Presence of melanocortin (MC4) receptor in spiny dogfish suggests an ancient vertebrate origin of central melanocortin system Aneta Ringholm 1 , Janis Klovins 1 , Robert Fredriksson 1 , Natalia Poliakova 1 , Earl T. Larson 1 , Jyrki P. Kukkonen 2 , Dan Larhammar 1 and Helgi B. Schio¨th 1 Department of Neuroscience, Division of 1 Pharmacology and Division of 2 Physiology, Uppsala University, Uppsala, Sweden We report the cloning, expression, pharmacological char- acterization and tissue distribution of a melanocortin (MC) receptor gene in a shark, the spiny dogfish (Squalus acanth- ias) (Sac). Phylogenetic analysis showed that this receptor is an ortholog of the MC4 subtype, sharing 71% overall amino acid identity with the human (Hsa) MC4 receptor. When expressed and characterized by radioligand binding assay for the natural MSH (melanocyte-stimulating hormone) pep- tides a-, b-, and c-MSH, the SacMC4 receptor showed pharmacological properties very similar to the HsaMC4 receptor. Stimulation of SacMC4 receptor transfected cells with a-MSH caused a dose-dependent increase in intracel- lular cAMP levels. The SacMC4 receptor has Ala in position 59 where all other cloned MC receptors have Glu. We con- firmed that this was not due to individual polymorphism and subsequently mutated the residue ÔbackÕ to Glu but the mutation did not affect the pharmacological properties of the receptor. SacMC4 receptor mRNA was detected by RT- PCR in the optic tectum, hypothalamus, brain stem, telen- cephalon and olfactory bulb but not in cerebellum or in peripheral tissues. This study describes the first characteri- zation of an MC receptor in a cartilaginous fish, the most distant MC receptor gene cloned to date. Conservation of gene structure, pharmacological properties and tissue dis- tribution suggests that this receptor may have similar roles in sharks as in mammals and that these were established more than 450 million years ago. Keywords: GPCR; melanocortin; melanocyte-stimulating hormone; receptor. The melanocortin (MC) receptor family consists of five subtypes, termed MC1-MC5 in mammals. The melanocortin system is unique because in addition to possessing endo- genous agonists, it also has an endogenous antagonist, Agrp. The agonists are the pro-opiomelanocortin (POMC) clea- vage products a-, b-andc-melanocyte-stimulating hormone (MSH) and adrenocorticotrophic hormone (ACTH). POMC has been used extensively as a model for studies of the evolution of neuropeptides and it is well established that the sequence of a-MSH is highly conserved between mam- mals and fishes [1,2]. The centrally expressed MC4 receptor received great attention by many researchers within the field of central regulation of food intake after it was Ôknocked-outÕ in mice [3], causing over-eating and obesity. Centrally administered MC4 receptor agonists have the ability of reducing appetite [4,5], while MC4 receptor antagonists are very effective in increasing food intake in rodents, both in acute and long-term studies [6–8]. These findings make the MC4 receptor very interesting for pharmacological research and drugs against this receptor may become helpful for people suffering from disorders like obesity and anorexia. The MC3 receptor has been found exclusively in the brain and it is involved with regulation of the energy balance [9]. The MC5 receptor is primarily expressed in a wide range of peripheral tissues and also in the mammalian brain [10]. The MC2 receptor subtype mediates the function of ACTH but does not bind a-, b-orc-MSH. This receptor has been found only in the adrenal gland. The MC1 receptor has a role in pigmentation and it also mediates the anti-inflammatory action of MSH [11]. Our understanding of the mechanisms of appetite regu- lation and metabolism in mammals is increasing rapidly. Many peptides such as neuropeptide Y, orexins, Agrp (agouti-related peptide), ghrelin and MSH are involved in the regulation of energy balance by binding to GPCRs in the central regions of the brain [12,13]. However, in ÔlowerÕ vertebrates, the molecular mechanisms for appetite regula- tion of these peptide binding receptors are poorly known. The spiny dogfish (Squalus acanthias)isasharkanda member of cartilaginous fishes (also called chondrichth- yans). Chondrichthyans are characterized by cartilaginous skeletons, placoid scales and pelvic claspers (in males) [14]. They arose from the Agnatha, the jawless fishes, in the late