Báo cáo khoa học: Cloning 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 toc-MSH ppt

Cloning 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 Klovins 1,2 , Tatjana Haitina 1 , Aneta Ringholm 1 , Maja Lo¨ wgren 1 , Davids Fridmanis 2 , Maija Slaidina 2 , Susanne Stier 1 and Helgi B. Schio¨th 1 1 Department of Neuroscience, Unit of Pharmacology, Uppsala University, Sweden; 2 Biomedical Research and Study Centre, University of Latvia, Riga, Latvia We report t he cloning and characterization of two melano- cortin receptors (MCRs) from the spiny dogfish (Squalus acanthias) (Sac). Phylo genetic analysis s hows t hat t hese shark r eceptors are orthologues of the MC3R and MC5R subtypes, sharing 65% and 70% overall amino acid identity with the human coun terparts, respectively. The SacMC3R was expressed and pharmacologically characterized in HEK293 cells. T he radioligand b inding results show that this receptor has high affinity for adrenocorticotropic hormone (ACTH)-derived peptides while it has comparable affinity for a-andb-melan ocyte stimulating hormone (MSH), and slightly lower a ffinity for c-MSH when compared with the human orthologue. ACTH(1–24) has high potency in a second-messenger cAMP assay w hile a-andc-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 S acMC5R 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 t he hypo- thalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum o f 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: A CTH; dogfish; melanocortin; MSH; POMC. The melanocortin r eceptors (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-andc-melanocyte stimulating hormones (MSH) and adrenocorticotropic hormone (ACTH), a ll o f 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 m ammals, MC1R i s expressed in melanocyte s and has an important role in determining skin a nd hair pigmentation [4]. Expression of MC1R is also detected in other skin cell t ypes and in a number of peripheral tissues, including leukocytes, where it mediates the broad anti- inflammatory actions of the m elanocortin p eptides [5]. MC2R is expressed in adrenal cortex, where it mediates the effects o f ACTH on s teroid 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 r egulation o f energy h omeostasis. M C4R is one of the best-characterized monogenic factors o f 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 e nergy balance, mice deficient in one of these receptors display s eparate phenotypes [10,11]. Knockout of MC3R causes an  50–60% increase in adipose mass, but these mice do not become significantly overweight [12]; t hey do, however, exhibit reduction in locomotory behaviour suggesting reduced energy expendi- ture. Mice lacking both MC3R a nd MC4R become significantly heavier than MC4R knockout mice [11]. MC3R and MC4R have also differences in pharmacology: MC3R has a unique preferen ce for c-MSH among different subtypes of MCRs in mammals. MC5R is expressed in a number of human peripheral tissues, including adr enal gland, ad ipocytes 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. H aitina contributed e qually to this work. (Received 2 5 May 2004, revised 27 August 2004, accepted 22 September 2004) Eur. J. Biochem. 271, 4320–4331 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04374.x the e xception of its participation in exocrine function, regulating sebaceous gland secretion in m ice [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 intak e 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 zeb rafish, 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, w ere 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]. Moreove r, there are some interesting differences in the pharmacology between teleosts and m ammals. A CTH s eems to play a more prominent role for the MCRs in Fugu as compared with a-MSH, which has t he highest a ffinity to the four MSH binding MCRs in mammals. The spiny dogfish ( Squalus acanthias)isasharkthathas 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 p rior t he split of teleosts, a nd can thus be considered in evolutionary term as ÔolderÕ 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 t eleost fishes however, are believed to have undergone another tetra- ploidization [22] followed by subsequent large g enome reorganizations. The teleosts have thus gained and lost many gene copies, which may reflect the different numbers of MCR genes i n the diffe rent teleost species. P OMC 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 