General and Comparative Endocrinology 161 (2009) 13–19

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General and Comparative Endocrinology

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Modeling the evolution of the MC2R and MC5R genes: Studieson the cartilaginous fish, Heterondotus francisci

Andrea Baron, Kristopher Veo, Joseph Angleson, Robert M. Dores *

University of Denver, Department of Biological Sciences, Olin Hall 102, 2190 E. Iliff, Denver Colorado 80210-5212, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 August 2008Revised 20 November 2008Accepted 21 November 2008Available online 7 December 2008

Keywords:MelanocortinsMelanocortin receptorsEvolutionCartilaginous fish

0016-6480/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.ygcen.2008.11.026

* Corresponding author. Fax: +1 303 871 3471.E-mail address: rdores@du.edu (Robert M. Dores).

Comparative studies support the hypothesis that the proliferation of melanocortin receptor genes (MCRs)in gnathostomes corresponds to the 2R hypothesis for the radiation of gene families in Phylum Chordata.This mini-review will initially focus on the distribution of MCRs in cartilaginous fish and the relationshipbetween the shark MC5R gene and the proposed ancestral MC5R/2R gene. This section will be followed bythe results of recent studies on the features of the ligand binding site common to all melanocortin recep-tors. These data will provide the background for a set of hypotheses to explain the unique ligand selec-tivity of the MC2 receptor in teleosts and tetrapods.

� 2008 Elsevier Inc. All rights reserved.

1. Background

The evolution of the chordates has been punctuated by at leasttwo genome duplication events that have shaped the radiation ofchemical communication systems in this phylum (Ohno et al.,1968; Holland et al., 1994). As a result, paralogous genes are presentwithin gnathostome vertebrate gene families that have either di-vided the function of the ancestral gene (subfunctionalization), haveevolved novel functions (neofunctionalization), or in some caseshave become pseudogenes (Force et al., 1999). This array of optionscan be seen for the opioid/orphanin gene family (Dores et al., 2002).Whereas, proenkephalin, prodynorphin, and proorphanin each pro-duce related, yet distinct opioid-like neuropeptides that function inthe central nervous system as inhibitory neurotransmitters, thefourth member of this family, proopiomelanocortin, produces notonly the opioid, b-endorphin, but also the structurally related mela-nocortin peptides, ACTH,a-MSH,b-MSH, and in some speciesc-MSHand d-MSH (Dores and Lecaude, 2005). The unifying feature of themelanocortins is the core sequence, HFRW; a linear internal motifessential for ligand specific activation of melanocortin receptors(MCRs). The MCRs, in turn, are members of the rhodopsin A familyof G-protein Coupled Receptors (GPCRs) and the rhodopin familyhas also proliferated as a result of the chordate genome duplicationevents (Fredriksson et al., 2003; Schioth and Fredriksson, 2005;Cone, 2006). GPCRs are single chain polypeptides that contain sevenmembrane spanning alpha-helical regions that are linked by extra-

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cellular and intracellular domains (Devi, 2005). In tetrapod verte-brates five distinct MCR genes are present, MC1R, MC2R, MC3R,MC4R, and MC5R (Cone, 2006). In mammals these receptors mediatepigmentation (MC1R), adrenocorticoid biosynthesis (MC2R), meta-bolic homeostasis (MC3R & MC4R), and exocrine secretion (MC5R;Cone, 2006). Given this background, this mini-review will considertwo issues: understanding the phylogeny of the MCRs through stud-ies on cartilaginous fish, and observations on the ligand selectivity ofthe MC2 receptor in tetrapods and teleosts.

2. Comparative studies on MCRs

Does the MCR gene family conform to the 2R hypothesis for theradiation of gene families in chordates? That is, did an ancestralgene undergo two genome duplication events to yield first twoparalogous genes (first duplication event) and then four paralogousgenes (second duplication event; Holland et al., 1994)? Since thereis considerable evidence that the MC2R and MC5R genes were theresult of a gene duplication event at some point in the early radi-ation of the gnathostomes (Fredriksson et al., 2003; Klovins et al.,2004a), it is possible to propose a scheme for the radiation of theMCR gene family that conforms to the 2R hypothesis (Fig. 1a). Insupport of this hypothesis, two distinct MCR genes (MCa & MCb)have been cloned from a lamprey (Haitina et al., 2007a; Fig. 1b).Among the gnathostomes, different combinations of MCR geneshave been detected in the genomes of bony fish. For example,MC1R, MC2R, MC4R, and MC5R have been detected in the fugugenome (Klovins et al., 2004a), and an apparent MC3R orthologhas been detected in the genome of the zebrafish along with ortho-

Gnathostomes MC1R MC5/2R MC3R MC4R

2nd Genome

Duplication

’’RCM’RCMsnahtangALamprey

1st Genome

Duplication

Protochordates Ancestral

MCR

Protochordates Lamprey Cartilaginous Fish Bony Fish Tetrapods

MC5R? MC5R+ MC5R+

MC4R+ MC4R+ MC4R+

MC3R+ MC3R+ MC3R MC3R+

MCb+ MC2R* MC2R*

( MCR) MCa+ MC1R MC1R+ MC1R+

a

b

Fig. 1. Modeling the evolution of melanocortin receptors in phylum chordata. (a) Hypothetical scheme for the radiation of the melanocortin receptors during the evolution ofthe chordates. Ancestral MCR—predicted MCR gene in protochordates. MCR’ and MCR’’—predicted MCR genes formed after the first genome duplication event. MC5R/MC2R—predicted ancestral gene for MC5R and MC2R. (b) Phylogeny of melanocortin receptors in extant vertebrates. (MCR) refers to the predicted ancestral MCR gene inprotochordates. (_____) indicates a gene that is predicted to have been secondarily lost. Within the bony fish an MC3R gene is found in the zebrafish genome, but not in thefugu genome. The prediction would be that the ancestral teleosts had an MC3R gene that was secondarily lost in the fugu lineage. (+) indicates a receptor that can activated byeither ACTH or MSH. (�) indicates a receptor that can only be activated by ACTH. (?) indicates that ligand selectivity has not been determined for the receptor.

14 A. Baron et al. / General and Comparative Endocrinology 161 (2009) 13–19

logs for MC1R, MC2R, MC4R, and two MC5Rs (Ringholm et al.,2002; Fig. 1b). In the genomes of an amphibian (Xenopus tropicalis),a bird (Gallus gallus), and among the mammals all five MCR genesare present (Cone, 2006; http://genome.jgi-psf.org/Xentr4/Xentr4.-home.html; http://www.chicken-genome.org; Fig. 1b). However,in the cartilaginous fish, Squalus acanthias, only three MCRs geneshave been detected, MC3R, MC4R, and MC5R (Ringholm et al.,2003; Klovins et al., 2004b; Fig. 1b). Given the phylogenetic posi-tion of the cartilaginous fish relative to the other lineages of gnat-hostomes, establishing a clearer understanding of the distributionof MCR genes in this group may not only reveal trends in the radi-ation of MCR genes in the gnathostomes, but may also help explainthe origin of the ligand selective properties of these receptors.

