Tai X , Zhang Y , Yao J , Li X , Liu J , Han J , Lyu J , Lin G , Zhang C .

	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, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.
	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, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.

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