m ammals. M oreover, two copies o f 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 a nd degenerate, e ither missing the c-MSH core motif [27,28] or even t he 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 a n additional M SH 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, intrace llular 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 ÔlowerÕ 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 w as homogenized in lysis buffer [ 100 m M EDTA, 10 m M Tris, 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 1 M NH 4 Ac 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 1 00 ng of dogfish genomic DNA. PCR was performed using the Taq DNA polymerase (Invitrogen) i n a reaction volume of 20 lL, containing 200 l M dNTP, 2 0 m M Tris/HCl pH 8.4, 50 m M KCl, 2 m M MgCl 2 and 20 pmol each primer. 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 followed b y 25 cycles of 30 s at 94 °C, 40 s at 50 °C, 1 min at 72 °C, w ith a final extension of 5 min at 72 °C. 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 t ransformed into TOP10 cells (TOPO TA-cloning k it, Invitrogen). Sequencing r eactions were performed using the A BI PRISM Big Dye Terminator c ycle sequencing kit according to the manufactures recommendations and a nalysed o n ABI PRIZM-310 or 3100 Automated Sequencers (Applied Biosystems). Sequences were compiled andalignedusingthe SEQMAN program from DNAstar (Lasergene). Sequences were compared against database assemblies using BLASTN and BLASTX . Screening of a phage genomic library and isolation of full-length gene The spiny do gfish 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 Ó FEBS 2004 MC3 and MC5 receptors of spiny dogfish (Eur. J. Biochem. 271) 4321 Petri dishes (roughly three genome equivalents) and grown at 37 °C for 8 h . Plaques were transferred to the nylon transfer membrane (Amersham Biosciences, U ppsala, Swe- den) and denatured for 1 min in 0.5 M NaOH; 1.5 M NaCl, then neutralized for 5 min in 1 M Tris/HCl pH 8.0, 1.5 M 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 [ 32 P]CTP[aP] using the Megaprime Labeling System (Amersham Biosciences) and used as a probe. Hybridization was carried out at 65 °C in 25% (v/v) formamide (Merck Eurolab AB), 6· NaCl/Cit, 10% (w/v) dextran sulfate (Amersham Biosciences), 5· Denhardt’s s olution, and 0.1% SDS overnight. The filters were washed five times in 0.2 · NaCl/Cit containing 0.1% ( w/v) S DS for 1 h at 65 °C. After exposure to autoradiographic films, one positive signal was chosen fo r further s election. T he procedure of s election and hybridiza- tion was repeated until a single phage clone was i solated (three times). The phage were g rown according to the protocol and phage DNA was isolated using k-purification kit ( Qiagen). Phage were sequenced as described above and found t o c ontain M C5R-like sequence, which was different from the probe s equence. We sequenced about 200 bp upstream and 100 bp downstream f rom of t he 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 37 °C. DNA from each digest was purified and ligated at 14 °Covernight with T4 DNA ligase and precipitated. ÔNested Õ PCR reaction was used to perform inverse PCR. Two s ets 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¢ (positio ns 692–710) and one pa ir of outer primers 5¢-CCATAC TGTATTTGTTAC-3¢ (positions 817 –831) and 5¢-ACT TACCAGCATGTCTGC-3¢ (positions 253–270). The primers w ere placed at the 3 ¢ and 5 ¢ parts of cloned fragment in o rientation 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: 95 °C for 15 s, 60 °C for 20 s, and 72 °C 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: 95 °Cfor15s,68°C for 20 s, and 72 °Cfor 2 min for 40 cycles. The products of second PCR r eactions were isolated from agarose gel, and w ere cloned into pCRII vector using TOPO cloning kit and transformed i nto 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 w ith other known MCRs was made using CLUSTALW 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), M C4R (NM_005912), MC5R (XM_008685); chicken Gallus gallus (Gga) MC1 R (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 an d 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 u sing 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 purifi ed prior t o ligation i nto modified p CEP expression vector [34] containing an enhanced green fluor- escence protein (EGFP) coding gene fused to the C terminus of the receptor gene. All constructs w ere 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 mod ified Eagle’s m edia: F-12 Nutrient Mixture (DMEM/F-12) (1 : 1) with G lutaMAX 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 )1 geneticin G-418 (Gibco) i n a humidified atmosphere of 95% a ir and 5% CO 2 (v/v) at 37 °C. 