RESUMO
BACKGROUND: The melanocortin (MC) receptors have a key role in regulating body weight and pigmentation. They belong to the rhodopsin family of G protein-coupled receptors (GPCRs). The purpose of this study was to identify ancestral MC receptors in agnathan, river lamprey. RESULTS: We report cloning of two MC receptors from river lamprey. The lamprey receptors, designated MCa and MCb, showed orthology to the MC1 and MC4 receptor subtypes, respectively. The molecular clock analysis suggested that lamprey MC receptor genes were not duplicated recently and diverged from each other more than 400 MYR ago. Expression and pharmacological characterization showed that the lamprey MCa receptor was able to bind and be activated by both lamprey and human MSH peptides. The lamprey MCa receptor had relatively high affinity for ACTH derived peptides similarly to the fish MC receptors. We found that both of the lamprey MC receptors were expressed in skin, while the MCb receptor was also found in liver, heart and skeletal muscle. CONCLUSION: This study shows presence of MC receptors in agnathans indicating early signs of specific functions of melanocortin receptor subtypes.
Assuntos
Evolução Molecular , Petromyzon/genética , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/genética , Hormônio Adrenocorticotrópico/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Transformada , Cosintropina/metabolismo , AMP Cíclico/metabolismo , Duplicação Gênica , Biblioteca Gênica , Feiticeiras (Peixe)/genética , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Fragmentos de Peptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Filogenia , Pró-Opiomelanocortina/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Receptores de Melanocortina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Pele/metabolismo , Especificidade da Espécie , Vísceras/metabolismo , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , beta-MSH/metabolismo , gama-MSH/metabolismoRESUMO
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.
