4, MC5R sequences from human, mouse, rat and chicken PCR products were purified from 1% agarose gel using Gel Extraction Kit and were cloned into pCRII vector and transformed into TOP10 cells (TOPO TA-cloning kit, Invitrogen) Sequencing reactions were performed using the ABI PRISM Big Dye Terminator cycle sequencing kit according to the manufactures recommendations and analysed on ABI PRIZM-310 or 3100 Automated Sequencers (Applied Biosystems) Sequences were compiled and aligned using the SEQMAN program from DNAstar (Lasergene) Sequences were compared against database assemblies usingBLASTNandBLASTX
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 (Promega) as a host was kindly supplied by A Johnsen (Rigshospitalet, Copenhagen, Denmark, [31]) The inserts in the phages were about 15–22 kb Approximately
50 000 phages were plated out on each of 12 different 15 cm
Trang 3Petri dishes (roughly three genome equivalents) and grown
at 37C for 8 h Plaques were transferred to the nylon
transfer membrane (Amersham Biosciences, Uppsala,
Swe-den) and denatured for 1 min in 0.5MNaOH; 1.5MNaCl,
then neutralized for 5 min in 1MTris/HCl pH 8.0, 1.5M
NaCl and equilibrated in 2· NaCl/Cit The filters were dried
and used for hybridization PCR product from the obtained
MCR-like sequence was labelled with [32P]CTP[aP] using
the Megaprime Labeling System (Amersham Biosciences)
and used as a probe Hybridization was carried out at 65C
in 25% (v/v) formamide (Merck Eurolab AB), 6· NaCl/Cit,
10% (w/v) dextran sulfate (Amersham Biosciences),
5· Denhardt’s solution, and 0.1% SDS overnight The
filters were washed five times in 0.2· NaCl/Cit containing
0.1% (w/v) SDS for 1 h at 65C After exposure to
autoradiographic films, one positive signal was chosen for
further selection The procedure of selection and
hybridiza-tion was repeated until a single phage clone was isolated
(three times) The phage were grown according to the
protocol and phage DNA was isolated using k-purification
kit (Qiagen) Phage were sequenced as described above and
found to contain MC5R-like sequence, which was different
from the probe sequence We sequenced about 200 bp
upstream and 100 bp downstream from of the coding
region, which did not include any introns
Inverse PCR
S acanthias genomic DNA was separately digested with
eight different restriction enzymes: HindIII, EcoRI, BamHI,
XhoI, BglII, XbaI, ClaI, KpnI overnight at 37C DNA
from each digest was purified and ligated at 14C overnight
with T4 DNA ligase and precipitated Nested PCR
reaction was used to perform inverse PCR Two sets of
PCR primers were designed for SacMC3R fragment
containing one inner primer pair 5¢-TCATTAGCCA
CTAAGTAGCCAT-3¢ (positions 314–335 on cDNA)
and 5¢-TGGTCCGCCAGAGGACTTG-3¢ (positions
692–710) and one pair of outer primers 5¢-CCATAC
TGTATTTGTTAC-3¢ (positions 817–831) and 5¢-ACT
TACCAGCATGTCTGC-3¢ (positions 253–270) The
primers were placed at the 3¢ and 5¢ parts of cloned
fragment in orientation to amplify nucleotide sequences
upstream and downstream of the known fragment
sequence A primary PCR reaction was performed with
the inner primer pairs using different ligations as a template,
with the following PCR conditions: 95C for 15 s, 60 C
for 20 s, and 72C for 2 min for 40 cycles One microlitre of
the primary PCR reactions was used as template in a second
PCR using the outer primer combination with the following
conditions: 95C for 15 s, 68 C for 20 s, and 72 C for
2 min for 40 cycles The products of second PCR reactions
were isolated from agarose gel, and were cloned into pCRII
vector using TOPO cloning kit and transformed into
Escherichia coli DH5a cells Inserts were sequenced with
vector-specific primers according to the sequencing protocol
above
Alignments and phylogenetic analysis
Alignment of predicted full-length amino acid sequences for
the new genes together with other known MCRs was made
usingCLUSTALW 1.8 software [32] The following receptor sequences (with their accession codes) were retrieved from GenBank for this analysis: human Homo sapiens (Hsa) MC1R (NM_002386), MC2R (NM_000529), MC3R (XM_009545), MC4R (NM_005912), MC5R (XM_008685); chicken Gallus gallus (Gga) MC1R (D78272), MC2R (AB009605), MC3R (AB017137), MC4R (AB012211), MC5R (AB012868), Danio rerio (Dre), MC1R (NM_180970), MC2R (NM_180971), MC3R (NM_180972), MC4R (AY078989), MC5aR (AY078990) and MC5bR (AY078991) and dogfish
S acanthias(Sac) MC4R (AAO39833) The identified genes have the following accession numbers: S acanthias MC3R (AY560605) and MC5R (AY562212) Phylogenetic trees were constructed by MEGA v.2.2 [33] using maximum parsimony and distance neighbor-joining methods Human NPY Y1R (P25929) was used to root the receptor tree Bootstrapping was performed with 1000 replicates Cloning and expression of receptors
