Fig. 8: Signaling profile of monomeric MC4R mutant and MC4R chimera are altered by co-expression of MRAP2. | Nature Communications

Fig. 8: Signaling profile of monomeric MC4R mutant and MC4R chimera are altered by co-expression of MRAP2.

From: MRAP2 modifies the signaling and oligomerization state of the melanocortin-4 receptor

Fig. 8: Signaling profile of monomeric MC4R mutant and MC4R chimera are altered by co-expression of MRAP2.The alternative text for this image may have been generated using AI.

A Average concentration-response curve of cAMP response using α-MSH in transiently transfected HEK293 cells expressing SNAP-MC4R and MC4R-H158R, in the presence or absence of co-transfection with MRAP2 in a 1 + 1 ratio. ΔFRET values are normalized to forskolin response in the presence of phosphodiesterase inhibitor IBMX. Mean ± SEM (n = 13) for independent experiments (containing each three technical replicates) are shown. B pEC50 values obtained using the Epac-SH187 FRET biosensor. Results are represented in a bar chart as mean ± SEM, in which individual points represent pEC50 from independent experiments (n = 13) are shown as overlaid dot plot. One-way ANOVA has been performed with Sidak’s multiple comparisons as post-hoc test (**: adjusted p-value = 0.0035, ***: adjusted p-value = 0.0002). C Constitutive activity of the MC4R-H158R and the MC4Rchim. Results are represented in a bar chart as mean ± SEM, in which individual points represent biological replicates expressed in donor/corrected FRET ratio from independent experiments (n = 8) are shown as overlaid dot plot. One-way ANOVA has been performed with Dunnett’s multiple comparisons as post-hoc test (***: adjusted p-value = 0.0007, ****: adjusted p-value < 0.0001). D Average concentration-response curves of Rluc8-miniGs recruitment to the plasma membrane localization sensor rGFP-CAAX upon stimulation of FLAG-hMC4R-WT, FLAG-hMC4R-H158R, or FLAG-MC4Rchim with α-MSH for 45 min, in absence or presence of MRAP2 or RAMP3 in transiently transfected HEK293-SL cells. BRET values are normalized to maximal response obtained with mock condition. Data are expressed as mean ± SEM of four independent experiments. E Average concentration-response curves of β-arrestin2 recruitment to the plasma membrane localization sensor rGFP-CAAX upon stimulation of FLAG-hMC4R(WT), FLAG-hMC4R-H158R, or FLAG-MC4Rchim with α-MSH for 2 min, in absence or presence of MRAP2 or RAMP3 in transiently transfected HEK293-SL cells. BRET values are normalized to maximal response obtained with FLAG-hMC4R-WT. MRAP2 or RAMP3 plasmid DNA is co-transfected with the same amount (1 + 1) or fourfold the amount (1 + 4) of the FLAG-hMC4R plasmid DNA in their respective assay. Data are expressed as mean ± SEM of three independent experiments. Parameters graphs are displayed in Supplementary Fig. 11.

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