Silurian period, approximately 420–430 million years ago. The spiny dogfish became popular as a research model among chondrichthyans and it has been extensively studied due to its peculiar rectal gland, an organ that regulates the secretion of chloride [15]. Cardiovascular control has also been widely studied in the spiny dogfish [16]. Several peptides have been characterized in the dogfish, including Correspondence to H. B. Schio ¨ th, Department of Neuroscience, Biomedical Center, Box 593, 75 124 Uppsala, Sweden. Fax: + 46 18 51 15 40, E-mail: helgis@bmc.uu.se Abbreviations: ACTH, adrenocorticotrophic hormone; GPCR, G-protein coupled receptor; MC, melanocortin; MSH, melanocyte- stimulating hormone; NDP-MSH, [Nle4, D -Phe7]a-MSH; NTS, nucleus of the solitary tract; POMC, pro-opiomelanocortin. (Received 18 August 2002, revised 7 November 2002, accepted 18 November 2002) Eur. J. Biochem. 270, 213–221 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03371.x peptide PYY [17] and gastrin/cholecystokinin-like peptides [18] but very few G-protein-coupled receptors (GPCRs) have been cloned in this species. Recently, we cloned one MC4 receptor and two MC5 receptors in a teleost fish, the zebrafish, Danio rerio [19]. The results showed high conser- vation in primary structure and pharmacology of these MC receptors as compared with the mammalian ones. The teleosts belong to the bony fishes which arose later in the evolution, after a split from cartilaginous fishes [20–22]. In this paper, we report the cloning, expression, phar- macological characterization and tissue distribution of a MC4 receptor in a cartilaginous fish, the spiny dogfish. This receptor is evolutionarily the most distant MC receptor from mammals cloned so far. Materials and methods Extraction of genomic DNA Spiny dogfish genomic DNA was extracted from muscle tissue of four different animals captured in the North Sea (Hambergs Fisk, Uppsala, Sweden). The muscle tissue (about 1 g) was homogenized in the lysis buffer [100 m M EDTA (TitriplexÒ, Merck, Stockholm, Sweden), 10 m M Tris (VWR International, Stockholm, Sweden) and 1% SDS (Scientific Imaging Systems, Eastman Kodak Com- pany)] and centrifuged for 5 min at 11 500 g. The superna- tant was purified first with saturated phenol (BDH Laboratory Supplies, Poole, UK), then with phenol/chlo- roform/isoamyl alcohol (1 : 1 : 1) (BDH Laboratory Sup- plies), and chloroform (KEBO Laboratory AB, Stockholm, Sweden). The DNA was precipitated with propan-2-ol and NH 4 Ac (2 : 1) and centrifuged for 30 min at 11 500 g. Finally the DNA was washed with 70% ethanol and centrifuged again. The DNA pellet was vacuum-dried and re-suspended in water. Cloning Degenerate primers based on conserved parts of the human, rat, mouse and chicken MC receptors were used in different pair-wise combinations. One hundred nanograms of dogfish genomic DNA was used as template in a low stringency PCR, using the AmpliTaq DNA polymerase Stoffel Frag- ment (Perkin Elmer, Roche, Langen, Germany) in a reaction volume of 20 lL, containing 4 m M dNTP, 1 · Stoffel buffer (Perkin Elmer), 60 m M MgCl 2 ,20pmol each primer and two units DNA polymerase, Stoffel Fragment. Touch-down PCR was performed, starting with an initial 1 min 95 °C denaturation, followed by 22 cycles of 45 s at 94 °C, 45 s at 52 °Cto42°C, 90 s at 72 °C. This was followedby25cyclesof30sat94 °C, 40 s at 50 °C, 1 min at 72 °C, with a final extension of 5 min at 72 °C. The PCR gave a product of the expected size, 600 bp. The 5¢ primer’s sequence was CAY TCN CCN ATG TAY TTY TT and the 3¢ primer ATN ACI GAR TTR CAC ATD AT. Y denotes C or T, R denotes A or G, D denotes, A, G or T, I denotes inosine and N denotes any base. The PCR product was purified from a 1% agarose gel using Gel Extraction Kit (Qiagen, Hilden, Germany). Re-amplification was performed by denaturating for 1 min, followed by 45 s at 95 °C, 45 s at 50 °C, 1 min at 72 °C and 40 cycles with a final extension of 72 °C for 5 min. An aliquot of the re-amplified product was cloned into a Topo-vector and transformed into TOP10 cells (TOPO TA-cloningÒ Kit, Invitrogen Corporation, Stockholm, Sweden). PCRs were performed on a GeneAmpÒ PCR System 9700 (Perkin Elmer). Screening of a phage genomic library and isolation of full-length gene The spiny dogfish phage-DNA library made in kGEM-11 (Promega, Falkenberg, Sweden) with Escherichia coli KW251-stem (Promega) as a host, was kindly supplied by