Full-length coding sequences were amplified by means of PCR either from phage DNA (MC5R) or from TOPO plasmid containing MC3R gene with Pfx polymerase using HindIII and XhoI restriction sites containing primers for the 5¢ and 3¢ termini of the gene, respectively The fragments obtained were then digested with both restriction enzymes and gel purified prior to ligation into modified pCEP expression vector [34] containing an enhanced green fluor-escence protein (EGFP) coding gene fused to the C terminus
of the receptor gene All constructs were verified by sequencing HEK293 cells, grown to 50–70% confluence, were transfected with 10 lg of the construct using FuGENE-6 Transfection Reagent (Roche) according to the manufacturer’s instruction The cells were grown in Dulbecco’s modified Eagle’s media: F-12 Nutrient Mixture (DMEM/F-12) (1 : 1) with GlutaMAX I containing 10% (v/v) fetal bovine serum, 100 lgÆmL)1 penicillin,
100 lgÆmL)1 streptomycin, 2.5 lgÆmL)1 amphotericin B, and 250 lgÆmL)1geneticin G-418 (Gibco) in a humidified atmosphere of 95% air and 5% CO2(v/v) at 37C Semi-stable cell lines, expressing target receptor, were obtained by selecting for growth in the presence of 100 lgÆmL)1 hygromycin B (Invitrogen), first added 24 h after transfec-tion The cells were grown continuously in the presence of hygromycin B
Fluorescent microscopy Chinese hamster ovary (CHO-1) cells were grown on glass coverslips and were transfected with 2 lg of appropriate plasmid according to FuGENE-6 Transfection Reagent protocol Cells were fixed and mounted 24 h post-transfec-ton Similarly, the semistable HEK293 cell lines expressing the dogfish receptors were plated on coverslips and grown for 48 h prior to fixation The cells were washed twice with ice-cold 1· NaCl/Pi before fixation in 4% (v/v) parafor-maldehyde containing 1· NaCl/Pifor 15 min The cover-slips were then washed twice with NaCl/Piand mounted in NaCl/Picontaining 1 lgÆmL)1DAPI (4,6-diamino-2-phe-nylindole) Cells were examined immediately using a Zeiss Axioplan 2 microscope The digitally acquired images were
Trang 4captured and analysed usingOPENLAB software
(Improvi-sion) At least three slides were analysed for each receptor
and cell type From all cells expressing receptor–EGFP
fusion, only the cells displaying single DAPI stained nucleus
were considered for analysis Fifteen randomly selected
cells were analysed from each slide
Radioligand binding assay
HEK293 cells expressing the dogfish receptors were
harves-ted from plates and resuspended in binding buffer
com-posed of 25 mMHepes, 2.5 mM CaCl2, 1 mMMgCl2and
0.2% (w/v) bacitracin, pH adjusted to 7.4 To obtain the
membranes, cells were homogenized using Ultra Turrax
The cell suspension was centrifuged for 3 min at 220 g and
membranes were collected from the supernatant by
centrif-ugation for 15 min at 20 000 g The pellet was resuspended
in binding buffer The binding was performed in a final
volume of 100 lL for 3 h at room temperature
Satura-tion experiments were carried out with serial diluSatura-tions of
125I-labelled (Nle4,D-Phe7)-a-MSH (NDP-MSH), labelled
by the Chloramine-T method Non-specific binding was
determined in the presence of 1 lMunlabeled NDP-MSH
Competition experiments were performed with constant
0.6 nMconcentration of125I-labelled NDP–MSH and serial
dilutions of competing unlabelled ligands: NDP-MSH,
a-MSH, b-MSH, c1-MSH, ACTH(1–24), ACTH(1–17),
MTII, and HS024 (Neosystem) The membranes were
collected by filtration on glass fibre filters, Filtermat A
(Wallac), using a TOMTEC Mach III cell harvester
(Orange, CT, USA) The filters were washed with 5 mL
per well of 50 mM Tris/HCl pH 7.4 and dried at 50C
MeltiLex A scintillator sheets (Wallac) were melted on dried
filters and radioactivity was counted with automatic
Microbeta counter 1450 (Wallac) Binding assay was
performed in duplicate from at least three independent
experiments Non-transfected cells did not show any specific
binding with 125I-labelled NDP-MSH The results were
analysed withPRISM3.0 software package (GraphPad)
cAMP assay
Cyclic adenosine monophosphate production was
deter-mined on semistable HEK293 cells expressing the target
MCR A confluent layer of cells was incubated for 3 h with
2.5 lCiÆmL)1 [3H]ATP (specific activity 29 CiÆmmol)1;
Amersham Biosciences) Cells were collected, washed with
buffer containing 137 mM NaCl, 5 mM KCl, 0.44 mM
KH2PO4, 4.2 mMNaHCO3, 1.2 mMMgCl2Æ6H2O, 20 mM
