MRAP2 regulates energy homeostasis by promoting primary cilia localization of MC4R

Abstract

The G protein-coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor-associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.

Keywords: Endocrinology; Melanocortin; Metabolism; Neuroendocrine regulation; Obesity.

Figure 1. MRAP2 functions in MC4R-expressing cells to regulate food intake and restrain body weight.

(A and B) Body weight curve of Mc4r^{t2aCre/t2aCre} Mrap2^fl/fl versus Mc4r^{t2aCre/t2aCre} Mrap2^+/+ female (A) and male (B) littermates. (C–R) Respective body composition at 4 and 12 weeks of Mc4r^{t2aCre/t2aCre} Mrap2^fl/fl versus Mc4r^{t2aCre/t2aCre} Mrap2^+/+ females (body weight [C and D], lean mass [G and H], fat mass [K and L], percent fat mass [O and P]) and males (body weight [E and F], lean mass [I and J], fat mass [M and N], percent fat mass [Q and R]). (S–V) Twenty-four–hour food intake at 4 and 12 weeks of Mc4r^{t2aCre/t2aCre} Mrap2^fl/fl versus Mc4r^{t2aCre/t2aCre} Mrap2^+/+ females (S and T) and males (U and V). n = 11–24 mice per group were used, individual values are displayed, and exact number of mice per panel is detailed in the methods. Data are represented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed Student’s unpaired t test (column analysis; C–V), mixed-effects model (REML), and Sidak’s multiple-comparison tests (weight curves; A and B). The exact number of mice displayed in each panel is as follows, with WT indicating Mc4r^{t2aCre/t2aCre} Mrap2^+/+ and mut indicating Mc4r^{t2aCre/t2aCre} Mrap2^fl/fl. Body weight: n = 19 WT females (F); 24 mut F; 20 WT males (M); 17mut M. EchoMRI at 4 weeks old: n = 13 WT F; 18 mut F; 17 WT M; 11 mut M. EchoMRI at 12 weeks old: n = 18 WT F; 20 mut F; 17 WT M; 14 mut M. Food intake at 4 weeks old: n = 13 WT F; 18 mut F; 17 WT M; 11 mut M. Food intake at 12 weeks old: n = 18 WT F; 20 mut F; 17 WT M; 14 mut M.

Figure 2. MRAP2 localizes MC4R to the IMCD3 primary cilium.

(A) Representative widefield micrographs of IMCD3 cells transiently transfected with GFP-tagged melanocortin receptors alone (left), or cotransfected with MRAP1-FLAG (center) or MRAP2-FLAG (right). Cells are stained for cilia (AcTub, magenta), GFP-tagged melanocortin receptors (yellow), MRAP1- or MRAP2-FLAG (white), and nuclei (Hoechst 33342, cyan). MC4R-GFP and MRAP2-FLAG colocalize at the primary cilium (top panel). Scale bars: 5 μm (low-magnification images) and 2 μm (inserts). (B) Melanocortin receptor enrichment at the cilium when transfected without MRAP (blue), with MRAP1 (purple), or with MRAP2 (magenta). MRAP2 expression increases ciliary localization of MC2R, MC3R, MC4R, and MC5R but not MC1R. MRAP1 coexpression has no effect on ciliary localization. n = 20–40 ciliated cells per condition were imaged and analyzed. Ciliary and cell body intensity of melanocortin receptors and MRAPs was measured using Fiji. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of GFP-tagged melanocortin receptor or MRAP at the primary cilium than at the cell body. Data are from 3 different replicates and are represented as a superplot, with their mean and SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using ordinary 2-way ANOVA, with Tukey’s multiple-comparison test performed on replicate averages.

Figure 3. MRAP2 colocalizes with MC4R at the primary cilia in vivo.

