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 gamma-MSH.
We report the cloning and characterization of two melanocortin receptors (MCRs) from the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis shows that these shark receptors are orthologues of the MC3R and MC5R subtypes, sharing 65% and 70% overall amino acid identity with the human counterparts, respectively. The SacMC3R was expressed and pharmacologically characterized in HEK293 cells. The radioligand binding results show that this receptor has high affinity for adrenocorticotropic hormone (ACTH)-derived peptides while it has comparable affinity for alpha- and beta-melanocyte stimulating hormone (MSH), and slightly lower affinity for gamma-MSH when compared with the human orthologue. ACTH(1-24) has high potency in a second-messenger cAMP assay while alpha- and gamma-MSH had slightly lower potency in cells expressing the SacMC3R. We used receptor-enhanced green fluorescence protein (EGFP) fusion to show the presence of SacMC3R in plasma membrane of Chinese hamster ovary and HEK293 cells but the SacMC5R was retained in intracellular compartments of these cells hindering pharmacological characterization. The anatomical distribution of the receptors were determined using reverse transcription PCR. The results showed that the SacMC3R is expressed in the hypothalamus, brain stem and telencephalon, optic tectum and olfactory bulbs, but not in the cerebellum of the spiny dogfish while the SacMC5R was found only in the same central regions. This report describes the first molecular characterization of a MC3R in fish. The study indicates that many of the important elements of the MC system existed before radiation of gnathostomes, early in vertebrate evolution, at least 450 million years ago.
Klovins
Janis
J
Department of Neuroscience, Unit of Pharmacology, Uppsala University, Sweden.
Haitina
Tatjana
T
Ringholm
Aneta
A
Löwgren
Maja
M
Fridmanis
Davids
D
Slaidina
Maija
M
Stier
Susanne
S
Schiöth
Helgi B
HB
D000324:Adrenocorticotropic Hormone / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages; D015139:Blotting, Southern; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004284:Dogfish; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007438:Introns; D007700:Kinetics; D009074:Melanocyte-Stimulating Hormones / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D044104:Receptor, Melanocortin, Type 3 / Q000235:genetics*; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D019825:gamma-MSH / Q000737:chemistry*
D010455:Peptides; D044104:Receptor, Melanocortin, Type 3; D044105:Receptor, Melanocortin, Type 4; D019825:gamma-MSH; D049452:Green Fluorescent Proteins; D000324:Adrenocorticotropic Hormone; D009074:Melanocyte-Stimulating Hormones; D000242:Cyclic AMP