Hepes, 1 mM CaCl2, and 10 mM glucose, pH adjusted to
7.4 All reaction components were resuspended in the
above-mentioned buffer containing 0.5 mM
isobutyl-methylxanthine (IBMX; Sigma) Resuspended cells were
incubated for 10 min at 37C Stimulation reaction was
performed for 20 min at 37C in a final volume of 150 lL
containing 2 · 105cells and various concentrations of a
a-MSH, c1-MSH, and ACTH(1–24) After incubation, cells
were centrifuged and 200 lL of 0.33Mperchloric acid was
added to pellets to lyse the cells The cells were frozen,
thawed and spun down Lysate (200 lL) was added to
Dowex 50 W-X4 resin columns (Bio-Rad), washed
previ-ously with 2· 10 mL HO As an internal standard, 750 lL
0.33Mperchloric acid containing 0.5 nCiÆmL)1[14C]cAMP (Amersham Biosciences) was added to each column Columns were washed with 2 mL H2O to remove ATP, which was collected in scintillation vials to estimate the amount of unconverted [3H]ATP Four millilitres of Ready Safe scintillation cocktail (Perkin Elmer) was added to the vials before counting Dowex columns were then placed over alumina (Sigma) columns (prewashed with 8 mL 0.1M
imidazole) and the cAMP was transferred onto the alumina column using 10 mL H2O cAMP was eluted from alumina column with 4 mL 0.1M imidazole and collected into scintillation vials to which 7 mL of scintillation fluid was added 3H and 14C were counted on Tri-carb liquid scintillation beta counter The amount of obtained [14C]cAMP was expressed as a fraction of total [14C]cAMP ([14C]cAMP/total[14C]cAMP) and was used to standardize [3H]cAMP to column efficiency Results were calculated as the percent of total [3H]ATP (obtained as a sum of [3H]ATP from first column and [3H]cAMP from second column) to [3H]cAMP and used to determine 50% effective concentra-tion (EC50) values by non-linear regression usingPRISM3.0 software All experiments were performed in duplicate and repeated three times
RT-PCR and Southern analysis Total RNA was isolated from number of peripheral tissues (muscle, heart, liver, kidney, rectal gland, spiral valve, eye, colon) and several brain regions (optic tectum, hypothala-mus, brain stem, telencephalon, cerebellum and olfactory bulbs) after dissection from frozen animals and homo-genizaton The total RNA was isolated using RNeasy Mini Kit (Qiagen) including processing with DNA shredder and DNAse I treatment (Qiagen) as recommended by the manufacturer As some of the samples retained genomic DNA, total RNA was exposed to a further treatment with
1 unitÆlL)1 RNase-free DNaseI (Roche) for 10 min, fol-lowed by heat inactivation of DNase for 5 min at 70C Due to absence of publicly available sequence of dogfish b-actin gene, we designed primers for conservative parts of b-actin genes from other vertebrate species: 5¢-CGTGCG TGACATCAAGGAGA-3¢ and 5¢-CTTGGTAGGGCTC CCAGCAC-3¢ The primers were tested on genomic DNA, the sequence of PCR product was determined as b-actin gene and submitted to GenBank (AY581300) Absence of genomic DNA in RNA preparations was confirmed in PCR reaction with these primers using 10–100 ng of total RNA
as a template and genomic DNA as a positive control Messenger RNA was reverse transcribed using the 1st Strand cDNA Synthesis kit (Amersham Biosciences) The quality of cDNA was tested in PCR with the above-mentioned b-actin primers The cDNA produced was used
as a template for PCR with the specific primers for the receptor genes (see below) The conditions for PCR on receptor genes were: 1 min initial denaturation, 95C for
15 s, 55C for 20 s, 72 C for 1 min for 30 cycles and ending with 5 min at 72C, using Taq polymerase (Invi-trogen) The following primers were used: 5¢-CTTCCT CTGTAGTTTAGCAG-3¢ (positions 231–250 on cDNA) and 5¢-CACATAATGAGGATCAGGTATG-3¢ (positions 848–869) for MC3R gene (expected size 639 bp); 5¢-GAC TGTAAAACGAGCCACTT-3¢ (positions 462–481) and
Trang 55¢-GCTCTCTGATGAATTGAGTT-3¢ (positions 676–
695) for MC5R gene (expected size 234 bp) The PCR
products were analysed on a 1% agarose gel DNA from the
gel was transferred onto nylon filters overnight using 0.4M
NaOH The filters were hybridized with a random-primed
32P-labelled, receptor specific probe Probes were generated
by Megaprime DNA labelling kit RPN1607 (Amersham
Biosciences), using sequence verified PCR products
ampli-fied from plasmids containing dogfish MCR genes
Hybrid-ization was carried out at 65C in 25% (v/v) formamide,
6· NaCl/Cit, 10% (w/v) dextran sulfate, 5· Denhardt’s
solution and 0.1% (w/v) SDS overnight The filters were
washed three times in 0.2· NaCl/Cit, 0.1% (w/v) SDS for
1 h at 65C and exposed to autoradiography film