3. Studies on the horn shark, Heterodontus francisci

Extant cartilaginous fish can be divided into two subclasses: theHolocephali (ratfish and related species) and the Elasmobranchii

(sharks and rays; Nelson, 1994). The extant Elasmobranchii forma monophyletic assemblage that can be further sub-divided intotwo superorders: Galea (galeoid sharks), and Squalea (the squaloidsharks and rays; Shirai, 1997). The dogfish, S. acanthias, is an exam-ple of a squaloid shark. Since recent studies (Ringholm et al., 2003;Klovins et al., 2004b) only detected the MC3R, MC4R, and MC5Rgenes in the genome of this species, do these results representthe ancestral condition for both squalid and galeoid sharks, orare these observations unique to the dogfish? To address this ques-tion an analysis was done to determine which MCR genes could bedetected in the genome of the galeoid shark, Heterodontus francisci(horn shark). Genus Heterodontus has a fossil record that extendedinto the Jurassic, and is one of the oldest lineages among the extantElasmobranchii (Carroll, 1988; Shirai, 1997), hence this speciesseemed particularly appropriate for this study. As described inthe legend to Fig. 2, a set of degenerate PCR primers was designedto the highly conserved regions in the MCR genes of a cartilaginousfish (dogfish), bony fish (fugu), and a tetrapod (mouse). Theoreti-

[-------- TM1 ------HFMC3R --------------------------------------------------------------- SAMC3R MD----------------------LNESVLNNRSSAGFCEQVPIKAEVFLILGILSLLENILV HFMC4R -------------------------------------------ISTEVFLTLGIISLLANILV SAMC4R MNSSFHHRLPETPQLRNHSVARFASANGSRSDGFSSGCYEQLWISTEVFLTLGIFSLLANILV HFMC5R -------------------------------------------IAVEVFLTLGILSLLENILV SAMC5R MN------LTGLQSREPWPKNLTPANDITNRTKSTSGLCEQVSIAVEVFLTLGIMSLLENILV

---] IC1 [--------- TM2 --------] EC1 [----------- HFMC3R --------NLHSPMYFFLCSLAVADMLVSVSNALETIVMGFLNNGYLVANDQFIQQMDNVFDS SAMC3R ILSILKNKNLHSPMYFFLCSLAVADMLVSVSNALETIVMAFLNNGYLVANDQFIQQMDNVFDS HFMC4R IAAIIKNKNLHSPMYFFICSLAVADMLVSVSNAWETIFIAMLKSRLLMAQDNLIKSMDNVFDS SAMC4R IAAIVKNNKLHSPMYFFICSLAVADMLVSVSNAWETIFIAMLKSRHLTAPENLIKNMDNVFDS HFMC5R ITAVIKNKNLHSPMYLFICSLAVADMLVSVSNAWETIMI-LLNNRHLIVEDSFAKQVDNVFDS SAMC5R ITAVIKNKNLHSPMYFFVSCLAVADMLVSASNAWETIVI-LLNSRHLIVEDSFVKQVDNVFDS * * *

TM3 ---------] IC2 [-------- TM4 --------] HFMC3R MICISLVASICNLLVIAIDRYITIFYALRYHSIMTVKRALILIIIIWIACIFCGIVFIIYSDS SAMC3R MICISLVASICNLLVIAIDRYITIFYALRYHSIMTVKRALILDRSYLDCLYFCGIIFIIYSDS HFMC4R MICTSLLASIWALLAIAVDRYITIFYALRYHNIMTVRRALTIITGIWAACTVSGILFIVYSES SAMC4R MICSSLLASICSLLAIAIDRYITIFYALRYHNIVTVRRALMIIAAIWAACTGSGILFIVYSES HFMC5R LICISVVASMCSLLAIAVDRYVTIFYALRYHHIMTVKRAAFI-AGIWMFCTGCGIIFIIYSES SAMC5R MICISVVASMCSLLAIAVDRYVTIFYALRYHHIMTVKRATFIIAGIWTFCIGCGIIFIIYSES ** + +

EC2 [------- TM5 -------] IC3 [------------ HFMC3R KTVIICLITMFFTMLFLMTTLYVHMFMLARLHIKRIATLPVNGIVRQRTCMKGAITITILLGI SAMC3R KTAIICLITMFFTMLSSMTTLYVHMFMLARLHIKRIATLPVNGMVRQRTCMKGAITITILLGI HFMC4R TTVIICLITMFFTMLVLMTSLYVHMFMLARLHVKRIAALPGNGAIRQAANMKGAITLTILLGV SAMC4R TAVIICLITMFFAMLALMASLYVHMFMLARLHVKRIAALPGNGAVRQAANMKGAITLTILLGV HFMC5R PTVVICLVTMFFIMLILMASLYSHMFLLARSHAKQIAALSGYNSIHQRASMKGAITLTILLGI SAMC5R PTVIICLIAMFFIMLVLMASLYSHMFLLARSHAKRIAALSSYNSIHQRASMKGAITLTILLGI

TM6 --------] EC3 [--------- TM7 ----------] HFMC3R FIVCWAPFFLHLILIISCPKNPYCICYTSHFNTYLILIMC----------------------- SAMC3R FIVCWAPFFLHLILIISCPKNPYCICYTSHFNTYLILIMCNSVIDPIIYAFRSQEMRKTFKEI HFMC4R FVVCWAPFFLHLILMISCPRNPYCLYFMSHFMNYLILIMCNS--------------------- SAMC4R FVVCWAPFFMHLILMISCPQNPYCVCFMSHFMNYLILIMCNSVIDPLIYAFRSQEMRKTFKEI HFMC5R FIICWAPFFLHLILMISCPGNLYCVCFMSHFNLYLILIMCNS--------------------- SAMC5R FIVCWAPFFLHLILMISCPGNLYCVCFMSHFNLYLILIMCNSIIDPLIYAFRSQEMRKTFKEI * * + *

HFMC3R ------------- SAMC3R ICCYCMNLNLRCK HFMC4R ---------------- SAMC4R ICCYSLPGLCDLTSEY HFMC5R --------------- SAMC5R ICCYSLRAACGLSGK