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 d ogfish receptors were plated on coverslips a nd grown for 48 h prior t o fi xation. The cells were washed twice with ice-cold 1· NaCl/P i before fixation i n 4% (v/v) parafor- maldehyde containing 1· NaCl/P i for 15 min. The cover- slips were then washed twice with NaCl/P i and mounted in NaCl/P i containing 1 lgÆmL )1 DAPI (4,6-diamino-2-phe- nylindole). Cells were examined immediately using a Zeiss Axioplan 2 microscope. The digitally acquired images were 4322 J. Klovins et al. (Eur. J. Biochem. 271) Ó FEBS 2004 captured and analysed using OPENLAB software (Improvi- sion). A t least three slides were analysed for each receptor and cell type. From all cells expressing re ceptor–EGFP fusion, only the cells displaying single DAPI stained nucleus were considered for analysis. Fifteen randomly s elected cells were analysed from each slide. Radioligand binding assay HEK293 cells expressing the d ogfish receptors were harves- ted from plates and resuspended in binding buffer c om- posed of 25 m M Hepes, 2.5 m M CaCl 2 ,1m M MgCl 2 and 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 a t 220 g and membranes were collected from the supernatant b y c entrif- 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 c arried out with serial dilutions of 125 I-labelled (Nle4, D -Phe7)-a-MSH (NDP-MSH), labelled by the Chloramine-T method. Non-specific binding was determined in the presence of 1 l M unlabeled NDP-MSH. Competition e xperiments were performed with constant 0.6 n M concentration of 125 I-labelled NDP–MSH and serial dilutions of competing u nlabelled ligands: N DP-MSH, a-MSH, b-MSH, c 1 -MSH, ACTH(1–24), ACTH(1–17), MTII, and HS024 (Neosystem). The membranes were collected by fi ltration on g lass fibre filters, Filtermat A (Wallac), using a TOMTEC Mach III cell harvester (Orange, CT, USA). The filters were washed with 5 mL per well o f 50 m M Tris/HCl pH 7.4 and dried a t 5 0 °C. MeltiLex A scintillator sheets (Wallac) were melted on dried filters and radioactivity was counted with automatic Microbeta counter 1450 (Wallac). Binding assay was performed i n duplicate from a t l east three independent experiments. Non-transfected cells did not show any specific binding with 125 I-labelled NDP-MS H. The results were analysed with PRISM 3.0 software package (GraphPad). cAMP assay Cyclic adenosine m onophosphate production was d eter- mined on semistable HEK293 cells expressing the target MCR. A con fluent layer of cells was incubated for 3 h with 2.5 lCiÆmL )1 [ 3 H]ATP (specific activity 29 CiÆmmol )1 ; Amersham Biosciences). Cells were c ollected, washed with buffer containing 137 m M NaCl, 5 m M KCl, 0.44 m M KH 2 PO 4 ,4.2m M NaHCO 3 ,1.2m M MgCl 2 Æ6H 2 O, 20 m M Hepes, 1 m M CaCl 2 ,and10m M glucose, pH adjusted to 7.4. All reaction components w ere r esuspended in t he above-mentioned buffer containing 0.5 m M isobutyl- methylxanthine (IBMX; Sigma). Resuspended cells were incubated for 10 min at 37 °C. Stimulation reaction was performed for 20 min a t 3 7 °C i n a final volume of 150 lL containing  2 · 10 5 cells and various concentrations of a a-MSH, c 1 -MSH, and ACTH(1–24). After incubation, cells were centrifuged and 200 lLof0.33 M perchloric acid was added to pellets to lyse the cells. The cells were frozen, thawed and spun down. Lysate (200 lL) was a dded to Dowex 50 W-X4 resin columns (Bio-Rad), washed previ- ously with 2· 10 mL H 2 O. As an internal standard, 750 lL 0.33 M perchloric acid containing 0.5 nCiÆmL )1 [ 14 C]cAMP (Amersham Biosciences) was added to each column. Columns wer e washed with 2 mL H 2 OtoremoveATP, which was collected in scintillation vials to estimate the amount o f unconverted [ 3 H]ATP. Four millilitres o f 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 0.1 M imidazole) and the cAMP was transferred onto the alumina column using 10 mL H 2 O. cAMP was e luted from alumina column with 4 mL 0.1 M imidazole and collected into scintillation vials to w hich 7 mL of scintillation fluid was a dded. 3 Hand 14 C were counted on Tri-carb liquid scintillation beta counter. The amount of obtained [ 14 C]cAMP was expressed as a fraction of total [ 14 C]cAMP ([ 14 C]cAMP/total[ 14 C]cAMP) and