One melanocortin 4 and two melanocortin 5 receptors from zebrafish show remarkable conservation in structure and pharmacology.
We report the cloning, genome mapping, functional expression, pharmacology and anatomical distribution of three melanocortin (MC) receptors from zebrafish (z). Phylogenetic analysis showed with high bootstrap support that these genes represent one MC4 receptor and two MC5 receptors. Chromosomal mapping showed conserved synteny between regions containing zMC4 and human (h) MC4 receptors, whereas the two zMC5 receptor genes map on chromosome segments in which the zebrafish has several genes with two orthologues of a single mammalian gene. It is likely that the two copies of zMC5 receptors arose through a separate duplication in the teleost lineage. The zMC4, zMC5a, and zMC5b receptors share 70-71% overall amino acid identity with the respective human orthologues and over 90% in three TM regions believed to be most important for ligand binding. All three zebrafish receptors also show pharmacological properties remarkably similar to their human orthologues, with similar affinities and the same potency order, when expressed and characterized in radioligand binding assay for the natural MSH) peptides alpha-, beta-, and gamma-MSH. Stimulation of transfected mammalian cells with alpha-MSH caused a dose-dependent increase in intracellular cAMP levels for all three zebrafish receptors. All three genes were expressed in the brain, eye, ovaries and gastrointestinal tract, whereas the zMC5b receptor was also found in the heart, as determined by RT-PCR. Our studies, which represent the first characterization of MC receptors in a nonamniote species, indicate that the MC receptor subtypes arose very early in vertebrate evolution. Important pharmacological and functional properties, as well as gene structure and syntenic relationships have been highly conserved over a period of more than 400 million years implying that these receptors participate in vital physiological functions.
Ringholm
Aneta
A
Department of Neuroscience, Unit of Pharmacology, Uppsala University, BMC, 75 124 Uppsala, Sweden.
Fredriksson
Robert
R
Poliakova
Natalia
N
Yan
Yi-Lin
YL
Postlethwait
John H
JH
Larhammar
Dan
D
Schiöth
Helgi B
HB
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D015139:Blotting, Southern; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010802:Phylogeny; D011869:Radioligand Assay; D044105:Receptor, Melanocortin, Type 4; D018043:Receptors, Corticotropin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D044101:Receptors, Melanocortin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000521:alpha-MSH / Q000031:analogs & derivatives / Q000493:pharmacokinetics
D008024:Ligands; D044105:Receptor, Melanocortin, Type 4; D018043:Receptors, Corticotropin; D044101:Receptors, Melanocortin; D029961:Zebrafish Proteins; C461479:mc4r protein, zebrafish; C461482:mc5ra protein, zebrafish; C461481:mc5rb protein, zebrafish; C086971:melanocortin 5 receptor; D000521:alpha-MSH