Fig. 2. Comparison of horn shark and dogfish melanocortin receptor 3, 4, and 5 sequences. The dogfish (Squalus acanthias) MCR 3, 4, and sequences were characterized byRingholm et al. (2003) and Klovins et al. (2004b). Horn sharks (Heterodontus francisci) were obtained from the Scripps Institute of Marine Biology, University of California atSan Diego (La Jolla, CA). The animals were sacrificed following a protocol approved by the University of Denver IUCAC committee. Genomic DNA extractions (PromegaWizard� Genomic DNA Purification Kit; Promega, Madison, WI) were done on muscle. Genomic extracts were initially screened with degenerate forward (50AAY CTB CAB TCNCCS ATG TAY TWY 30) and reverse (50RTG GTA NYK NAR VGC RTA RAA KAT 30) primers designed from an analysis of universally conserved sites in mouse, fugu, and dogfishMC1R, MC2R, MC3R, MC4R, and MC5R. The PCR parameters were: initial denature at 94 �C for 3 min, denaturation at 94 �C for 1 min, annealing at 55 �C for 30 s, extension at72 �C for 1 min and 30 s, 32 repetitive cycles, and a final extension of 72 �C for 10 min. This approach yielded amplicons corresponding to MC3R, MC4R, and MC5R. Byanalyzing MCR gene sequences in fugu, dogfish, and Xenopus tropicalis it was possible to design new sets of degenerate primers that were specific for orthologous MC3R,MC4R, and MC5R genes. These primer pairs were: MC3R FWD (50TTG GAG ACG ATC GTG ATG GGG 30) and MC3/4/5R REV (50YGA RTT RCA CAT RAT GAG RAT 30); MC4R FWD(50ATC TCG ACG GAG GTC TTC CTC 30) and MC3/4/5R REV; MC5R FWD (50ATA GCA GTT GAG GTG TTT CTG 30) and MC3/4/5R REV. The PCR parameters were modified for eachprimer set in the following way: 57 �C for MC3R, 62 �C for MC4R, and 55 �C for MC5R. Subcloning and sequencing of the PCR products was performed as described in Robertset al., 2007). Shading indicates residues that are identical in at least five of the six sequences. The (�) indicates residues predicted to be in the HFRW binding site (Table 1). The(+) indicates positions in the predicted HFRW binding site (Table 1) that have undergone apparent neutral mutations. Abbreviations: TM, transmembrane domain; IC,intracellular loop; EC, extracellular loop; HF, Heterodontus francisci; SA, Squalus acanthias.

A. Baron et al. / General and Comparative Endocrinology 161 (2009) 13–19 15

cally, these primers should anneal to any of the five MCR genes.However, when this degenerate primer set was used to screen hornshark genomic DNA, only gene sequences corresponding to MC3R,MC4R, and MC5R were detected. The use of additional sets of prim-ers designed, respectively, from the sequences of the dogfish, thefugu fish, Takifugi rubripes, and the amphibian, X. tropicalis MCR3R,MC4R, and MC5R genes were used to amplify and characterize par-

tial sequences for horn shark MC3R, MC4R, and MC5R (Fig. 2).When sequences of the horn shark receptors are compared to thecorresponding sequences in the dogfish receptors, 65% of the resi-dues are identical in at least five of the six shark receptor se-quences presented in Fig. 2. Perhaps what is more noteworthy,however, was that neither the dogfish studies nor the currentstudy on the horn shark revealed gene sequences corresponding

16 A. Baron et al. / General and Comparative Endocrinology 161 (2009) 13–19

to an MC1R gene or an MC2R gene in the genomic DNA of thesecartilaginous fish. Given the hypothetical scheme for MCR geneevolution presented in Fig. 1a, it might be reasonable to proposethat cartilaginous fish have secondarily lost the MC1R gene afterthe divergence of the ancestral gnathostomes into the cartilaginousfish and the bony fish (Fig. 1b). Of course it is recognized that onlycharacterization of the complete shark genome can confirm thatthe MC1R gene has been lost in cartilaginous fish. Assuming thatgene loss has occurred, the apparent absence of a MC2R gene raisesa more perplexing issue.

Cartilaginous fish have a hypothalamus/pituitary/interrenalaxis (Denning-Kendall et al., 1982; Vallarino and Ottonello,1987), and injections of ACTH do induce the production of a gluco-corticoid by the interrenal cells (Idler and Truscott, 1966). Since thereceptor for ACTH on mammalian adrenal cells (Cone, 2006) andteleost interrenal cells (Klovins et al., 2004a) is the MC2R, the ques-tion is which MCR is the ‘‘ACTH receptor” on shark interrenal cells?To address this question, mRNAs were extracted from several hornshark tissues, and each tissue set was screened with primers spe-cific for either MC3R, MC4R or MC5R, respectively. While it waspossible to demonstrate expression of MC3R and MC4R in variousregions of the central nervous system, the expression of neithergene was detected in extracts of the interrenal/kidney of the hornshark (data not shown). However, as shown in Fig. 3, MC5R geneexpression was detected in the interrenal/kidney tissue in additionto being expressed in the optic tectum, hind brain, pituitary and li-ver. Based on these observations, it would appear that MC5R maybe the ‘‘ACTH receptor” on horn shark interrenal cells. If this con-clusion is correct then the extant shark MC5R gene may be theortholog of the proposed ancestral MC5R/MC2R gene (Fig. 1a).Since recent ligand activation studies indicate that the lampreyMCa and MCb receptors and the dogfish MC3R and MC4R receptorscan be activated by either ACTH or a-MSH; the proposed ancestralcondition for ligand activation of the MCRs (Haitina et al., 2007b),it is highly likely that the ancestral MC5R/MC2R gene could also beactivated by these two melanocortins. The evolutionary hypothesisthat emerges from these observations is that cartilaginous fishhave retained the ortholog (MC5R) of the ancestral MC5R/MC2Rgene (Fig. 1a), and that the duplication of the ancestral MC5R/MC2R gene occurred after the cartilaginous fish diverged from

Fig. 3. RT PCR analysis of MC5R expression in horn shark tissues. To analyze theexpression pattern for the MC5R gene, individual mRNA extracts were made fromforebrain (FB), optic tectum (OT), cerebellum (CB), hindbrain (H), pituitary (PT),liver (L), pancreas, PN), interrenal/kidney (IR), and integument (IN) and converted tocDNA following the RNA extraction and cDNA first strand synthesis protocolsoutlined in Shoureshi et al., 2007. PCR reactions were performed using the MC5Rforward primer and the MC3R/MC4R/MC5R reverse primer as described in thelegend to Fig. 2. The PCR products and a Low Range Plus DNA Ladder (S: FisherScientific) were run on 1.5% TBE agarose gel (0.5 lg/ml ethidium bromide) at110 mV. The gel was visualized using the GelDoc UV Transilluminator System (Bio-Rad, Hercules, CA). For each chromatography run a positive control (PC; genomicDNA) and a negative control (NC; no DNA in the PCR reaction) were run. PCRproducts were isolated, subcloned and sequence to confirm the identity of theproduct. Sequence analysis showed that the PCR product migrating close to the400 bp standard (B) was a false positive; whereas, the PCR product migrating closeto the 800 bp standard (A) was a 753 bp region of horn shark MC5R.

the ancestral gnathostomes, but early in the radiation of the bonyfish. This proposed duplication event yielded a MC5R paralog thatretained the ability to be activated by either ACTH or a-MSH (Cone,2006), and a MC2R paralog that evolved exclusive ligand selectivityfor ACTH (Fig. 1b).