(Amer-sham Biosciences) As positive controls in the Southern
blots, the genomic DNA and the PCR products obtained
from it were used PCR products from other SacMCR
subtypes were used to show the absence of
cross-hybridiza-tion for probes used in Southern blots The RT-PCR
reactions and Southern blotting were performed at least
twice each To confirm the single-strand conformational
nature of the apparent double bands, PCR products were
denatured in 3% (v/v) formaldehyde, 25% (v/v) formamide
solution and separated on 1.4% agarose gel using Mops
buffer (20 mM Mops, 2 mM sodium acetate and 1 mM
EDTA); this resulted in a single band in Southern blots
To estimate the length of the bands on the Southern blots
images were overlaid with images of the ethidium
bromide-stained gel and length was assigned according to marker
positions
Results
Degenerate PCR was performed on genomic DNA of spiny
dogfish resulting in a PCR product of 600 bp; this was
then cloned and about 10 clones were sequenced Sequences
were compared with sequences in GenBank by using
BLASTNandBLASTX Three identical clones displayed high
identity to the MCRs, with the highest score to the chicken
MC3R This sequence was used as a probe for high
stringency screening of a dogfish genomic phage library
Three positive phages were selected and these were found to
include the entire sequence of a full-length MCR gene This
sequence was however, different from probe sequence and
showed highest similarity to the zebrafish MC5aR when
compared with the GenBank database Repeated screening
with the MC3R probe did not result in a phage clone from
this library In order to obtain the full-length MC3R gene
sequence, we performed inverse PCR on circularized
fragments of dogfish genomic DNA using number of
different restriction enzymes for DNA fragmentation PCR
products obtained from ClaI and KpnI fragments contained
a MC3R sequence and we were able to extend the sequence
in both 3¢ and 5¢ directions to overlap the stop codon and
initiating ATG codon, respectively The genes were designed
S acanthias (Sac) MC3R and MC5R according to their
sequence similarity with previously cloned MCRs from
human, mouse, chicken, zebrafish and Fugu and the
phylogenetic analysis Protein sequence alignment of these
receptors is shown in Fig 1 Maximum parsimony analysis
of this group of receptors is shown in Fig 2, representing a
consensus tree The dogfish MC3R sequence has highest
identity to the MC3 subtype receptors (63–75%), while the dogfish MC5R gene displayed 72–77% identity to other receptors of the MC5R subtype Both maximum parsimony and neighbor-joining methods gave consistent topology of trees supporting the designation of the new receptors as subtypes of the MC3R and MC5R, respectively As expected, the SacMCRs branched out at basal to node dividing the teleost (zebrafish) and two tetrapod MCRs sequences in MC3R, MC4R and MC5R clades
The coding sequences of the SacMC3R and SacMC5R were recloned into an expression vector containing the cytomegalovirus (CMV) promoter and sequenced to con-firm The receptors expressed in semistable HEK293 cells were tested in radioligand-binding assays on prepared membranes Transfection of SacMC3R gave rise to specific binding of the radioligand while transfection of SacMC5R did not, despite repeated attempts SacMC5R has an alternative ATG start coding upstream of the one we considered to be the start codon We reconstructed the clone and added an additional sequence including the alternative start codon, but transfection with this clone still did not give rise to any specific binding This was very surprising to us as this was the first wild-type MCR clone in any species that has not been readily expressed to give binding with
125I-labelledNDP–MSH (except the MC2R) In order to control the expression levels and cell surface location of the protein, we fused an EGFP gene to the C terminus of the receptor gene for both the clones The constructs were transfected in HEK293 cells and semistable cell lines were created Moreover, transient expression of hybrid receptors
in CHO-1 cells was performed to estimate the localization
of receptors in cell detecting the EGFP in a fluorescent microscopy In 80–90% of the receptor–EGFP fusion expressing cells, analysed in several experiments, the SacMC3R showed diffuse staining, which was also present
in plasma membrane of CHO-1 (Fig 3) and semistable HEK293 cells (data not shown) The cells expressing the SacMC5R, however, showed staining concentrated around the nucleus and was not present in plasma membrane (Fig 3) None of the analysed cells expressing SacMC5R displayed EGFP presence around the plasma membrane The absence of SacMC5R from plasma membrane may explain why 125I-labelled NDP–MSH did not show any specific binding with this receptor
