Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors

This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs) related to research published in “Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish” (Takahashi et al., 2016) [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.


Specifications
Type of data Text files, graphs How data was acquired Amino acid sequences were aligned using MEGA 6.0. Ligand selectivity assays were done using the CRE/Luciferase reporter assay [2]. Luminescence was measured using a Bio-Tek Synergy HT plate reader (Bio Tek, Winooski, VT, USA), and the data were analyzed and graphed using Kaleidagraph software (Synergy Software, Reading, PA, USA) Data format Raw Experimental factors Melanocortin DNAs were cloned from stingray genomic DNA or brain mRNA. Cloned DNA were expressed in Chinese Hamster Ovary cells Experimental features Sequence alignment was done using MEGA 6.0. The ligand selectivity assays were done as described in reference [3]. Data source location Kitasato University, Sagamihara, Kanagawa, Japan. University of Denver, Denver, Colorado, USA Data accessibility Data is within this article Value of the data These data are valuable for researchers participated in endocrinology of primitive fish and evolution of melanocortin systems.
These could be used as probes to explore orthologs in other cartilaginous fish such as skates, sharks and chimaeras.
The data on ligand selectivity could be useful tools for structure-function relationship studies in endocrinology and pharmacology.

Experimental design, materials and methods
In order to align the amino acid sequences of the melanocortin receptors for the Japanese stingray, D. akajei, the dogfish, S. acanthias, the elephant shark, C. milii, the goldfish, C. auratus, and the mouse, M. musculus, it was essential to identify putative transmembrane domains in each receptor sequence. To this end, the program ''MEMSAT3'' (http://bioinf.cs.ucl.ac.uk/psipred/) was used. The amino acid sequences where then aligned using the program MEGA 6.0.
To functionally express and determine the ligand selectivity of the stingray (sr) MC1R, srMC2R, srMC3R, srMC4R, and srMC5R paralogs, the nucleotide sequences for the srmcrs were separately synthesized with a V-5 epitope tag at the N-terminal of the receptor, and inserted into a pcDNA3.1 expression vector (GenScript; Picataway, NJ, USA). Each srmcr cDNA was separately transiently transfected into Chinese Hamster Ovary (CHO) cells. The CHO cells were grown at 37°C in a humidified 5%     CO 2 incubator in DMEM/F12 with 5% fetal calf serum. Each sr cDNA was co-expressed with a CRE/ Luciferase reporter plasmid [2] using the Solution T Cell Line Nucleofector Kit (Amaxa Inc., Gaithersburg, MD, USA) and program U-23 [4]. The transiently transfected cells were seeded on a 96-well plate at a density of 1 Â 10 À 5 cells/well. After 48 h in culture, the transfected cells were stimulated with either synthetic srACTH(1-24), srDes-acetyl-α-MSH, srβ-MSH, srγ-MSH, srδ-MSH, srβ-endorphin or hACTH (1-24), or NDP-MSH at concentrations ranging from 10 À 6 M to 10 À 12 M, in serum-free CHO media for four hours at 37°C. At the end of the incubation period, 100 ml of Bright-Glo luciferase assay reagent (Promega Inc., Madison, WI, USA) was added to each well, and incubated for 5 min at room temperature. Luminescence was measured with a Bio-Tek Synergy HT plate reader (Bio Tek, Winooski, VT, USA), and the dose response curves were analyzed by using Kaleidagraph software (Synergy Software, Reading, PA, USA). All experimental treatments were performed in triplicate.