Support for this hypothesis should come from ligand bindingand activation studies on the dogfish MC5 receptor. However, pre-vious attempts at expressing the dogfish MC5 receptor were notsuccessful in HEK 293 (Haitina et al., 2007b). This was surprisinggiven that both zebrafish MC5R (Ringholm et al., 2002) and fuguMC5R (Klovins et al., 2004a) were successfully expressed in HEK293 cells. However, this perplexing observation actually providessupport for the hypothesis that shark MC5R represents the ances-tral condition for the proposed MC5R/MC2R ancestral gene. Inmammalian cell lines MC2R translocation to the plasma membraneand activation requires the presence of a MC2R Accessory Protein(MRAP; Rachel et al., 2005; Metherell et al., 2005; Forti et al.,2006; Roy et al., 2007). It would be reasonable to predict that theancestral MC5/MC2 receptor also required an MRAP, and that thisancestral requirement has been retained in the dogfish MC5 recep-tor, but secondarily lost for the teleost and tetrapod MC5 receptors.

Collectively, then, comparative studies on the MCR gene familyhave revealed that apparent gene loss (MC1R; the cartilaginousfish), gene duplication (MC5R/MC2R; ancestral bony fish), andindependent genome duplication events (zebrafish) have occurredduring the radiation of the gnathostomes. When these occurrencesare taken into account, the radiation of the MCR genes in chordates(Fig. 1b) does appear to conform to the 2R hypothesis.

4. Modeling the HFRW binding site in melanocortin receptors

While comparative studies on the distribution of the MCR genesprovide insights into the evolution of the gene family, the evolu-tion of ligand selectivity among these receptors appears to be amore complex issue. In vitro receptor activation studies (de novoproduction of cAMP by cell lines expressing the respective MCR)indicate that for the lamprey MCRs and the spiny dogfish MCRseither ACTH or the various forms of MSH can induce cAMP produc-tion by any receptor (Ringholm et al., 2003; Klovins et al., 2004b;Haitina et al., 2007a; Fig. 1b). Furthermore when these same ana-lyzes were done on fugu or mammalian MCRs, ACTH and the MSHscould activate MC1R, MC3R, MC4R, and MCR5R with varying de-grees of efficacy (Klovins et al., 2004a; Cone, 2006; Fig. 1b). How-ever, fugu MC2R and mammalian MC2R could only be activatedby ACTH, but not by a-MSH (Mountjoy et al., 1992; Klovins et al.,2004a; Fig. 1b). Although the ligand selectivity of MC1R, MC3R,MC4R, and MC5R would appear to be an example of subfunctional-ization, these receptors mediate different functions in gnathosto-mes (Cone, 2006). However, what is striking about the evolutionof the MCR gene family is the emergence of the strict ligand selec-tivity of MC2R for ACTH.

5. Activation scenarios for the MC2 receptor

Since all melanocortin ligands share the HFRW motif (Dores andLecaude, 2005), it is possible that the HFRW site binding in bonyfish and mammalian MC2Rs has evolved in such a way that onlyACTH(1–39) or the fully functional analog, ACTH(1–24), can prop-erly fit into the binding site to induce cAMP production. If thishypothesis is correct then in addition to a site on the receptor forthe binding of the HFRW motif (residues 5–8 in the ACTH primarysequence), there must be an extension of the binding site in MC2Rto accommodate residues 15–24 in the biologically active portionof ACTH. Alternatively, initial contact of ACTH with the inactiveform of MC2R at some specific site may result in a conformational

A. Baron et al. / General and Comparative Endocrinology 161 (2009) 13–19 17

change that exposes the HFRW binding site in the active form ofthis receptor. The corollary to the latter hypothesis would be thatthe MSHs (a, b, c, or d) lack the C-terminal extension required tounmask the HFRW binding site in bony fish or mammalianMC2R. These hypotheses beg two questions: a) what is knownabout the HFRW binding site in MCRs, and is there evidence thatthe MSHs do not bind to MC2R?

Ligand binding to GPCRs could involve interaction with the N-terminal region of the receptor that is oriented on the outer planeof the plasma membrane, interaction with the extracellular loopsof the GPCR, interaction with residues on the transmembranespanning domains that form hydrophilic pockets near the mem-brane surface, or a combination of regions in the structure of theGPCR that are exposed to the external environment (Hoffmannet al., 2008). However, an understanding of the binding site onthe MCRs has been hampered by the lack of a crystal structurefor any of these receptors. Although the crystal structure for theinactive form of the rhodopsin receptor has been determined, whatis really needed is a model for the active conformation of a MCR. Toevaluate the ligand binding site for human MC4R, Mosberg and col-leagues approached this problem in a novel manner (Pogozhevaet al., 2005; Chai et al., 2005) by first using distance constraintsfrom the crystal structure of the inactive conformation of rhodop-sin (Li et al., 2004) to generate a model for the active conformationof the rhodopsin-related opioid mu receptor (Fowler et al., 2004).Based on the assumption that rhodopsin-related GPCRs share com-mon activation mechanisms (Meng and Bourne, 2001; Karnik et al.,2003), the active conformation of the mu receptor was used as thetemplate for modeling the active conformation of human MC4R.This model predicted a cleft between TM regions 2, 3, 6, and 7 thatcould accommodate the thirteen amino acids in the a-MSH se-quence (S1Y2S3M4E5H6F7R8W9G10K11P12V13). This model took intoconsideration that a-MSH has a b-hairpin-like structure with a re-verse turn spanning H6 and F7 residues in the HFRW motif (Haslachet al., 2008). The model indicated that as a-MSH is positioned inthe predicted binding site, there would be the opportunity for sev-eral H-bonding patterns between residues in TM 2, 3, 6, and 7 andthe R-groups on the residues in the HFRW motif. Based on thesepredictions, site directed mutagenesis was performed on the indi-vidual residues in the predicted ligand binding site, and the mutantreceptor constructs were tested for the efficacy of cAMP produc-tion following a-MSH binding (Pogozheva et al., 2005). The alaninesubstitution experiments identified eleven residues (Table 1) in thepredicted binding site that affected a-MSH activation of the recep-tor. In the human MCRs, 10 of the 11 predicted residues from Table1 are conserved (Pogozheva et al., 2005). Furthermore in the se-quences of MC3R, MC4R, and MC5R for the dogfish shark and thehorn shark, 8 of the 11 predicted residues from Table 1 are presentat the same sites in these MCRs (Fig. 2). In the MCRs of a frog (X.tropicalis), a teleost fish (T. rubripes), and the two lamprey (Lampe-tra fluviatilis) MCRs that have been sequenced, 9 of the 11 pre-dicted residues have been conserved in 89% of the sequences

Table 1HFRW binding site.