was used to standardize [ 3 H]cAMP to column effic iency. Results were calculated as the perce nt of total [ 3 H]ATP (obtained as a sum of [ 3 H]ATP from first column and [ 3 H]cAMP from second column) to [ 3 H]cAMP and used to d etermine 50% effective concentra- tion (EC 50 ) values by non-linear r egression using PRISM 3.0 software. All experiments were performed in duplicate and repeated three times. RT-PCR and Southern analysis Total RNA was isolated f rom 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 w as 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 wa s exposed to a further treatment with 1 unit ÆlL )1 RNase-free DNaseI (Roche) for 10 min, fol- lowedbyheatinactivationofDNasefor5minat70°C. Due to absence of publicly available sequence of dogfish b-a ctin gene, we designed primers for c onservative parts of b-a ctin genes from other vertebrate species: 5¢-C GTGCG TGACATCAAGGAGA-3¢ and 5¢-CTTGGTAGGGCTC CCAGCAC-3¢. The primers w ere tested on genomic DNA, the s equence o f 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 n g of total RNA as a template and genomic DNA as a positive control. Messenger RNA w as reverse transcribed u sing the 1st Strand cDNA Synthesis kit (Amersham Biosciences). The quality of cDNA was t ested in PCR with the a bove- mentioned b-actin primers. The cDNA produced was u sed as a template for PCR with t he specific primers for the receptor genes (see below). The conditions for PCR on receptor genes were: 1 min initial denaturation, 95 °Cfor 15 s, 55 °Cfor20s,72°C for 1 min for 30 cycles and ending with 5 m in at 72 °C, 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 Ó FEBS 2004 MC3 and MC5 receptors of spiny dogfish (Eur. J. Biochem. 271) 4323 5¢-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 0.4 M NaOH. The filters were hybridized with a random-primed 32 P-labelled, receptor specific probe. P robes w ere g enerated 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 65 °C 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· Na Cl/Cit, 0.1% (w/v) SDS for 1 h at 65 °C and exposed to autoradiography fi lm (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 p robes 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 a pparent double bands, PCR products were denatured in 3% (v/v) formaldehyde, 25% (v/v) formamide solution and separated on 1.4% agarose g el using Mops buffer (20 m M Mops, 2 m M sodium acetate and 1 m M EDTA); this re sulted 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 BLASTN and BLASTX . Three identical clones displayed high identity to the MCRs, with the highest score to t he chicken MC3R. This sequence was used as a p robe for high stringency screening of a dogfish genomic phage library. Three positive phages were selected an d these were found to include the entire sequence of a full-length MCR gene. This sequence was however, different from probe seq uence and showed highest s imilarity to the zebrafish MC5aR when compared with the GenBank database. Repeated s creening 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 fragme ntation. PCR products obtained from ClaIandKpnI fragments contained a MC3R s equence and we were able to extend the sequence in bo th 3¢ and 5 ¢ directions to overlap the st op codon and initiating ATG codon, respectively. The genes were designed S. acanthias (Sac) MC3R and MC5R according to their sequence similarity with previously cloned MCR sfrom 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 i s shown in Fig. 2, r epresenting a consensus t ree. The dogfish M C3R sequence h as highest identity to the MC3 subtype r eceptors (6 3–75%), w hile 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 o f the new receptors as subtypes of the M C3R and MC 5R, r espectively. 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 i nto 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 w hile transfection of SacMC5R did not, despite repeated attempts. SacMC5R has an alternative ATG start c oding upstream of t he one we considered to be the start codon. We reconstructed the clone and added an additional sequence i ncluding 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 t he first wild-type MCR clone in any s pecies that has not been readily expressed to g ive binding with 125 I-labelledNDP–MSH (except the MC2R). In order to control t he expression levels and cell surface location of the protein, we fused an EGFP gene to the C terminus of the receptor g ene for both the clones. The constructs were