Journal: bioRxiv

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

doi: 10.1101/2024.04.09.588099

Figure Lengend Snippet: (A) Representative images from TIRF microscopy of the basolateral membrane of HEK293AD cells expressing SNAP-tagged MC4R labeled using Alexa Fluor 647 (gray) and Nb35-eYFP (green). Images were taken pre-(top) and 4 min post-(bottom) stimulation with 1 µM NDP-α-MSH for cells expressing SNAP-MC4R alone or with MRAP2 (at a ratio of 1:4) and indicate recruitment of Nb35 (green intensity). (B) Kinetics of recruitment of Nb35 to the SNAP-MC4R–Gs complex, without (black; 5 transfections, 51 single cells) and with MRAP2 (1+4) (turquoise; 5 transfections, 55 cells). Shown are the normalized fluorescence intensities recorded at the membrane in the eYFP channel; the shaded area represents SEM. (C) Mean concentration-response curves of Rluc8-miniGs recruitment to the plasma membrane localization sensor rGFP-CAAX upon MC4R stimulation with α-MSH for 45 minutes, in the presence or absence of MRAP2 or RAMP3, at the indicated ratio with respect to MC4R, in transiently transfected HEK293-SL cells. Normalized data are expressed as mean ± SEM of three independent experiments. (D) EC50 values from the miniGs recruitment BRET experiments. Data are expressed as mean ± SEM of three independent experiments. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons post-hoc test (*** = p < 0.001).

Article Snippet: Prior to microscopy imaging experiments, coverslips with cells expressing SNAP-MC4R and MRAP2 were labeled with 1 μM SNAP-Surface Alexa Fluor 647 dye in complete DMEM (Pan Biotech) for 25 min and kept in the incubator at 37 °C and 5 % CO 2 .

Techniques: Microscopy, Membrane, Expressing, Labeling, Transfection, Fluorescence, Concentration Assay