Amino positionin human MC4R

Region withinhuman MC4R

Amino acid positiona-MSH

H264 TM6 E5, W9

E100 TM2 W9

D122, D126 TM3 H6, R8

D189 EC2 K11

I129, C130, L133 TM3 F7

F261 TM6 F7

N285, L288 TM7 F7

Adapted from Pogozheva et al. (2005).

(Fig. 4). Moreover, in those sites where there was not an exactmatch (L133, D189, N285) apparent neutral substitutions have oc-curred. Hence, over nearly 450 million years of vertebrate evolu-tion 73% of the residues listed in Table 1 have been rigorouslyconserved in MCRs. Based on these observations, it seems reason-able to propose that all vertebrate MCRs have identical HFRWbinding sites that are demarcated by the amino acid positionspresented in Table 1. The model, then, that emerges for a-MSHbinding to human MC1R, MC3R, MC4R, and MC5R is that thedocking of a-MSH to the HFRW binding site in the inactivereceptor induces the conformational change of the receptor tothe active state. A similar model has been independently proposedfor the HFRW binding site in mouse MC1R (Haskell-Luevano et al.,1996).

Since fugu and human MC2Rs have virtually the same residuesfor the HFRW binding site in the same TM regions as the otherMCRs (Fig. 4), can a-MSH bind to an MC2R? The studies on thein vitro activation of fugu MC2R (Klovins et al., 2004a) and humanMC2R (Mountjoy et al., 1992) clearly state that only ACTH, not a-MSH, could initiate the production of cAMP, but neither study pre-sented data on whether a-MSH competitively bound to the respec-tive receptor. However, binding studies done nearly a decade priorto the Mountjoy et al. study had shown that a-MSH could not dis-place iodinated ACTH(1–39) from rat adrenal membranes (Buckleyet al., 1981); MC2R is the predominate MCR on mammalian adre-nal cells. Subsequent in vitro binding studies on mouse MC2R ex-pressed in different cell lines were in complete agreement withthe early studies on rat adrenal membrane preparations (Schiothet al., 1996; Kapas et al., 1996). These observations would indicatethat activation of the MC2 receptor follows a sequences of eventsthat are distinct from the other vertebrate MCRs. Assume, forexample that the HFRW binding site on MC2R were in a ‘‘open”conformation in the inactive form of the MC2R—the apparent con-dition for the inactive states of MC1R, MC3R, MC4R, or MC5R. Inthis scenario, as the HFRW portion of the ACTH(1–24) becomespositioned in the HFRW binding site the C-terminal extension ofACTH(1–24) could now make contact with a region of the receptoroutside of the HFRW binding site that induces the active conforma-tion of the receptor, and as a result cAMP is producted. In this sce-nario, a-MSH could enter the HFRW binding site on the receptor,but because this ligand lacks the C-terminal extension, the receptordoes not change to the active conformation. The flaw in thishypothesis is the assumption that when the MC2R receptor is inthe inactivate conformation, a-MSH could bind to the HFRW bind-ing site on MC2R. The study by Buckley et al., 1981 clearly showsthat a-MSH does not compete with ACTH(1–24) for the HFRWbinding site on the receptor. Since C-terminally truncated formsof ACTH(1–24) loose the ability to activate the MC2 receptor (as re-viewed by Schwyzer, 1977) it is clear that a multi-step mechanismfor MC2R activation is required.

For the multi-step model, it is assumed that the HFRW bindingsite in MC2R is ‘‘closed” when the receptor is in the inactive confor-mation. In this three-step scenario, (1) the C-terminal region ofACTH(1–24) would first make contact with a region of the receptoroutside the HFRW binding site; (2) interaction at this secondarybinding site would induce the HFRW binding site on the receptorto move to its ‘‘open” conformation; and (3) finally, the HFRW motifof ACTH(1–24) would dock with the ‘‘open” HFRW binding site onthe receptor and this binding event would trigger G-Protein activa-tion. Since a-MSH lacks a C-terminal extension, this ligand can notinduce the ‘‘opening” of the HFRW binding site in the receptor andas a result can not activate the MC2 receptor. When ACTH(1–24)dissociates from the active receptor, the receptor reverts to itsinactive conformation. The three-step model provides not only anexplanation for the importance of the C-terminal extension ofACTH(1–24) as compared to the truncated nature of N-acetyl-

N-terminal [---------TM1-------- xMC1R M-----------LHSTVNSTNAT-----INVGTELKPTNTS------DTVMDVPEELFLFLCVFSLLENILVV 73 fMC1R M-----ELFNRSLHGSYILHMFSPLIEFMDDNET----NITGEQNLDCVQILIPQELFLTLGLISLVENILVI xMC2R MN------------------------------------------STKCSSVHVPEVVYLTVSAIGLLENLLVL fMC2R MN--------------ATTVNRS-----------------------DCPEVNVPIHVFFTIGFVSLLENLLVI xMC3R MNTT------NVFSVQAVLANAT----LDPNNETLFLSNLS--RIGFCEQVLIKTEVFLTLGIISLLENILVI xMC4R MNFT----HHHREPHHLHYRNHSRT-VGAGANDTKE---KGHHSGGCYEQLFVSPEVFVILGIVSLMENILVI fMC4R MN------ATDPPGRVQDFSNGSQTPETDFPNEEKE------SSTGCYEQMLISTEVFLTLGIISLLENILVV xMC5R MNLS--------LQQSTLELNIS----SLLGNNT---VPTGKTKSSACEQVVIAAEVFLTLGIVSLLENILVI fMC5R MNTSHRSSDPQEDPQEGIMGNSTWNPLSYQPNFTLSPPLLPKTKTAACEQLHIAIEVFLTLGIISLLENILVI LMCaR MNLSEALFPNPFVGTSGPDDNGT---ASASANRT---------RFSPCHNFSIPTEVFLALGIVSLVENALVI LMCbR M------TFSAGGVGGVVNNHHHGANHQGGGNHSGHGNATGGGHGRPCEQVLIPIEVFLILGVISLLENILVI