transfected in HEK293 ce lls and semistable c ell lines were created. Moreover, transient expression of hybrid receptors in CHO-1 cells was p erformed 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, s howed st aining concentrated around the nucleus and w as not present i n plasma m embrane (Fig. 3). None of the analysed cells expressing SacMC5R displayed EGFP presence a round the plasma membrane. The absence of SacMC5R from plasma membrane may explain why 125 I-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 125 I-labelled NDP–MSH was used f or saturation analysis. Competition binding analysis was performed using NDP-MSH, a-MSH, b-MSH, c 1 -MSH, ACTH(1–17), ACTH(1–24), MTII and HS024 as competitors. Figure 4 shows saturation and competition curves for SacMC3R. The K d and K i values 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. R esults show that 125 I-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 1 -MSH 4324 J. Klovins et al. (Eur. J. Biochem. 271) Ó FEBS 2004 showed 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 r eceptor than for the human MCRs, except f or HsaMC1R. A CTH(1–17) had an 8 .7-, and A CTH(1–24) had a 21.6-fold higher affinity. The synthetic ligands MTII and HS024 bound to SacMC3R w ith approximately the same affinities as shown for the HsaMC3R. HEK293 ce lls expressing SacMC3R were a lso tested in a cAMP assay in order to determine the ability of t hese Fig. 1. The amino acid sequence alignment of MCRs was made using CLUSTALW 1.8. Putative transmem brane (T M) regions are marked with lines. Black boxes show identical amino acid p ositions, 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 predicte d using the NETNGLYC 1.0 Server ( http://ww w.cbs.dtu.dk/se rvices/NetN Glyc/), is underlined. Ó FEBS 2004 MC3 and MC5 receptors of spiny dogfish (Eur. J. Biochem. 271) 4325 receptors to couple to G-prote ins and induce the accumu- lation of cAMP upon stimulation with a-MSH, ACTH and c 1 -MSH. The results are shown in Fig. 5. These three substances were able to activate the a ccumulation of cAMP with considerable EC 50 values. a-MSH and ACTH had EC 50 values of 0.4 and 0.8 nmolÆL )1 , respectively, while c 1 -MSH displayed s lightly lower potency (EC 50 ¼ 2.6 nmolÆL )1 ). The tissue distribution of the two do gfish MCRs was determined by RT-PCR. Total RNA from a number of tissues, including different brain regions, was isolated from an adult female dogfish a nd 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 t o b e a dogfish b-actin. T he results of t he RT-PCR for both receptors are sho wn in Fig. 6. Bands on Southern blots were observed for b oth 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 c DNA synthesis were carried out twice, PCR and Southern blotting were repeated at least three times. Discussion We d escribe the cloning of two MCRs in spiny dogfish. Based on phylogenetic analysis we show that these receptors are orthologues of the M C3R and MC5R and w ere 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 h ave 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 pharmaco logical or an anatomical perspective. The presence of a SacMC3R in chondrichthyans, which diverged prior to the split of ray- finned fishes and t etrapods, suggests that this receptor was created and took on important functions very early in vertebrate evolution a nd is likely to be found in most vertebrate species, despite not being found in t he teleost Fugu. The phylogenetic analysis p resented in Fig. 2, indeed, assigns high bootstrap values to the SacMC4R cloned by u s previously [30], and puts both the SacMC3R and SacMC5R in positions that are in agreement w ith the split of cartilaginous fishes, confirming that these are the m ost Ôancie ntÕ 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 re ceptors exhibit structural c harac- teristics typical for the mammalian MCRs, inclu ding 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 r eceptor [36,37], such a s t he 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 p ick 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. A bb reviations: H sa, human; Dre, zebrafish;Gga,chicken;Sac,dogfishTru,Fugu. The cloned dogfish MC3R and MC5R are underlin ed. Dogfish MC receptors are in bold. Fig. 3. Microscope images of CHO cells transiently expressing S acMC3R (left) and SacMC5R (right). The fl uorescent images were obtained by illumination at 488 nm. Arrows indicate plasma membrane localiza- tion of the r eceptor–EGFP fusion proteins. 