Interaction of RMRAP2 and RMrap2a/b with mMC4R and zMc4r. (A,B) Multiple sequence alignment by MUSCLE (3.8) of wild (A) and reversed (B) zebrafish Mrap2a/b, mouse MRAP2 and human MRAP2. Red frame indicates the transmembrane region. (C,G) Negative control. Mouse RAMP3 did not interact with MC4R. (D–F) Interactions of MRAP2 with MC4R proteins. Coimmunoprecipitation of 3xHA-Mc4r with 2xFlag-RMrap2a (D) or 2xFlag-RMrap2b (E) or mouse MC4R and RMRAP2 (F) in HEK293T cells. MC4R was detected with mouse anti-HA antibody; MRAP2 was detected with mouse anti-Flag antibody. IP: protein samples with anti-HA immunoprecipitation. Lysate: relevant protein samples to the samples in the IP group but without any immunoprecipitation. (G–J) Corresponding immunofluorescence of co-localization of protein complexes on the cell surface. Green indicates zMc4r or mMC4R, and red indicates RMRAP2s. Scale bar = 10 μm. mMC4R: mouse MC4R, zMc4r: zebrafish Mc4r, RMrap2a: reversed zebrafish mrap2a, RMrap2b: reversed zebrafish mrap2b, mRMRAP2: mouse MC4R. The uncropped Western blot figures were presented in Figure S2.

Formation of homodimers and heterodimers of RMRAP2 and RMrap2a/b. (A,B) The dimerization of RMrap2a with itself (A) or with Mrap2a (B). The blue marker indicates approximate Mol. wt. of RMrap2a and Mrap2a. (C,D) The dimerization of RMrap2b with itself (C) or with Mrap2b (D). (E) Co-immunofluorescence of RMrap2a and RMrap2b. (F,G) The dimerization of mouse RMRAP2 with itself (J) or with MRAP2 (K). (H–N) Immunofluorescence of the co-localization of the CoIP protein complexes on the plasma membrane. Scale bar = 10 μm. The uncropped Western blot figures were presented in Figure S2.

RMrap2a/b and RMRAP2 form antiparallel dimers on the plasma membrane. (A) Schematic illustration of parallel and antiparallel dimers of WT MRAP2s (left), RMRAP2s (middle) as well as WT MRAP2 and RMRAP2 dimers (right). (B–E) The schematic diagrams illustrate the principle of YFP fluorescence emission and the localization of YFP-F1/F2 on the fused protein. Red: RMrap2a, blue: RMrap2b, yellow: RMRAP2. (F–I) YFP fluorescent and DAPI under confocal microscope. Scale bar = 10 μm.

Inhibition of the membrane trafficking of zMc4r by RMrap2a and RMrap2b. (A–C) The surface expression of zMc4r (A,B) and mMC4R (C) with the increasing dosages of RMrap2a/b and RMRAP2. Mock: blank control transfected with pcDNA3.1. Data were analyzed by one-way ANOVA and are shown as mean ± SEM of three replicates. ns (not significant difference), * p < 0.05, ** p < 0.01 and *** p < 0.001.

Pharmacological modulation of zMc4r signaling by RMrap2a/b. (A–C) Dose–response stimulation of MC4R by α−MSH−induced cAMP production in the presence of different amounts of RMrap2a/b (A,B) or RMRAP2 (C). (D–F) Dose–response inhibition of MC4R by antagonist SHU9119 in the presence of different amounts of RMrap2a/b (D,E) or RMRAP2 (F). (G–I) The constitutive activity of MC4R in different dosages of RMRAP2. Data were analyzed by one−way ANOVA and shown as mean ± SEM of three replicates. ns (not significant difference), * p < 0.05, *** p < 0.001 and **** p < 0.0001.

Wild−type and reversed Mrap2a/b differently affect the pharmacological profiles of zMc4r. (A–C) Dose−responsive stimulation of MC4R by α−MSH−induced cAMP production with different ratios of wild-type and reversed Mrap2a/b (A,B) or MRAP2 (C). (D–F) Dose−responsive inhibition of MC4R by antagonist SHU9119 with different ratios of wild-type and reversed Mrap2a/b (D,E) or MRAP2 (F). (G–I) The basal cAMP level caused by transfected MC4R, MRAP2 and RMRAP2 in the absence of an agonist. Data were analyzed by one−way ANOVA and shown as mean ± SEM of three replicates. ns (not significant difference), * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. 2a: Mrap2a, R2a: RMrap2a, 2b: Mrap2b, R2b: RMrap2b, M2: MRAP2, RM2: RMRAP2.