Anders Johnsen, Rigshospitalet, Denmark [23]. The inserts in the phages were about 15–22 kb. Approximately 50 000 phages were plated out on each of 12 different 15-cm Petri dishes (roughly three genome equivalents) and grown at 37 °C for 8 h. Plaques were lifted over to the nylon transfer membrane (Amersham Biosciences, Uppsala, Sweden) and denatured for 1 min in Soak I solution (0.5 M NaOH + 1.5 M NaCl), then neutralized for 5 min in Soak II solution [1 M Tris/HCl (pH 8.0) + 1.5 NaCl] and equilibrated in 2 · saline sodium citrate buffer (NaCl/Cit). The filters were dried and used for hybridization. The MC receptor-like sequence PCR product was labeled with 32 P using Mega- prime Labeling System (Amersham Biosciences) and used as a probe. Hybridization was carried out at 65 °Cin25% formamide (Merck Eurolab AB, Stockholm, Sweden), 6 · NaCl/Cit, 10% dextran sulfate (Amersham Biosci- ences), 5 · Denhardt’s solution, and 0.1% SDS overnight. Thefilterswerewashedfivetimesin0.2· NaCl/ Cit + 0.1% SDS for 1 h at 65 °C. After exposure to autoradiographic films, one positive signal was selected for further selection. The procedure of selection and hybridiza- tion was repeated until a single phage was isolated (three times). The phage was grown according to the protocol and the phage DNA was isolated using k-purification kit (Qiagen, Hilden, Germany). The phage was confirmed to be true positive and used as template for sequencing to obtain the full-length receptor. We sequenced about 200– 300 bp both upstream and downstream of the coding region which did not include any introns. Sequencing Sequence determinations were performed using ABI PRISM Dye Terminator cycle sequencing kits according to the manufacture’s recommendations (Applied Biosys- tems, Stockholm, Sweden) and analyzed on an ABI PRISM-310 Automated Sequencher (Applied Biosystems). Sequences were compiled and aligned in Sequencher (Gene Codes). Sequences were compared with National Center for Biotechnology Information (NCBI) database using BlastX. Alignments and phylogenetic analysis The full-length sequence of the SacMC4 receptor was aligned with other MC receptor sequences using ClustalW (1.7) software [24] and edited manually after visual inspec- tion. The sequences (see Figs 1 and 2) were retrieved from GenBank and have accession codes as follows: Homo sapiens (Hsa) MC1 (NM_002386), MC2 (NM_000529), 214 A. Ringholm et al. (Eur. J. Biochem. 270) Ó FEBS 2003 MC3 (XM_009545), MC4 (NM_005912), MC5 (XM_008685), Mus musculus (Mmu) MC4 (AF201662), MC5 (NM_013596), Gallus gallus (Gga) MC4 (AB012211), MC5 (AB012868), Danio rerio (Dre) MC4 (AY078989), MC5a (AY078990), MC5b (AY078991) receptors. The Squalus acanthias (Sac) MC4 receptor gained the accession number AY169401. Its phylogenetic tree was generated by using the MEGA v.2.1 software [25] applying maximum parsimony methods. Human cannabinoid 2 receptor (hCB2) (accession code S36750) was used as an out-group. A bootstrap consensus tree assessing the robustness of the nodes was made with 100 replicates. Cloning into expression vector The entire coding region of the receptor sequence was amplified with Pfu Turbo DNA polymerase (Stratagene, AH Diagnostic, Stockholm, Sweden). Specific primers containing HindIII and XhoI sites were used under the following conditions: 60 s at 95 °C for one cycle, then 30 s at 95 °C, 30 s at 53 °Cand70sat72°C for 35 cycles. The PCR fragments were purified by QIAquick PCR Purifica- tion Kit (Qiagen) and digested by HindIII and XhoI. The full-length receptor sequence was re-purified and ligated into a modified pCEP4 expression vector containing the CMV promotor [26]. The new construct was sequenced and found to be identical to the genomic clone. Site-directed mutagenesis The A59E mutation was introduced into the SacMC4 receptor coding sequence by PCR. Two complementary oligonucleotides were designed to contain the required mutation. The SacMC4-A59E primers were 5¢-CAG CCT CTT GGA AAA TAT TTT GGT CAT TG and 3¢-GAC CAA AAT ATT TTC CAA GAG GCT GAA GAT G. The primers were hybridized to opposite strands of the receptor gene and the complete coding sequence was amplified. The end primer was complementary to 3¢ or 5¢ depending on which mutagenesis primer was used (forward or reverse). The two products were used as templates and linked together in a second PCR, in which only the end- primers were used. Transfection HEK 293-EBNA cells were transiently transfected