--] IC1 [---------TM2----------] EC1 [----------TM3--------- xMC1R IAIFRNHNLHSPMYYFICCLAASDMLVSSSNLGETLIIFMLKQGIIKSEPLLVKKMDYIFDTMICCSLVTSLS 146 fMCIR LAIMKNRNLHSPMYYFICCLALSDMLVSNVSVSETVFMLLNDHGLMDMYPGMLRHLDNVIDVMICSSVVSSLS xMC2R LAVIKNKNLHLPMYFFICSLAVSDMLFSLYKILETIIIILANIGFLDRNGPFEKKMDDVMDWIFVLSLLGSIF fMC2R GAISWNRNLHSPMYCFIGSLAAFNTVASVTKTWENLMITFAEVGHLRKVGFSERKADDVVDSLLCMSFLGSIF xMC3R LAILKNKNLHSPMYFFLCSLAVADMLVSVSNALETIVIA-IQNKYLVIGDYLLQHLDDVFDSMICISLVASIC xMC4R AAISRNKNLHSPMYFFICSLAVADMLVSVSNGFETVVITLFNTTD-KNTQHIIVNVDNILDSVICSSLLASIC fMC4R AAIVKNKNLHSPMYFFICSLAVADMLVSVSNASETIVIALINSGTLTIPATLIKSMDNVFDSMICSSLLASIC xMC5R FAIILNKNLHSPMYFFVCSLAVADMLVSVSNAWETITINLINNRHLIMEETFVRHIDNVFDSMICISVVASMC fMC5R MAIVKNKNLHSPMYFFVCSLAVADMLVSVSNASETIIIYLLNNKQL1AEDHLIRQLDNVFDSMICISVVASMC LMCaR AAIARNRNMHSPMYCFICSLAVADMLVSVSNAWETIIMALLQNGSLAMQEDTLKQMDNIMDSMICTSVVASMC LMCbR TAILKNKNLHSPMYYFICSLAVADMLVSVSNAWETIIMALLQNGSLAMQEDTLKQMDNIMDSMICTSVVASMC * * * ** +

---] IC2 [--------TM4---------] EC2 [-------TM5----- xMC1R FLGAIAIDRYITIFYALRYHSIMTLRRVVIAIGVIWSVSLVCAAIFIVYHESRAVILCLIVFFLFMLALMVAL 219 fMC1R FLCTIAADRYITIFYALRYHSIMTTPRAITIIVIVWCASIASSILFIVYHTDNAVIVCLVTFFCITLVFNAVL xMC2R SISAIAADRYITVFHALHYHNIMTVKRASVILAVIWTFCGGSGIAIIMLFHDTAMIICLTVMFLLLLVLIVCL fMC2R SFLAIAVDRYITIFHALRYHNIMTMQRTGAILGLIWTTCGVSAMLMVRFFDSNLIMSCFVVFFIISLAIIYIL xMC3R NLLVIAIDRYITIFYALRYHSIMTVKKAIALIVVIWTSCIICGIVFIVFSESKTVIVCLITMFFTMLVLMATM xMC4R SLLSIAVDRYFTIFYALQYHNIITVRRAVVIISCIWTACSISGVLFIIYYDSAVVIICLISIIFTMLALMASL fMC4R SLLAIAVDRYITIFYALRYHNIVTLRRASLVISSIWTCCTVSGVLFIVYSESTTVLICLITMFFTMLVLMASL xMC5R SLLAIAVDRYVTIFYALRYHNIMTMRRAGIIIACIWTFCTGCGIIFILYYESTYVIICLITMFFTMLFLMVSL fMC5R SLLAIAVDRYVTIFYALRYHNIMTVRRAGCIIGGIWTFCTGCGIVFIIYSDITPVIICLVCMFFAMLLIMASL LMCaR SLLAIAVDRYVTIFYALRYHNIMTVRRAASIIGAIWGACVVSGTLFITYWDHRTVIVCLIALFVTMLVLMASL LMCbR SLLAIAVDRYVTIFYALRYHNIMTVRRAASIIGAIWGTCTLCGVIFIVYSDSTAVIICLITMFFTMLVLMASL +

----] IC3 [-----------TM6-----------] EC3 xMC1R YIHMFALARQHARSISALQKGKSRRITPHQARANMKGAITLTLLLGVFFLCWGPLFLHLTLFVSCPGHHICN 289 fMC1R YVHMFVLAHVHSRRIMAFHK-------NRRQSTSMKGAITLTILLGVFILCWGPFFLHLILILTCPTSVFCN xMC2R YIHMFLLARSHAKKIASL----SGQWNSVQQRANINGAITLTILLGLFICCWSPFVLHLLLYVLCRYNPYCA fMC2R YVYMFILARVHARKIAALPNGSGKHQHQRRWGHGMRGILTLTILFGAFMVCWAPFFLHLIFLMACPMNPYCE xMC3R YVHMFLFARLHVKRIAALP-----VDGVVQQRTCMKGAITITILLGVFVVCWAPFFLHLILIISCPSNSYCV xMC4R YVHMFMLARLHIKRIAVLPG-----TNSVRQVTNMKGAITLTILLGVFVACWSPFFLHLIFYVSCPRNPYCV fMC4R YVHMFLLARLHMKRIAAMPG-----NAFIHQRANLKGAITLTILLGVFVVCWAPFFLHLILMITCPKNPYCT xMC5R YIHMFLLARTHVKRIAALPGYNS-----VHQRTSMKGAITLTILLGIFIVCWAPFFLHLILMISCPQNLYCV fMC5R YSHMFMLARSHVKRIAALPGSNS-----IHQRASMKGAITLTILLGIFIICWAPFFLHLILMISCPRNLYCM LMCaR YAHMFALARSHAQRISAQP--RSSRQGQQNGAASLKGAVTLSILLGVFVFCWAPFFLHLTFIISCPANPYCC LMCbR YVHMFMLARLHAKRIAALP-----ASGIIQHKTSMRGAITLTILLGVFIVCWAPFFLHLILIVSCPRSPYCV

