Molecular cloning and expression of two melanin-concentrating hormone receptors in goldfish.
Melanin-concentrating hormone (MCH) is a neurohypophysial hormone and induces melanin aggregation in the skin in teleosts. MCH also has multiple roles in the central regulation of food intake in teleosts and mammals. MCH receptors (MCH-R) are among type I G-protein-coupled receptors. Here, we cloned two MCH receptors from goldfish, Carassius auratus. The amino acid sequence of goldfish MCH-R1 had 57-88% homology with fish MCH-R1 and 49-50% homology with mammalian MCH-R1, while the amino acid sequence of goldfish MCH-R2 had 72-92% homology with fish MCH-R2 and 32% homology with human MCH-R2. Phylogenetic analysis showed that these two MCH-Rs are orthologous to the respective mammalian MCH-Rs. The common amino acid residues for ligand binding, signal transduction, and receptor conformation were well conserved in these receptors, although some intracellular basic-amino-acid-rich domains, which have been shown to exist in human MCH-R1 and MCH-R2, were absent in goldfish MCH-R2. When stably expressed in HEK293 cells, both goldfish MCH-R1 and MCH-R2 displayed a strong, dose-dependent, transient elevation of intracellular calcium in response to salmon MCH (EC(50)=0.8nM and 31.8nM, respectively). In contrast to goldfish MCH-R2, goldfish MCH-R1 signaling is not sensitive to pertussis toxin, suggesting an exclusive Galphaq coupling of goldfish MCH-R1 in the mammalian cell-based assay. Reverse transcriptase PCR revealed that both MCH-R1 and MCH-R2 mRNA are distributed in various tissues in goldfish. The various tissues including the brain and skin express both MCH-R1 and MCH-R2. These results suggest that these functional receptors mediate multiple effects of MCH in goldfish.
Mizusawa
Kanta
K
Kitasato University, Sanriku, Ofunato, Iwate, Japan. mizusawa@kitasato-u.ac.jp
Saito
Yumiko
Y
Wang
Zhiwei
Z
Kobayashi
Yuki
Y
Matsuda
Kouhei
K
Takahashi
Akiyoshi
A
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005786:Gene Expression Regulation; D006054:Goldfish / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D011979:Receptors, Pituitary Hormone / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000378:metabolism
D000199:Actins; D043562:Receptors, G-Protein-Coupled; D011979:Receptors, Pituitary Hormone; C092274:melanin-concentrating hormone receptor; D002118:Calcium