4326 J. Klovins et al. (Eur. J. Biochem. 271) Ó FEBS 2004 either 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 t hey 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 t o 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 t han the human counterpart, the ACTH peptides indeed had the highest affinity to the SacMC3R among the natural peptides. This is particularly interesting as p revious findings on o ther MCR subtypes in non-mammalian species indicate the same.TheMC1R,MC4RandMC5RinFugu 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 A CTH- derived peptides. Our new results on SacMC3R provide additional evidence for the hypothesis that we presented earlier [15] that ACTH c ould be the ÔoriginalÕ 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. T his could mean that there was an increased specification o f the roles of the different subtypes in mammals, as compared with ÔlowerÕ vertebrates. It is also interesting i n this c ontext to note t hat t he difference in the affinity of c-MSH a nd a-MSH for SacMC4R is not as high as for t he 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 thou gh 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 c artilaginous fishes, while this specifica- tion is more prominent i n mammals. It is also notable that Fig. 4. Saturation curves with Scatchard plots and competition curves for SacMC3R expressed in HEK293 ce lls. The saturation curves (left) were obtained with 125 I-labelled NDP–MSH and the figure sho ws 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 (*) a nd HS024 (r) were obtained by using a fixed conc entration of  0.6 n M 125 I-labelled NDP–MSH and varying concentrations of t he unlabe lled 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)andhuman(Hsa) M C3 and M C4 receptors. ND , Not determined. Ligand SacMC3 (nmolÆL )1 ) HsaMC3 a (nmolÆL )1 ) SacMC4 d (nmolÆL )1 ) HsaMC4 a (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 a-MSH (K i ) 11.6 ± 1.2 21.2 ± 5.3 198 ± 42 522 ± 122 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(1–24) (K i ) 1.52 ± 0.23 32.8 ± 6.7 ND 755 ± 151 ACTH(1–17) (K i ) 1.61 ± 0.59 14.0 ± 4.5 b ND 419 ± 62 b MTII (K i ) 15.9 ± 1.0 34.1 ± 4.4 c ND 6.60 ± 0.82 c HS024 (K i ) 7.38 ± 0.49 15.1 ± 3.0 ND 0.341 ± 0.089 Data are from a Schio ¨ th et al. [44]; b Schio ¨ th et al. [45]; c Schio ¨ th et al. [46]; d Ringholm et al. [30]. Ó FEBS 2004 MC3 and MC5 receptors of spiny dogfish (Eur. J. Biochem. 271) 4327 the overall potency of the MSH peptides in the second messenger assay is very high, which is in good agreement with the fact t hat the core regions of POMC encoding these peptides is very well conserved. It should, however, be kept in mind that we used human MSH p eptides 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 i n the plasma membrane of CHO and HEK293 cells, while SacMC5Rs was r etained 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 a drenal cortex cells in which it r egulates 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 r eceptor to plasma m embrane [39,40]. Analysis of SacMC5R sequence u sing N-linked g lycosyla- tion prediction software r evealed a reduced potency for one of the glycosylation sites at the middle of N terminus of SacMC5R, which is p resent in all other MCRs (Fig. 1). SacMC5R also has Trp instead of Arg324 in a position in the C-terminal region which is h ighly conserved for all known MCRs, and this may influence t he 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 S acMC3R expressed in HEK293 cells. Untransfected cells showed no adenyl ate 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 a s determined by RT-PCR. Autoradiographs of Southern blots, hybridized with gene specific probes a re shown. The tissues, controls and expected sizes of the PCR products are denoted at the top of each panel. OT, o ptic tectum; HT, hypotha- lamus; BS, b rain stem; Tel., telencephalon; CB, cerebellum; OB, olfactory bulb. 4328 J. Klovins et al. (Eur. J. Biochem. 