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Gen Comp Endocrinol
2015 Feb 01;212:114-23. doi: 10.1016/j.ygcen.2014.03.005.
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Molecular cloning, expression, and signaling pathway of four melanin-concentrating hormone receptors from Xenopus tropicalis.
Kobayashi Y
,
Hamamoto A
,
Hirayama T
,
Saito Y
.
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Melanin-concentrating hormone (MCH) mainly regulates feeding in mammals and pigmentation in teleosts. It acts via two G-protein-coupled receptors, MCH receptor 1 (MCHR1) and MCHR2. Although many studies exploring the MCH system in teleosts and mammals have been carried out, studies on other organisms are limited. In this study, we cloned and characterized four MCHR subtypes from the diploid species Xenopus tropicalis (X-MCHRs; X-MCHR1a, R1b, R2a, and R2b). According to a phylogenetic tree of the X-MCHRs, X-MCHR1a and R2a are close to mammalian MCHRs, while X-MCHR1b and R2b are close to teleostean MCHRs. We previously reported that the G-protein coupling capacity of the MCHR subtypes differed between mammals (R1: Gαi/o and Gαq; R2: Gαq) and teleosts (R1: Gαq; R2: Gαi/o and Gαq) in mammalian cell-based assays. By using Ca(2+) mobilization assays with pertussis toxin in CHO dhfr(-) cells, we found that X-MCHR1a promiscuously coupled to both Gαi/o and Gαq, while X-MCHR1b and R2a exclusively coupled to Gαq. However, no Ca(2+) influx was detected in cells transfected with X-MCHR2b. Reverse transcription-PCR showed that the X-MCHR mRNAs were expressed in various tissues. In particular, both X-MCHR1b and R2b were exclusively found in melanophores of the dorsal skin. In skin pigment migration assays, melanophores were weakly aggregated at low concentrations but dispersed at high concentrations of MCH, suggesting possible interactions between X-MCHR1b and R2b for the regulation of body color. These findings demonstrate that X. tropicalis has four characteristic MCHRs and will be useful for elucidating the nature of MCHR evolution among vertebrates.
Fig. 1.
Schematic diagrams depicting the relative positions of the DNA fragments of Mchr1b (A), r2a (B), and r2b (C) in Xenopus tropicalis. The DNA fragments were amplified from brain cDNAs. The boxes show the reading frames. The horizontal arrows show the relative positions and directions of the primers. The numbers show the positions on each cDNA. For details on the nucleotide sequences, see the following accession numbers in the DNA Data Bank of Japan database: AB897693 for X-Mchr1b, AB897694 for r2a, and AB897695 for r2b
Fig. 2.
Amino acid sequences of the four subtypes of X-MCHRs. Common amino acids with X-MCHR1a are shaded. Transmembrane domains (TMs; underlined) were deduced for each X-MCHR subtype. The colored amino acids show individual motifs: orange, N-glycosylation site; blue, DRY motif; red, NPxxY motif. The numbers in parentheses show the sequence identity with X-MCHR1a. The asterisks indicate other amino acid residues involved in receptor functions referred to in this paper. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3.
Phylogenetic tree for the MCHRs of fish, chicken, and mammals, including the four MCHR subtypes of Xenopus tropicalis, constructed by the neighbor-joining method.
Fig. 4.
Doseâresponse relationships of the MCH-mediated calcium mobilization in viable CHO dhfr(-) cells transfected with the four X-MCHRs. These cells were stimulated with the indicated concentrations of MCH, and the subsequent changes in the intracellular free Ca2+ levels were measured using a Flexstation Microplate Reader. (AâC) Cells expressing the XMCHR1a, R1b and R2a (filled markers) and cells pretreated with 200 ng/ml pertussis toxin (PTX; open markers). (D) Cells transfected with X-MCHR2b caused no calcium mobilization by the addition of MCH. All experiments were independently performed three times in duplicate. The representative results shown are expressed as the mean ± SEM.
Fig. 5.
Tissue distributions of Mch, Mchr1a, r1b, r2a, and r2b of Xenopus tropicalis. Total RNAs extracted from various tissues were subjected to RT-PCR using specific primers for X-Mch, X-Mchr subtypes, or β-actin as a positive control. Negative controls confirmed that the RT-PCR results were from the RNAs and not from contaminating DNA (direct PCR of the total RNA for β-actin). b, brain; p, pituitary; e, eyeball; lu, lung; a, atrium; v, ventricle; liv, liver; st, stomach; pa, pancreas; si, small intestine; li, large intestine; sp, spleen; k, kidney; m, muscle; f, fatbody: vs. ventralskin; ds, dorsal skin; t, testis; o, ovary.
Fig. 6.
Pigment-dispersing activity of Xenopus tropicalis MCH on melanophores. The asterisks indicate significant differences compared with the control value by a post hoc multiple comparison test for the KruskalâWallis test at P < 0.05.
Fig. 7.
Expression of X-Mchr1a, r1b, r2a, and r2b in cells isolated from the dorsal skin. RT-PCR was performed using total RNA extracted from melanophores (A) and non-chromatophoric dermal cells (B). The numbers indicate the Mchr subtypes. The total RNA extracts prepared from five single cells were combined. β-actin was evaluated as an internal control (C). ââMâ and ââDâ indicate melanophores and non-chromatophoric dermal cells, respectively.
Fig. 8.
Hypothesis about the molecular evolution of MCHR subtypes. On the basis of G protein selectivity and the phylogenetic tree, we have proposed a model for the molecular evolution of MCHR subtypes.