* *

[-----------TM7----------] C-terminal xMCR1 SYFYYFNIYLLLVICNSVIDPLIYAFRSQELRKT 353 fMC1R CYFRNFNLFLILIICNSLIDPLIYAYRSQELRKT-LQELVLCSWCFGP xMC2R CYLSMLNVNGTLILFSSVIDPLIYAFRSPELRNTFKKML-CC fMC2R CYRSMFQLHLVLLMSHALIDPVIYAFRIPELRHTFRRMLPCLNWRWR xMC3R CYTSYFNTYLILIMCNSIIDPLIYAFRSLEMRKTFKEI-ICCYGMNFGKCG xMC4R CFMSHFNIYLILIMCNSVIDPLIYALRSQELRKTFKEM-MCCYCMGGIWDFSSSY fMC4R CFMSHFNMYLILIMCNSVIDPIIYAFRSQEMRKTFKEI-FCCSQMLVCM xMC5R CFMSHFNMYLILIMCNSVIDPMIYAFRSQEMRKTFKEI-ICCYSLRMFCDLPSKY fMC5R CFMSHFNMYLILIMCNSVIDPLIYAFRSQEMRKTFKEI-IFCYSLRNTCSTICTLPGKY LMCaR AYIAYFPLYLLLIMINSVIDPLIYAFRSPELRVIIRDTLRKCGRGRGRGANGTRGSSCCCVQVR LMCbR CYMSHFNLYLVLIMLSSVIDPIIYAFRSHEMRHTFKEI-VCCYSGSLYCALPATWKY * *

Fig. 4. Alignment of selected chordate MCR amino acid sequences. The amino acid sequences of Xenopus tropicalis (x; http://genome.jgi-psf.org/Xentr4/Xentr4.home.html),Takifugu rubripes (f; Klovins et al., 2004a) and Lampetra fluviatilis (L; Haitina et al., 2007a) MCRs are presented. Shaded residues identify sites in which at least eight of residuesare identical. Underlined residues highlight predicted N-linked glycosylation sites in the N-terminal regions of each receptor. The (�) indicates residues predicted to be in theHFRW binding site (Table 1). The (+) indicates positions in the predicted HFRW binding site (Table 1) that have undergone apparent neutral mutations. Abbreviations: TM,transmembrane domain; IC, intracellular loop; EC, extracellular loop.

18 A. Baron et al. / General and Comparative Endocrinology 161 (2009) 13–19

A. Baron et al. / General and Comparative Endocrinology 161 (2009) 13–19 19

ACTH(1–13)NH2 [a-MSH], but also provides an explanation for whya-MSH can not compete with ACTH(1–24) for binding to MC2R.

6. Deciphering the activation of the MC2 receptor

Support for the three-step activation hypothesis requires theidentification of the proposed secondary binding site on MC2Rfor the C-terminal portion of ACTH(1–24) and identification ofthe critical residues in the C-terminal of ACTH(1–24) that bind atthis putative site. Recent studies indicate that a solution to the lat-ter issue will come from more detailed structure/function studieson the tetrabasic residues (positions 15,16, 17, and 18) and theproline residue at position 19 in ACTH(1–24) (Costa et al., 2004).Site-directed mutagensis studies on regions of teleost and tetrapodMC2Rs should help resolve the former issue. While both of theseissues should be straightforward to address, the challenge whenworking with MC2R is to find a cell line that can efficiently expressthe receptor on the plasma membrane. As noted previously, mam-malian MC2Rs requires the presence of MRAPs in order to be func-tionally expressed in cell lines (Rachel et al., 2005; Metherell et al.,2005; Forti et al., 2006; Roy et al., 2007). Since comparative studieson the expression of non-mammalian MC2Rs have largely beenunsuccessful up to this point, it appears that other tetrapod MC2Rsand fish MC2Rs may also require interaction with an MRAP. Oncethe parameters for in vitro expression of non-mammalian MC2Rshave been determined, conducting structure/function studies toresolve the activation mechanism for non-mammalian MC2Rs willbe feasible.

Acknowledgment

This research was supported by NSF Grant IOB 0516958(R.M.D.).

References

Buckley, D.I., Yamashiro, D., Ramachandran, J., 1981. Synthesis of a corticotropin analoguethat retains full biological activity after iodination. Endocrinology 109, 5–9.

Carroll, R.L., 1988. Vertebrate Paleontology and Evolution. W.H. Freeman, New York.pp. 62–82.

Chai, B.-N., Pogozheva, I.D., Lai, Y.-M., Li, J.-Y., Neubig, R.R., Mosberg, H.I., Gantz, I.,2005. Receptor-antagonist interactions in the complexes of agouti and agouti-related protein with human melanocortin 1 and 4 receptors. Biochemistry 44,3418–3431.

Cone, R.D., 2006. Studies on the physiological studies of the melanocortin system.Endocr. Rev. 27, 736–749.

Costa, J.L., Bui, S., Reed, P., Dores, R.M., Hochgeschwender, U., Brennan, M.B., 2004.Mutational analysis of evolutionarily conserved ACTH residues. Gen. Comp.Endocrinol. 136, 12–16.

Denning-Kendall, P.A., Sumpter, J.P., Lowry, P.J., 1982. Peptides derived from pro-opiomelanocortin in the pituitary gland of the dogfish, Squalus acanthias. J.Endocrinol. 93, 381–390.

Devi, L.A. (Ed.), 2005. The G-Protein-Coupled Receptors Handbook. Humana Press,New Jersey.

Dores, R.M., Lecaude, S., 2005. Trends in the Evolution of the Proopiomelanocortin.Gen. Comp. Endocrinol. 142, 81–93.

Dores, R.M., Lecaude, S., Bauer, D., Danielson, P.B., 2002. Analyzing the evolution ofthe opioid/orphanin gene family. Mass. Spec. Rev. 21, 220–243.

Force, A., Lynch, M., Pickett, F.B., Amores, A., Yan, Y.L., Postlewait, J., 1999.Preservation of duplicate genes by complementary, degenerative mutations.Genetics 151, 1531–1545.

Forti, F.L., Dias, M.H.S., Armelin, H.A., 2006. ACTH receptor: ectopic expression,activity and signaling. Mol. Cell. Biochem. 293, 147–160.

Fowler, C., Pogozheva, I.D., LeVine, H., Mosberg, H.I., 2004. Refinement of ahomology model of the mu-opioid receptor using distance constraints fromintrinsic and engineered zinc-binding sites. Biochemistry 43, 8700–8710.

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. Phylogeneticanalysis, paralogon groups, and fingerprints. Mol. Pharmacol. 63, 1256–1272.

Haitina, T., Klovins, J., Takahashi, A., Löwgren, M., Ringholm, A., Enberg, J., Kawauchi,H., Larson, E.T., Fredriksson, R., Schiöth, H.B. 2007a. Functional characterizationof two melanocortin (MC) receptors in lamprey showing orthology to the MC1and MC4 receptor subtypes. BMC Evol. Biol. 7, 101. Available from: <http://www.biomedcentral.com/1471-2148/7/101>.

Haitina, T., Takahashi, A., Holmen, L., Enberg, J., Shioth, H.B., 2007b. Further evidencefor ancient role of ACTH peptides at melanocortin (MC) receptors;pharmacology of dogfish and lamprey peptides at dogfish MC receptors.Peptides 28, 798–805.