with the constructs using FuGENE TM Transfection Reagent (Boeh- ringer Mannheim, Roche, Stockholm, Sweden) diluted in OptiMEM medium (Invitrogen Corporation) according to the manufacturer’s recommendations. The cells were grown in DMEM/Nut Mix F-12 with 10% fetal bovine serum (Invitrogen Corporation) containing 0.2 m ML -glutamate (Invitrogen Corporation) and 250 lg/ml G-418 (Invitrogen Corporation), 100 U penicillin and 100 lgstrepto- mycinÆmL )1 (Invitrogen Corporation). Radioligand binding Intact transfected cells were re-suspended in 25 m M Hepes buffer (pH 7.4) containing 2.5 m M CaCl 2 ,1m M MgCl 2 and 2 gÆL )1 bacitracin. Saturation experiments were performed in a final volume of 100 lL for 3 h at 37 °C and carried out with serial dilutions of 125 I-labelled [Nle4, D -Phe7]a-MSH (NDP-MSH). Non-specific binding was defined as the amount of radioactivity remaining bound to the cells after incubation in the presence of 2000 n M unlabelled NDP-MSH. Competition experiments were performed in a final volume of 100 lL. The cells were incubated in the well plates for 3 h at 37 °Cwith 0.05 ml binding buffer in each well containing a constant concentration of 125 I-labelled NDP-MSH and appropriate concentrations of competing unlabelled ligands NDP-, a-, b-, c-MSH or HS014. The incubations were terminated by filtration through Filtermat A, glass fiber filters (Wallac Oy, Turku, Finland), which had been presoaked in 0.3% polyethylenimine, using a TOMTEC Mach III cell harvester (Orange, CT, USA). The filters were washed with 5.0 mL of 50 m M Tris/HCl (pH 7.4) at 4 °Cand dried at 60 °C. The dried filters were then treated with MeltiLex A (Perkin Elmer) melt-on scintillator sheets and counted with Wallac 1450 (Wizard automatic Microbeta counter). The results were analyzed with a software package suitable for radioligand binding data analysis ( PRISM 3.0, Graphpad Software, San Diego, CA, USA). Data were analyzed by fitting to formulas derived from the law of mass action by the method generally referred to as computer modeling. The binding assays were per- formed in duplicate wells and repeated three times. Nontransfected HEK293-EBNA cells did not show any specific binding for 125 I-labelled NDP-MSH. NDP-MSH was radio-iodinated by the chloramine T method and purified by HPLC. NDP-, a-, b-, c-MSH or HS014 were purchased from Neosystem, France. cAMP assay The experiments were performed essentially as described in earlier [27]. Briefly, the cells were incubated for 2 h with 5 lCiÆmL )1 [8- 3 H]adenine (Amersham Biosciences, Upp- sala, Sweden) and then washed and harvested in a medium composed of 137 m M NaCl, 5 m M KCl, 0.44 m M KH 2 PO 4 , 4.2 m M NaHCO 3 ,1.2m M MgCl 2 ,20m M Hepes, 1 m M CaCl 2 and 10 m M glucose, pH adjusted to 7.4. The pelleted cells were resuspended in the medium as above containing 0.5 m M isobutylmethylxantine (Sigma) and preincubated for 10 min at 37 °C before adding to appropriate concen- trations of the stimulant (a-MSH) in 96-well plates (Nunc, VWR International, Stockholm, Sweden). After an addi- tional 10 min of incubation in 37 °C with the hormone, the reactions were stopped by rapid centrifugation at 1000 g for 1 min, removal of supernatants, and addition of 200 lL ice-cold 0.33 M perchloric acid per well. The plates were frozen down to )20 °C, thawed, and the cell debri were spun down (1000 g for 10 min). The extent of conversion of [ 3 H]ATP to [ 3 H]cAMP was determined by Dowex/alumina sequential chromatography [28] [ 14 C]cAMP (Amersham Biosciences, Uppsala, Sweden) tracer in 0.75 mL 0.33 perchloric acid (about 1000 cpm) was added to each column together with the samples. The ATP/ADP and cAMP fractions were dissolved in an appropriate volume of scintillation cocktail (Optiphase Hisafe 3, Wallac, Turku, Finland) and analyzed in a b-counter. The conversion to [ 3 H]cAMP was calculated as a percentage of total eluted Ó FEBS 2003 Dogfish melanocortin 4 receptor (Eur. J. Biochem. 