Membranes containing SacMC3R were used to test the binding properties of the endogenous melanocortin peptides
of human origin and the synthetic ligands The high-affinity ligand 125I-labelled NDP–MSH was used for saturation analysis Competition binding analysis was performed using NDP-MSH, a-MSH, b-MSH, c1-MSH, ACTH(1–17), ACTH(1–24), MTII and HS024 as competitors Figure 4 shows saturation and competition curves for SacMC3R The Kdand Kivalues obtained from these experiments are shown in Table 1, which also includes results for the human MCRs (published previously, see Table 1) for comparison, tested with the same methodological approach Results show that125I-labelled NDP–MSH binds to single saturable site on SacMC3R with an affinity very similar to that of HsaMC3R The affinity of NDP–MSH in competition experiments was slightly lower than for the human ortho-logue a-MSH and b-MSH had similar affinities (1.8 and 2.4-fold higher, respectively) to HsaMC3R, while c-MSH
Trang 6showed a 7.5-fold lower affinity than HsaMC3R Of note is
that both the 1–17 and 1–24 truncated versions of ACTH
showed considerably higher affinities for this receptor than
for the human MCRs, except for HsaMC1R ACTH(1–17)
had an 8.7-, and ACTH(1–24) had a 21.6-fold higher
affinity The synthetic ligands MTII and HS024 bound to SacMC3R with approximately the same affinities as shown for the HsaMC3R
HEK293 cells expressing SacMC3R were also tested in a cAMP assay in order to determine the ability of these
Fig 1 The amino acid sequence alignment of MCRs was made using CLUSTALW 1.8 Putative transmembrane (TM) regions are marked with lines Black boxes show identical amino acid positions, conserved amino acids are marked with grey boxes Abbreviations: Hsa, human; Dre, zebrafish; Gga, chicken; Sac, dogfish The accession numbers and Latin names are listed in Materials and methods Putative glycosylation sites in the N-terminal region are boxed A site with reduced potency of glycosylation in the N-terminal sequence of SacMC5R, as predicted using the
NETNGLYC 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/), is underlined.
Trang 7receptors to couple to G-proteins and induce the
accumu-lation of cAMP upon stimuaccumu-lation with a-MSH, ACTH and
c1-MSH The results are shown in Fig 5 These three
substances were able to activate the accumulation of cAMP
with considerable EC50 values a-MSH and ACTH had
EC50 values of 0.4 and 0.8 nmolÆL)1, respectively, while
c1-MSH displayed slightly lower potency (EC50¼ 2.6
nmolÆL)1)
The tissue distribution of the two dogfish MCRs was
determined by RT-PCR Total RNA from a number of
tissues, including different brain regions, was isolated from
an adult female dogfish and used to generate cDNA It
should be noted that the RT-PCR assay was not designed
for quantitative analysis The integrity of the mRNA was
confirmed by PCR using b-actin primers based on
consen-sus sequence for b-actin from different fish species PCR
product of the expected length of the b-actin was observed
on EtBr stained agarose gels from all of the different tissues
The sequence of the PCR product was determined and
confirmed to be a dogfish b-actin The results of the
RT-PCR for both receptors are shown in Fig 6 Bands on Southern blots were observed for both receptors in the hypothalamus, brain stem and telencephalon and a signal was also detected in optic tectum A band was seen in olfactory bulb for the SacMC3R The RNA preparation and cDNA synthesis were carried out twice, PCR and Southern blotting were repeated at least three times
Discussion
We describe the cloning of two MCRs in spiny dogfish Based on phylogenetic analysis we show that these receptors are orthologues of the MC3R and MC5R and were therefore designed as SacMC3R and SacMC5R Presence
of three MCRs in dogfish (together with previously cloned SacMC4R) suggests that the full repertoire of MCRs is likely to have been present prior to the radiation of the gnathostomes The MCRs that have been cloned previously and characterized from fish include all five mammalian subtypes except the MC3R subtype The only known MC3R in fishes is zebrafish MC3R [15,35] which has still not been characterized from either a pharmacological or an anatomical perspective The presence of a SacMC3R in chondrichthyans, which diverged prior to the split of ray-finned fishes and tetrapods, suggests that this receptor was created and took on important functions very early in vertebrate evolution and is likely to be found in most vertebrate species, despite not being found in the teleost Fugu The phylogenetic analysis presented in Fig 2, indeed, assigns high bootstrap values to the SacMC4R cloned by us previously [30], and puts both the SacMC3R and SacMC5R
