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The melanocortin system consists of five G protein-coupled receptors (MC1R-MC5R), the bidirectional endogenous ligands (MSH and Agouti families), and accessory proteins (MRAP1 and MRAP2). Accumulative studies of vertebrate species find high expression level of melanocortin 1 receptor (MC1R) in the dermal melanocyte and elucidate the essential roles in the skin and fur pigmentation, morphological background adaptation, and stress response. The diploid amphibian Xenopus tropicalis (xt) has been utilized as a fantastic animal model for embryonic development and studies of physiological cryptic colouring and environmental adaptiveness. However, the interaction of xtMc1r signaling with xtMrap proteins has not been assessed yet. In this study, we carried out in silico evolutionary analysis of protein alignment and genetic phylogenetic and genomic synteny of mc1r among various vertebrates. Ubiquitous expression of mrap1 and mrap2 and the co-expression with mc1r transcripts in the skin were clearly observed. Co-immunoprecipitation (ip) and fluorescent complementary approach validated the direct functional interaction of xtMc1r with xtMrap1 or xtMrap2 proteins on the plasma membrane. Pharmacological assay showed the improvement of the constitutive activity and alpha melanocyte-stimulating hormone (α-MSH) stimulated plateau without dramatic alteration of the cell surface translocation of xtMc1r in the presence of xtMrap proteins. Overall, the pharmacological modulation of xtMc1r by dual xtMrap2 proteins elucidated the potential role of this protein complex in the regulation of proper dermal function in amphibian species.
FIGURE 1 Protein alignment and phylogenetic analysis of Xenopus tropicalis Mc1r. (A) Sequence alignments of xtMc1r (XP_012817790.1) and other Mc1rs from human (NP_002377.4), mouse (NP_032585.2), monkey (NP_032585.2), bovine (NP_776533.1), chicken (NP_001026633.1), pigeon (OPJ78282.1), turtle (XP_005308247.1), European common frog (ACA28876.1); zebrafish (NP_851301.1), torafugu (AAO65548.1), coelacanth (XP_005999265.1), red stingray (BAU98230.1), elephant shark (ENSCMIT00000036457.1), lamprey Mca receptor (XP_032816350.1), and lamprey Mcb receptor (ABB36647.1).The blue, red, and yellow represent a homology over 50%, 75%, and 100%, respectively. (B) Dendrogram of Mc1rs was generated by the NJ analysis with Molecular Evolutionary Genetics Analysis (MEGA) software. Asterisk (*) indicates xtMC1R with bold letters.
FIGURE 2 Synteny analysis of Xenopus tropicalis mc1r. Synteny mapping of mc1rs among with Callorhinchus milii (elephant shark), Danio rerio (zebrafish), Xenopus tropicalis, Chrysemys picta bellii (turtle), Gallus gallus (chicken), Mus musculus (house mouse), and Homo sapiens (human). Positional conserved genes among multiple species are marked with color.
FIGURE 3 Expressional analysis of mc1r transcript in multiple tissues of Xenopus tropicalis. Expression profiles of mc1r, mrap2, and mrap2 transcript in 19 tissues from an adult female Xenopus tropicalis. Housekeeping gene β-actin was used as an internal control.
FIGURE 4 Investigation of the direct Protein interaction of xtMraps and xtMc1r proteins in vitro. (A) Co-immunoprecipitation of the HA-xtMc1r and Flag-xtMrap1 protein complex. (B) Co-immunoprecipitation of HA-xtMc1r and Flag-xtMrap2 protein complex.
FIGURE 5 Functional protein complex of xtMc1r and xtMraps on plasma membrane. (A) Formation of functional protein complex of xtMc1r and xtMrap1 on the plasma membrane. (B) Formation of functional protein complex of xtMc1r and xtMrap2 on the plasma membrane. Nuclei were shown in blue (DAPI). Scale bar = 50 μm.
FIGURE 6 Pharmacological modulation of xtMc1r signaling by xtMrap proteins. (A–D) Dose-responsive cAMP level of α-MSH (0 M, 10−11 to 10−6 M) and ACTH (0 M, 10−11 to 10−6 M) stimulated xtMc1r in presence of different amounts of xtMrap1(A, C) and xtMrap2 (B, D). Data were represented as the mean ± SEM from three independent experiments (E–H). The antagonistic effect of AgRP (10−11 to 10−6 M) to the EC80 dosage of α-MSH (E, F) or ACTH (G, H) induced xtMc1r signaling in presence of different amounts of xtMrap1(E, G) or xtMrap2 (F, H). Data were represented as the mean ± SEM from three independent experiments.
FIGURE 7 Measurement of the constitutive activity and surface translocation of xtMcar by xtMrap proteins. The constitutive activity of xtMc1r in the presence of xtMrap1 (A) or xtMrap2 (B) at ratio of 1:0, 1:1, 1:3, and 1:6. Surface expression level of the HA-tagged xtMc1r in the presence of xtMrap1 (C) or xtMrap2 (D) at ratio of 1:0, 1:1, 1:3, and 1:6. Data were represented as the mean ± SEM from three independent experiments. **p < 0.01, âââp < 0.001 and ââââp < 0.0001.
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