270) 215 [ 3 H]ATP and was normalized to the recovery of [ 14 C]cAMP. The cAMP assay was performed in triplicates and repeated twice for each receptor. RT-PCR and Southern analysis Fresh periphery tissues (muscle, heart, liver, kidney, rectal gland, spiral valve, eye, colon) and several brain regions (optic tectum, hypothalamus, brain stem, telencephalon, cerebellum and olfactory bulb) were collected from spiny dogfish. The total RNA was isolated according to the RNeasy Mini Kit (Qiagen) protocol. The RNA prepara- tions were then DNaseI treated for 20 min at room temperature using the RNase-Free DNase Set protocol (Qiagen). Absence of genomic DNA in all RNA prepara- tions was confirmed in PCR reaction using 10–100 ng of total RNA as a template. Messenger RNA was reverse transcribed using the 1st Strand cDNA Synthesis kit (Amersham Pharmacia Biotech) with a reverse primer specific to dogfish MC4 receptor. The produced cDNA was used as a template for PCR with the specific primers for the receptor gene. The conditions for PCR were: 1 min initial denaturation, then 30 s at 95 °C, 40 s at 55 °C, 60 s at 72 °C for 35 cycles and finished by 5 min at 72 °C, using Taq polymerase (Invitrogen Corporation). The following prim- ers were used: 5¢-AGG CAC TTA ACG GCC CCG GA-3¢ and 5¢-AGA GCG AGG CCA TGA GGG CG-3¢ giving the expected size of the PCR product of around 300 bp. The PCR products were analyzed on a 1% agarose gel. The DNA products on the gel were transferred to nylon filters over night using 0.4 M NaOH. The filter was hybridized with a random-primed 32 P-labeled, species and receptor specific probe (Megaprime kit, Amersham Biosciences) at 65 °C in 25% formamide, 6 · NaCl/Cit, 10% dextran sulfate, 5 · Denhardt’s solution and 0.1% SDS over night. Thefilterwasthenwashedfivetimesin0.2· NaCl/ Cit + 0.1% SDS for 1 h at 65 °C and exposed to autoradiography film (Amersham Biosciences). Due to the appearance of double bands (not shown), the PCR products were denatured in 3% formaldehyde, 25% formamide solution and separated on 1.4% agarose gel using Mops buffer (20 m M Mops, 2 m M sodium acetate and 1 m M EDTA) resulting in a single band. The original double band had different relative intensity in the ethidium bromide staining and the hybridization signal. It is thus likely that the double band was caused by an extra signal of single- stranded DNA. As positive control for the Southern blot, the genomic DNA was used in PCR reaction. Sequence analysis of this PCR product confirmed the presence of SacMC4 receptor sequence. Water was used as negative control. The RT-PCR reactions and Southern blotting were performed three times. Results Several 600-bp PCR products were cloned and about 25 clones were sequenced of which two identical clones showed high identity to the MC receptors. After library screening, a single phage was confirmed to contain a MC receptor-like protein of 331 amino acids. Among the mammalian and chicken MC receptor subtypes, our clone had highest identity to the MC4 receptors (69–71%) and was designated as the SacMC4 receptor. The protein sequence of the spiny dogfish receptor are shown in Fig. 1, aligned with the human MC receptors, the GgaMC4 and GgaMC5 recep- tors, the MmuMC4 receptor and the recently cloned DreMC4, DreMC5a and DreMC5b receptors [19]. The SacMC4 receptor has 71% identity to the HsaMC4, 69% to the GgaMC4 and 75.8% to the DreMC4 receptor. Phylogenetic analysis was performed using the maximum parsimony method (MP) (Fig. 2). The MC3, MC4 and MC5 receptors are more similar to each other in amino acid sequence comparison, than the MC2 and MC1 receptors are (see also previous analysis in [19]). The Hsa and MmuMC4 receptors are most similar to each other, followed by the Gga- and DreMC4 receptors. As expected the SacMC4 branched out at basal to the DreMC4 receptor. As an out-group, we used the human cannabinoid 2 (hCB2) receptor. The SacMC4 receptor had an unusual amino acid in a very conserved region (TM1). All previously cloned MC receptors regardless of subtype, share the acidic amino acid Glu in position 59 in transmembrane (TM) region 1, whereas the new SacMC4 receptor surprisingly had Ala in this position (see Fig. 1). In order to investigate if this was a single amino acid polymorphism, we prepared genomic DNA from three additional individuals, ran PCR to generate an  490 bp fragment containing position 59, cloned it and sequenced. All the individuals had identical sequence in this region including the Ala in position 59. Subsequently we inserted Glu into position 59 by site- directed mutagenesis in order to enable pharmacological characterization (see below). The coding sequence of the genomic clone SacMC4 and mutant SacMC4-A59E receptors were transferred to the expression vector and control sequenced. The constructs were transiently expressed in mammalian cells and the receptors were tested in radioligand binding assay on intact cells