271) Ó FEBS 2004 As a consequence, neither SacMC5R–EGFP fusion protein nor the n ative SacMC5R s howed any detectable bind ing 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 M C2R and MC5R, that coincidentally are found in tandem on the chromosomes for both fish (zebrafish, Fugu), chickens and mammals. If our hypothesis that A CTH is the ÔoriginalÕ 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 ÔhigherÕ 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 t he adrenal gland [41]. The e xact functional r ole 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 ha s been sugge sted to serv e 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 th e other MCRs, and that fact could fit well to the idea that the MC3R has a ÔsupportingÕ 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 inte grated 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 p resence of a c-MSH seems however, not be a prerequisite for t he presence of a MC3R, as this receptor is found in zebrafish, despite that t he zebrafish P OMC does not have c-MSH. Unfortunately, there is no data on pharmacology of zebrafish MC3R, the only known MC3R f rom l ower vertebrates missing the sequence of c-MSH. We have found that SacMC5R is expressed exclusively in the brain tiss ue 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 a nd tro ut ind icated localiz ation of MC5R in brain and some p eripheral 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 m any mammals indicate that it took on both central and peripheral r oles early i n vertebrate evolution and it was thus a little s urprising that this recepto r was not found in peripheral t issue in the spiny dogfish. The more restricted a natomical d istribution o f this receptor in the spiny dogfish c ould 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 t o themembraneincelltypesthatworkwellformostGPCRs. The central expression pattern could a lso mean t hat 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 remark able that despite relatively high conservation of the primary sequence elements, the five MCRs seem to have very diverse functional roles, at least in mammals. A ccord- ing t o our pr evious suggestion about evolution o f MC system, these three receptors share common ancestral genes and arose by both genome a nd local duplications [15]. I t 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 i s 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 t he evolution o f new functions [43]. According to this, the divergence and functional specificity for the MCRs were acquire d later in evolution, partially in fish but to a greater extent in the tetrapod lineages. In conclusion, we have shown that the sp iny dogfish h as both MC3R and MC5R. We have performed thorough characterization of the se 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 ÔlowerÕ vertebrates. It is now almost certain that appearance of the most important elements of th e 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 o f the dogfish. J. Klovins w as supported by the Wenner-Gren foundation and by a Marie Curie Fellowship of the European Community programme ÔImproving the Human Research potential and the Socio-Economic Knowledge BaseÕ under contract number HPMF-CT-2002–01786.¢ The studies were supported by the Swedish Research Council (V.R., m edicine), the Swedish S ociety for Medical Research (S.S.M.F.) and Svenska La ¨ karesa ¨ llskapet. References 1. Fredriksson, R., Lagerstrom, M.C., Lundin, L.G. & Schioth, H.B. (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272. 2. Schioth, H.B. (2001) The physiological role of melanocortin receptors. Vitam. Horm. 63, 195–232. 3. Gantz, I. & Fong, T.M. (2003) The melanocortin system. Am. J. Physiol. Endocrinol. Metab. 284, E468–E474. 4. 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Larhammar, D (2001) Cloning and characterization of the guinea pig neuropeptide Y receptor Y5 Peptides 22, 357–363 35 Logan, D.W., Bryson-Richardson, R.J., Pagan, K.E., Taylor, M.S., Currie, P.D & Jackson, I.J (2003) The structure and evolution of the melanocortin and MCH receptors in fish and mammals Genomics 81, 184–191 Ó FEBS 2004 MC3 and MC5 receptors of spiny dogfish (Eur J Biochem 271) 4331 36 Schioth,... regions of ray-finned fish POMC Gen Comp Endocrinol 116, 164–177 27 Amemiya, Y., Takahashi, A., Dores, R.M & Kawauchi, H (1997) Sturgeon proopiomelanocortin has a remnant of gammamelanotropin Biochem Biophys Res Commun 230, 452–456 28 Dores, R.M., Smith, T.R., Rubin, D.A., Danielson, P., Marra, L.E & Youson, J.H (1997) Deciphering posttranslational processing events in the pituitary of a neopterygian fish: cloning . Cloning 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 Klovins 1,2 ,. 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 125 I-labelled. vector and t ransformed into TOP10 cells (TOPO TA -cloning k it, Invitrogen). Sequencing r eactions were performed using the A BI PRISM Big Dye Terminator c ycle sequencing kit according to the manufactures