in positions that are in agreement with the split of cartilaginous fishes, confirming that these are the most
ancient MCRs described so far It is interesting to note that SacMC3R and SacMC5R show very high sequence identity with the other orthologues from corresponding MCR subgroups This is remarkable because cartilaginous fishes diverged from the lineage leading to mammals over 450 million years ago Both receptors exhibit structural charac-teristics typical for the mammalian MCRs, including the presence of highly conserved subgroup-specific amino acid positions, short extra- and intra-cellular loops, and short divergent C and N termini (Fig 1) Moreover, many of the regions within the transmembrane (TM) bundle that have been suggested to be crucial for the binding and activation of the receptor [36,37], such as the TM2 and TM3, have 88–92% and 89–96% amino acid identity between the dogfish and human MC3R and MC5R orthologues, respect-ively It should be mentioned that we were not able to pick up
Fig 2 Phylogenetic analysis of MCRs using full-length amino acid
sequences The consensus tree was generated by using maximum
parsimony analysis ( MEGA 2.2) The numbers at the nodes indicate the
percentage of bootstrap replicates Abbreviations: Hsa, human; Dre,
zebrafish; Gga, chicken; Sac, dogfish Tru, Fugu The cloned dogfish
MC3R and MC5R are underlined Dogfish MC receptors are in bold.
Fig 3 Microscope images of CHO cells transiently expressing SacMC3R (left) and SacMC5R (right) The fluorescent images were obtained by illumination at 488 nm Arrows indicate plasma membrane localiza-tion of the receptor–EGFP fusion proteins.
Trang 8either MC1R or MC2R sequences from the libraries using our probes Comparison of already known Fugu and zebrafish sequences has revealed that these subtypes have diverged more than other subtypes as they have only 52–53% and 46–47% amino acid identity, respectively, with the human orthologues This could be the reason why we were not able to detect such genes during our screening The pharmacological characterization of the SacMC3R shows that it has slightly higher affinities for a-MSH and b-MSH, but a lower affinity for c-MSH as compared with the human orthologue ACTH showed, however, much higher affinity (21-fold) for SacMC3R than the human counterpart, the ACTH peptides indeed had the highest affinity to the SacMC3R among the natural peptides This
is particularly interesting as previous findings on other MCR subtypes in non-mammalian species indicate the same The MC1R, MC4R and MC5R in Fugu have higher affinity for ACTH-derived peptides than a-, b-, and c-MSH Our unpublished results on characterization of the chicken MCRs suggest similar preference to ACTH-derived peptides Our new results on SacMC3R provide additional evidence for the hypothesis that we presented earlier [15] that ACTH could be the original ligand for MCRs in lower vertebrates This hypothesis suggests that the subtype-dependent characteristics of the MCR subtypes such as the relatively high potency of c-MSH to MC3R, the slightly higher affinity for b-MSH and very low affinity of c-MSH for MC4R, were developed later during the evolution of vertebrates This could mean that there was
an increased specification of the roles of the different subtypes in mammals, as compared with lower vertebrates
It is also interesting in this context to note that the difference in the affinity of c-MSH and a-MSH for SacMC4R is not as high as for the human counterparts The second messenger results also show that ACTH has good ability to activate the receptor, while a-MSH and c-MSH had slightly lower potency It seems clear, therefore, that even though c-MSH does not have the same import-ance for SacMC3R as it does for human MC3R, c-MSH has still a clear preference for MC3R over MC4R in the spiny dogfish Taken together, it is important to note that there seems to be clear evidence for subtype specificity at the different MCRs in cartilaginous fishes, while this specifica-tion is more prominent in mammals It is also notable that
Fig 4 Saturation curves with Scatchard plots and competition curves
for SacMC3R expressed in HEK293 cells The saturation curves (left)
were obtained with 125 I-labelled NDP–MSH and the figure shows total
binding (j) and binding in the presence of 2 l M cold NDP–MSH (m).