using radioligand-binding assay. Figure 3 shows saturation and competition curves for these dogfish recep- tors. Table 1 shows the K d and the K i values obtained from saturation and competition analysis, respectively. Our results suggest that 125 I-labelled NDP-MSH bound to the SacMC4 receptor with indistinguishable affinity as com- pared with the Hsa- and DreMC4 receptors. Both the wild- type SacMC4 receptor and the SacMC4-A59E mutant receptor had also the same affinities for the endogenous peptides a-MSH, and the high potency synthetic ligand NDP-MSH, as compared to the Hsa- and DreMC4 receptors. c1-MSH had also the same affinity to the Hsa- and SacMC4 receptors. The SacMC4 receptor had how- ever, slightly lower affinity for b-MSH, as compared with the Hsa- and DreMC4 receptors. The synthetic compound HS014 had the same affinity for the Dre- and SacMC4 receptors, while it had 90-fold lower affinity for the HsaMC4 receptor. The binding profile for the mutant A59E receptor was not determined for the low affinity ligands b-, c1-MSH and HS014. In order to investigate if the SacMC4 and SacMC4-A59E receptors were able to influence intracellular cAMP after stimulation of a-MSH, we tested both receptors in a cAMP assay. The results are shown in Fig. 4. We found that both SacMC4 and SacMC4-A59E receptors responded to the stimulation by a-MSH with the same potency. These results 216 A. Ringholm et al. (Eur. J. Biochem. 270) Ó FEBS 2003 are in line with that which we have observed for the HsaMC4 receptor in response to a-MSH [11]. Non- transfected HEK293-EBNA cells, which showed no response to the a-MSH, were used as controls. Tissue distribution was determined by RT-PCR. The results of the RT-PCR are shown in Fig. 5. The SacMC4 receptor was expressed in five brain regions (Fig. 6) but not in any of the peripheral tissues. There were strong signals in the brain stem and the hypothalamus and slightly weaker signals in the optic tectum, olfactory bulb and telencepha- lon. However, it should be noted that the PCR assay was not designed for quantification. The experiments were performed three times and there were no qualitative differences between the runs. The integrity of the mRNAs was tested by using zebrafish and fugu based actin primers for RT-PCRs. We received a distinct product of the expected length for the cDNA from all the different tissues. This PCR product was confirmed to be dogfish actin by sequencing. Discussion We describe here the cloning of the first MC receptor in spiny dogfish. The phylogenetic analysis indicates that the new receptor is an ortholog of the MC4 receptor and we use thus the nomenclature SacMC4 receptor and the gene is entered in the gene database under this name. The SacMC4 receptor appears basal in the MC4 receptor phylogenetic cluster (see Fig. 2). The spiny dogfish belongs to the chondrichthyans, the cartilaginous fishes, which diverged prior to the split leading to ray-finned and lobe finned fishes. The sharks are thus more distant from mammals than the bony fishes, including zebrafish which we cloned earlier [19]. The phylogenetic positioning of the SacMC4 receptor is Fig. 1. Amino acid sequence alignment made using CLUSTALW (1.7) software and edited by manual inspection. TheSacMC4receptorservedasa master for the HsaMC1-5, MmuMC4, GgaMC4, GgaMC5, DreMC4, DreMC5a and DreMC5b sequences. The lines mark putative trans- membrane (TM) regions (according to [34]). The accession numbers are listed in Material and methods. Fig. 2. Phylogenetic analysis of the MC-receptor family using the full- length amino acid sequences. The tree was generated by maximum parsimony analysis ( PAUP 4.0). The human cannabinoid 2 receptor (hCB2) sequence was used to root the tree. The numbers above the nodes indicate percentage of bootstrap replicates. The accession numbers are listed in Material and methods. Ó FEBS 2003 Dogfish melanocortin 4 receptor (Eur. J. Biochem. 