Haskell-Luevano, C., Sawyer, T.K., Trumpp-Kallmeyer, S., Bikker, J.A., Humblet, C.,Gantz, I., Hruby, V.J., 1996. Three-dimensional molecular model of the hMC1Rreceptor melanocortin receptor: complexes with melanotropin peptideagonists. Drug Des. Discov. 14, 197–211.

Haslach, E.M., Schaub, J.W., Haskell-Luevano, A., 2008. Beta-turn secondarystructure and melanocortin ligands. Bioorganic Med. Chem.. doi:10.1016/j.bmc.02.090 (Pre-print online).

Hoffmann, C., Zurn, A., Bunemann, M., Lohse, M.J., 2008. Review: Conformationalchanges in G-protein-coupled receptors—the quest for functionally selectiveconformations is open. Br. J. Pharmacol. 153, S358–S366.

Holland, P.W., Garcia-Fernandez, J., Williams, N.A., Sidow, A., 1994. Geneduplications and the origins of vertebrate development. Development(Suppl.), 125–133.

Idler, D.R., Truscott, B., 1966. 1a-hydroxycorticosterone from cartilaginous fish: anew adrenal steroid in blood. J. Fish. Res. Board Can. 23, 615–619.

Kapas, S., Hinson, F.M.C.J.P., Clark, A.J.L., 1996. Agonist and receptor bindingproperties of adrenocorticotropin peptides using cloned mouseadrenocorticotropin receptor expressed in a stabily transfected HeLa cell line.Endocrinology 137, 3291–3294.

Karnik, S.S., Gogonea, C., Patil, S., Saad, Y., Takezako, T., 2003. Activation of G-proteinCoupled Receptors: a common molecular mechanism. Trends Endocrinol.Metab. 14, 431–437.

Klovins, J., Haitina, T., Fridmanis, D., Kilianova, Z., Kapa, I., Fredriksson, R., Gallo-Payet, N., Schioth, H.B., 2004a. The melanocortin system in fugu: determinationof POMC/AGRP/MCR gene repertoire and synteny, as well as pharmacology andanatomical distribution of the MCRs. Mol. Biol. Evol. 21, 563–579.

Klovins, J., Haitina, T., Ringholm, A., Lowgren, M., Fridmanis, D., Slaidina, M., et al.,2004b. Cloning of two melanocortin (MC) receptors in spiny dogfish. Eur. J.Biochem. 271, 4320–4331.

Li, J., Edwards, P.C., Burghammer, M., Villa, C., Schertler, G.F., 2004. Structure ofbovine rhodopsin in a trigonal crystal form. J. Mol. Biol. 343, 1409–1438.

Meng, E.C., Bourne, H.R., 2001. Receptor activation: what does the rhodopsinstructure tell us? Trends Pharmacol. Sci. 22, 587–593.

Metherell, L.A., Chapple, J.P., Cooray, S., David, A., Becker, C., Ruschendorf, F., Naville,D., Begeot, M., Khoo, B., Nurnberg, P., Huebner, A., Cheethan, M.E., Clark, A.J.,2005. Mutations in MRAP, encoding a new interacting partner of the ACTHreceptor, cause familial glucocorticoid deficiency type 2. Nat. Genet. 37, 166–170.

Mountjoy, K.G., Robbins, L.S., Mortrud, M.T., Cone, R.D., 1992. The cloning of a familyof genes that encode the melanocortin receptors. Science 257, 1248–1251.

Nelson, J.S., 1994. Fishes of the World. Wiley, New York. pp. 37–65.Ohno, S., Wolf, U., Atkins, N.B., 1968. Evolution from fish to mammals by gene

duplication. Hereditas 59, 169–187.Pogozheva, I.D., Chai, B.-X., Lomize, A.L., Fong, T.M., Weinberg, D.H., Nargund, R.P.,

Mulholland, M.W., Grantz, I., Mosberg, H.I., 2005. Interactions of humanmelanocortin 4 receptors with nonpeptide and peptide agonists. Biochemistry44, 11329–11341.

Rachel, M., El Mourabit, H., Buronfosse, A., Blondet, A., Naville, D., Begeot, M.,Penhoat, A., 2005. Expression of the human melanocortin-2 receptor in differenteukaryotic cells. Peptides 26, 1842–1847.

Ringholm, A., Fredriksson, R., Poliakova, N., Yan, Y.L., Postlewait, J.H., Larhammar, D.,Schioth, H.B., 2002. One melanocortin 4 and two melanocortin 5 reeceptorsfrom zebrafish show remarkable conservation in structure and pharmacology. J.Neurochem. 82, 6–18.

Ringholm, A., Klovins, J., Fredriksson, R., Poliakova, N., Larson, E.T., Kukkonen, J.P.,Larhammar, D., Schioth, H.B., 2003. Presence of melanocortin (MC4) receptor inspiny dogfish suggests an ancient vertebrate origin of central melanocortinsystem. Eur. J. Biochem. 270, 213–221.

Roberts, E., Shoureshi, P., Kozak, K., Baron, A., Lecaude, S., Dores, R.M., 2007.Tracking the evolution of the proenkephalin gene in tetrapods. Gen. Comp.Endocrinol. 153, 189–197.

Roy, S., Rachel, M., Gallo-Payet, N., 2007. Differntial regulation of the humanadrenocorticotropin receptor [melanocortin-2 receptor (MC2R)] by humanMC2R accessory protein isoforms a and b in isogenic human embryonic kidney293 cells. Mol. Endocrinol. 21, 1656–1669.

Schioth, H.B., Chhajlani, V., Muceniece, R., Klusa, V., Wikberg, J.E., 1996. Majorpharmacological distinction of the ACTH receptor from other melanocortinreceptors. Life Sci. 59, 797–801.

Schioth, H.B., Fredriksson, R., 2005. The repertoire of G-Protein-coupled receptors infully sequenced genomes. Mol. Pharmacol. 67, 1414–1425.

Schwyzer, R., 1977. ACTH: a short introductory review. Ann. NY Acad. Sci. 297, 3–26.

Shirai, S., 1997. Pylogenetic interrelations of neoselachians. In: Stiassny, M.L.J.,Parenti, L.R., Johnson, G.D. (Eds.), Interrelationships of Fishes. Academic Press,San Diego, pp. 9–34.

Shoureshi, P., Baron, A., Szynskie, L., Dores, R.M., 2007. Analyzing the evolution of b-endorphin posttranslational processing events: studies on reptiles. Gen. Comp.Endocrinol. 153, 189–197.

Vallarino, M., Ottonello, I., 1987. Neuronal localization of immunoreactiveadrenocorticotropin-like substance in the hypothalamus of elasmobranchs.Neurosci. Lett. 80, 1–6.