The lines represent the computer-modelled best fit of the data
assu-ming that ligands bound to one site The competition curves (right) for
NDP–MSH (s), a-MSH (m), b-MSH (h), c 1 -MSH (d), ACTH(1–24)
(j), ACTH(1–17) (,), MTII (*) and HS024 (r) were obtained by
using a fixed concentration of 0.6 n M125I-labelled NDP–MSH and
varying concentrations of the unlabelled competing peptide.
Table 1 K d and K i values (mean ± ± SEM) obtained from the saturation and competition curves, respectively, for melanocortin peptide analogues of the dogfish (Sac) and human (Hsa) MC3 and MC4 receptors ND, Not determined.
Ligand
SacMC3 (nmolÆL)1)
HsaMC3a (nmolÆL)1)
SacMC4d (nmolÆL)1)
HsaMC4a (nmolÆL)1)
125
I-labelled NDP–MSH (K d ) 0.597 ± 0.007 0.412 ± 0.121 1.21 ± 0.44 1.78 ± 0.36 NDP–MSH (K i ) 0.927 ± 0.158 0.319 ± 0.064 1.50 ± 0.05 1.96 ± 0.39
b-MSH (K i ) 6.22 ± 0.44 15.1 ± 3.4 570 ± 278 387 ± 208 c-MSH (K i ) 55.5 ± 3.7 7.45 ± 2.55 1950 ± 70 51800 ± 12000
ACTH(1–17) (K i ) 1.61 ± 0.59 14.0 ± 4.5 b ND 419 ± 62 b
Data are fromaSchio¨th et al [44];bSchio¨th et al [45];cSchio¨th et al [46];dRingholm et al [30].
Trang 9the overall potency of the MSH peptides in the second
messenger assay is very high, which is in good agreement
with the fact that the core regions of POMC encoding these
peptides is very well conserved It should, however, be kept
in mind that we used human MSH peptides in our study
Even thought there is a high similarity between fish and
mammalian MSH sequences it would be interesting to test dogfish MSH peptides at the SacMCRs
The new receptors were characterized by their ability undergo proper folding and transport to the cell surface Using receptor–EGFP fusion we were able to show the presence of SacMC3R in the plasma membrane of CHO and HEK293 cells, while SacMC5Rs was retained in intracellular compartments of these cells It is not clear whether this unusual property of SacMC5R has a functional meaning There is however, another example among MCRs where the receptor is not transported to the cell membrane The MC2R
is arrested in the endoplasmatic reticulum of most cell lines This is likely to play a significant role in the functional properties of this receptor [38] as it seems to be functional only in cell types of adrenocortical origin MC2R is transported to the plasma membrane only in adrenal cortex cells in which it regulates steroid synthesis The sequence or structural determinants of receptors responsible for this phenomenon is, however, not known, but is being studied further in our laboratory It has been shown for some GPCRs, that removal of glycosylation signals may alter the proper transport of receptor to plasma membrane [39,40] Analysis of SacMC5R sequence using N-linked glycosyla-tion predicglycosyla-tion software revealed a reduced potency for one
of the glycosylation sites at the middle of N terminus of SacMC5R, which is present in all other MCRs (Fig 1) SacMC5R also has Trp instead of Arg324 in a position in the C-terminal region which is highly conserved for all known MCRs, and this may influence the structural properties of SacMC5R and subsequently its transport to the membrane
Fig 5 Generation of cAMP in response to a–MSH (m), ACTH(1–24)
(j) and c 1 –MSH (h) for the SacMC3R expressed in HEK293 cells.
Untransfected cells showed no adenylate cyclase activity in response to
ligands (data not shown) The cAMP assay was performed in duplicate
and repeated at least twice for each receptor subtype.
A
B
Fig 6 Expression of the SacMC3R (A), SacMC5R (B) mRNA as determined by RT-PCR Autoradiographs of Southern blots, hybridized with gene specific probes are shown The tissues, controls and expected sizes
of the PCR products are denoted at the top of each panel OT, optic tectum; HT, hypotha-lamus; BS, brain stem; Tel., telencephalon;
CB, cerebellum; OB, olfactory bulb.