270) 217 thus in agreement with that of the cartilaginous fishes. This shows that the MC4 receptor originated before the radiation of gnathostomes. It is interesting that despite the fact that cartilaginous fishes diverged from the lineage leading to mammals over 450 million years ago, the SacMC4 receptor shows very similar pharmacological properties as the HsaMC4 receptor. The characteristics of the HsaMC4 receptor, such as relatively low affinity for c-MSH and a slightly higher affinity for b-MSH as compared with a-MSH, seem to be conserved. The ligand b-MSH, whose physiological roles are still obscure, also has a higher affinity for the SacMC4 receptor than a-MSH, which is in line with our previous results for the human and rat MC4 receptors [29,30]. This Fig. 3. Saturation binding with Scatchard plots (left) and competition curves for the SacMC4 and SacMC4-A59E receptors expressed in intact transfected cells. The figure shows competition curves (right) for 125 I-labelled NDP-MSH (m), a-MSH (j), c-MSH (h) and HS014 (d)tothe SacMC4 receptors, and the two first mentioned for the SacMC4-A59E receptor. The binding curves were obtained by using a fixed concentration of 2n M 125 I-labelled NDP-MSH and varying concentrations of the nonlabeled competing peptide. Lines represent the computer-modeled best fit of the data assuming that ligands bound to one-site. Table 1. K i and K d values (mean±SEM) obtained from competition and saturation curves, respectively, for melanocortin peptides analogs on SacMC4, SacMC4-A59E, DreMC4, HsaMC4 receptor transfected EBNA cells. Ligand SacMC4 (nmolÆL )1 ) SacMC4-A59E (nmolÆL )1 ) DreMC4 a (nmolÆL )1 ) HsaMC4 a (nmolÆL )1 ) 125 I-labelled NDP- MSH 1.21 ± 0.44 1.93 ± 0.30 2.39 ± 0.96 2.35 ± 1.18 NDP-MSH 1.50 ± 0.054 1.41 ± 0.070 3.35 ± 0.31 3.57 ± 0.30 a-MSH 198 ± 42 198 ± 28 243 ± 27 289 ± 29 b-MSH 570 ± 278 ND 163 ± 14 126 ± 15 c1-MSH 1950 ± 70 ND 2200 ± 550 3690 ± 260 HS014 368 ± 110 ND 493 ± 40 5.60 ± 0.22 a Data taken from Ringholm et al. [19]. ND, not done. 218 A. Ringholm et al. (Eur. J. Biochem. 270) Ó FEBS 2003 feature was also conserved in the zebrafish receptors (see Table 1) and our new results thus provide additional evidence for our speculation that b-MSH may have a specific and also an evolutionarily conserved role for this receptor subtype. HS014, an antagonistic substance, is a synthetic cyclic peptide and was developed to be selective for the human MC4 receptor [31]. This substance is the only one that has lower affinity for the SacMC4 receptor as compared with the human ones. It could be speculated that even though the ability of the receptors to bind the natural peptides is highly conserved, the 3D binding cavity of the SacMC4 receptor may not be as well conserved to fit this synthetic ligand. Moreover, our results also show that the SacMC4 receptor is a functional receptor and able to activate the Gs pathway when stimulated with a-MSH, in agreement with the other MC receptors from mammals and zebrafish. The SacMC4 receptor exhibits high sequence identity with the mammalian orthologs. It also displays several of the structural characteristics typical for the mammalian MC receptors such as conserved Cys in the first extra-cellular loop, lack of Pro in TM5, short extra-cellular and intracel- lular loops, and short divergent C- and N-terminals (see Fig. 1). This is in line with what we found for the DreMC receptors. The SacMC4 receptor sequence had however, Ala in position 59 in TM1. We found this remarkable as all the five MC receptor subtypes in all the species cloned so far have Glu in this position. TM1 is believed to contribute to the main binding region in MC receptors [32,33]. Some earlier studies suggested that this acidic and hydrophilic Glu may play an important role in the ligand binding [34] while other studies have indicated that this residue may not be participating in the ligand binding [35] for the mammalian receptors. It was possible that this was a mutation that was only found in the genome of the individual we cloned. We investigated if this residue was conserved in the SacMC4 receptor gene by sequencing additional three individuals. The results show that the missing Glu was not due to polymorphism. The putative importance of the unique amino acid exchange in the SacMC4 receptor was investi- gatedbymutatingtheAlaÔbackÕ to Glu. We investigated the mutant receptor both regarding binding of NDP-MSH and a-MSH, and the ability to increase production of adenylate cyclase when stimulated with a-MSH. The SacMC4 and the SacMC4-A59E had indistinguishable K d , K i and EC 50 -values, suggesting that this exchange is not important for the pharmacological profile of the receptor. It is difficult to speculate why this Glu59 has been replaced in the SacMC4 receptor but cloning of other MC4 receptors from other distant species as well as other MC receptors from Fig. 4. Generation of cAMP in response to a-MSH forSacMC4 (h)and SacMC4-A59E (j) receptors in intact transfected cells. Each point represents the mean ± SEM. Untransfected HEK293-EBNA (m) cells showed no adenylate cyclase-activity in response to a-MSH. The cAMP assaywasperformedintriplicatesandrepeatedtwiceforeachreceptor. Fig. 5. Expression of SacMC4 receptor mRNA as determined by RT- PCR on total RNA preparations from spiny dogfish tissues. The tissues and the controls are denoted at the top of the figure. The figure shows 4-h exposure on an X-ray film after hybridization with the SacMC4 receptor probe. The size (bp) of the ladder is shown on the right. The PCR reactions and the hybridization were performed three times with qualitatively similar results. Fig. 6. Side view of spiny dogfish (Squalus acanthias) brain. Shaded sections represent regions expressing the SacMC4 receptor. Dashed lines indicate incisions made to divide brain regions for collecting tis- sues for RT-PCR. OB, olfactory bulb; Tel, telencephalon; OT, optic tectum; Hyp, hypothalamus; Cb, cerebellum; BS, brain stem. Ó FEBS 2003 Dogfish melanocortin 4 receptor (Eur. J. Biochem. 270) 219 dogfish may provide additional information about this unusual replacement. The results show that the SacMC4 receptor was expressed in olfactory bulb, telencephalon, optic tectum, hypothala- mus, and brain stem but not in cerebellum (Fig. 6). The presence of MC4 in the hypothalamus is not surprising. This receptor is expressed in hypothalamus in all species inves- tigated and is important in the regulation of appetite. In the telencephalon, especially the limbic system, the MC4 receptor is possibly playing a role in the communication between the melanocortin system and the serotonergic system and stress effects on appetite [36]. The presence of SacMC4 receptor in the olfactory bulb may suggest a role in chemosensory mechanisms, possibly those associated with feeding [37,38]. In the brainstem, the SacMC4 receptor could be involved in visceral afferent signals from the gut via the nucleus of the solitary tract (NTS). The NTS could be sending signals to the hypothalamus from the gut to regulate energy balance [39]. a-MSH immunoreactivity has been found in the spotted dogfish (Scyliorhinus canicula) [40], showing that perikarya were largely located in the hypothalamus with projections running to various places in the diencephalon. The SacMC4 receptor was found both in the hypothalamus and diencephalon, indicating that this receptor is expressed in brain regions of sharks where a-MSH can be found. The SacMC4 receptor was expressed only in the brain but not in peripheral tissues. This is in agreement with the mammalian MC4 receptors that have only been found in brain tissue [8,41]. The DreMC4 receptor was also found in the brain but rather surprisingly also in the eye, gastro- intestinal tract and ovaries. In chicken, the MC4 receptor is expressed in a wide variety of peripheral tissues, including the heart, adrenal glands, ovaries, testes, spleen, adipose tissues and eye, as well as the brain [42,43]. In our previous report, we speculated that the expression pattern of the MC4 receptor in mammals had become more confined to central regions, as compared with zebrafish and chicken [19]. Our new results from the dogfish indicate rather that the MC4 receptor had a predominant and important function in the CNS very early in vertebrate evolution and later became more widely expressed in zebrafish and chicken. In conclusion, our data show that the MC4 receptor had already arisen before the radiation of gnathostomes. The spiny dogfish receptor clone will facilitate cloning in cyclostomes and amphioxus as well as additional gnathos- tomes. Our results provide an understanding of the evolutionary origin of the MC receptor system and enhance further studies on their many important physiological roles. The high degree of conservation of MC receptors in teleosts and cartilaginous fish may indicate that the MC receptors arose even before the appearance of vertebrates. The conservation of the pharmacological properties and tissue distribution pattern could demonstrate an early develop- ment of a mechanism where a peptide binds to a GPCR for central regulation of the energy balance. Acknowledgments We thank Flemming Cornelius, University of Aarhus, Denmark for providing dogfish tissues and Anders Johnsen, Rigshospitalet, Den- mark for providing a dogfish phage library. 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