Trang 10As a consequence, neither SacMC5R–EGFP fusion protein
nor the native SacMC5R showed any detectable binding or
stimulation of cAMP synthesis It is interesting to speculate
that this could be a specific regulatory feature of some of the
MCRs, such as MC2R and MC5R, that coincidentally are
found in tandem on the chromosomes for both fish
(zebrafish, Fugu), chickens and mammals If our hypothesis
that ACTH is the original ligand for the MCR family is true,
suggesting that MC2R may have maintained the original
function of the MCR system to a greater extent than the
other receptors, it is possible to speculate that such a
cell-specific regulatory element could have been maintained also
by MC5R in the spiny dogfish while it was lost in higher
vertebrates
Anatomical charting revealed that SacMC3R is expressed
in different brain regions including the hypothalamus, brain
stem and telencephalon, olfactory bulbs and optic tectum
but not in the cerebellum The presence of SacMC3R in the
brain is not surprising, as the mammalian MC3R is found
primarily in the brain The mammalian MC3R has also
been found in some peripheral tissues such as placenta and
gut MC3R has however, not been found in the brain of
chicken but only in the adrenal gland [41] The exact
functional role of MC3R is not fully known but it is clear
that it has important role in regulation of the energy balance
(as does MC4R) Moreover, it has been suggested to serve
as an auto-receptor for MC4R in regulation of the energy
balance [11] The fact that the MC3R gene is missing in a
thriving vertebrate such as Fugu could suggest that this
receptor may be less important than the other MCRs, and
that fact could fit well to the idea that the MC3R has a
supporting role for the very important MC4R The
expression patterns of SacMC3R and SacMC4R are fairly
similar, being in the central regions of the brain and not in
the cerebellum This could support the idea that these two
receptors may have an integrated physiological role The
presence of MC3R and its clear expression at several sites in
the spiny dogfish may indicate that the absence of this
receptor in Fugu is a feature of that particular species This
should also be considered in light of the fact that c-MSH
sequence is absent from the POMC gene of teleost fishes
including both Fugu and zebrafish The presence of a
c-MSH seems however, not be a prerequisite for the
presence of a MC3R, as this receptor is found in zebrafish,
despite that the zebrafish POMC does not have c-MSH
Unfortunately, there is no data on pharmacology of
zebrafish MC3R, the only known MC3R from lower
vertebrates missing the sequence of c-MSH
We have found that SacMC5R is expressed exclusively in
the brain tissue of the spiny dogfish In mammals and
chickens MC5R is expressed in a wide range of tissues
including peripheral ones [5,42] Our previous results on
different teleost fish species such as the zebrafish, Fugu,
goldfish and trout indicated localization of MC5R in brain
and some peripheral tissues including eye, ovary and
gastrointestinal tract The functional role of MC5R is much
less well defined than that of the other subtypes Results
from the teleost, chicken and many mammals indicate that
it took on both central and peripheral roles early in
vertebrate evolution and it was thus a little surprising that
this receptor was not found in peripheral tissue in the spiny
dogfish The more restricted anatomical distribution of this
receptor in the spiny dogfish could be mean that this receptor has a role only in some specific cell types which may be reflected by the fact that it cannot be transported to the membrane in cell types that work well for most GPCRs The central expression pattern could also mean that MCRs
in general had roles predominantly in the central nervous system in early vertebrate evolution considering that all three MCRs in the dogfish are found mainly in the brain The MCR family is interesting for understanding how closely related genes have gained their specific functions It is quite remarkable that despite relatively high conservation of the primary sequence elements, the five MCRs seem to have very diverse functional roles, at least in mammals Accord-ing to our previous suggestion about evolution of MC system, these three receptors share common ancestral genes and arose by both genome and local duplications [15] It is thus possible that the receptors have similar functions and were localized in similar tissues shortly after they emerged, and that their evolution displays gradual partitioning or complementation of their ancestral functions This is in agreement with the recently developed degeneration com-plementation (DDC) model, which suggests that the functions of a new gene reflects the partitioning of ancestral functions, rather than the evolution of new functions [43] According to this, the divergence and functional specificity for the MCRs were acquired later in evolution, partially in fish but to a greater extent in the tetrapod lineages
In conclusion, we have shown that the spiny dogfish has both MC3R and MC5R We have performed thorough characterization of these receptors from different aspects including phylogenetic analysis, expression, intracellular localization, pharmacology and tissue distribution MC3R
is the first receptor of its subtype that has been characterized
in lower vertebrates It is now almost certain that appearance of the most important elements of the MC system dates to before the radiation of gnathostomes, early
in vertebrate evolution
Acknowledgements
We thank E T Larsson, Uppsala University for expert assistance during dissection of the dogfish J Klovins was supported by the Wenner-Gren foundation and by a Marie Curie Fellowship of the European Community programme Improving the Human Research potential and the Socio-Economic Knowledge Base under contract number HPMF-CT-2002–01786.¢ The studies were supported by the Swedish Research Council (V.R., medicine), the Swedish Society for Medical Research (S.S.M.F.) and Svenska La¨karesa¨llskapet.
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