(A–C) MRAP2 localizes to primary cilia in vivo. (A) Mice expressing a transgene encoding a ciliary GFP (Arl13-GFP) were used in this experiment. (B) Representative low-magnification image of the PVN (representative of n = 3) and nuclei (Hoescht, cyan). Magenta square indicates higher-magnification image, depicted in C. Scale bar: 200 μm. (C) Immunofluorescence image of cilia (Arl13b-GFP^tg, yellow), MRAP2 (magenta), and nuclei (Hoechst, cyan) in the mouse PVN, showing that MRAP2 localizes to primary cilia (arrows). (D–F) MRAP2 colocalizes with MC4R in vivo (arrows and boxes). (D) Mouse line expressing a GFP tag in frame at the C-terminus of the endogenous Mc4r locus. (E) Representative low-magnification image of the PVN (representative of n = 3) and nuclei (Hoescht, cyan). Magenta square indicates higher magnification image, depicted in F. Scale bar: 200 μm. (F) Immunofluorescence image of MC4R-GFP (yellow), MRAP2 (Magenta). and nuclei (Hoechst, cyan) in the mouse PVN. Indicated boxed regions are shown to the right at higher magnification. Scale bars: 20 μm (low-powered images) and 5 μm (high-powered images). Brains were collected from P6 mice. 3V, third ventricle; PVN, paraventricular nucleus of the hypothalamus. Arrowheads indicate cilia expressing both MRAP2 and arl13-GFP (A–C) or MRAP2 and MC4R (D–F).

Figure 4. MRAP2 is required for MC4R localization to the primary cilia.

(A and B) Immunofluorescence images of MC4R-GFP (yellow), cilia (ADCY3, magenta) and nuclei (Hoechst, cyan). Top panels: dashed lines delineate the PVNs. Scale bar: 50 μm. Bottom panels: High-magnification images. Scale: 5 μm. Arrowheads indicate MC4R-GFP⁺ cilia. (C) Quantitation of MC4R-GFP fluorescence intensity at the primary cilium (in arbitrary units). MC4R ciliary localization is decreased in the absence of MRAP2. (D) Quantitation of MC4R enrichment at the primary cilium as: (integrated density at the cilium)/(integrated density in an equal area of the cell body). Enrichment > 1 indicates higher localization of MC4R-GFP at the primary cilium than at the cell body. MC4R localization was quantified from 2 PVNs per P6 Mc4r^gfp Mrap2^+/+ (n = 2) and Mc4r^gfp Mrap2^–/– (n = 3) mice; 60–90 cilia we quantified per mouse. Data are represented as superplots displaying individual cilia intensity and enrichment values (small dots), and 2-tailed Student’s t tests were performed on the averages per mouse (big dots), represented as mean ± SEM; *P < 0.05, **P < 0.01.

Figure 5. MRAP2 is required in the adult PVN for MC4R ciliary localization.

(A) Experimental design. Mrap2^fl/fl Mc4r^gfp mice were injected unilaterally with AAV encoding mCherry-IRES-Cre (n = 4, 8-week-old females) and analyzed 3 weeks following injection. (B) Representative low-magnification image of PVN, mCherry (magenta), and nuclei (Hoescht, cyan). Orange squares indicate higher-magnification images depicted in C. Scale bar: 200 μm. (C) Higher-magnification images of inserts from B (orange squares). Immunofluorescence staining of MRAP2 (white) of the control contralateral and experimental, ipsilateral PVN with mCherry (magenta), and nuclei (Hoescht, cyan). Arrows indicate MRAP2⁺ cilia, absent from mCherry-expressing cells. Scale bar: 50 μm. (D) Immunofluorescence staining of control, contralateral PVN, and experimental, ipsilateral PVN for, on the left, mCherry (magenta) and nuclei (Hoescht, cyan); in the center, ADCY3 (white); and, on the right, MC4R-GFP (yellow). White squares indicate regions imaged for higher-magnification images in the middle and right. ADCY3 is not altered by loss of MRAP2, while MC4R localization to neuronal primary cilia is abrogated by loss of MRAP2. Arrows indicate MC4R⁺ cilia. Scale bars: 20 μm. (E) Quantification of the total number of cilia (ADCY3⁺) and MC4R⁺ cilia per image in the control, contralateral PVN, and experimental, ipsilateral PVN. (F) Quantification of the percentage of MC4R⁺ cilia remaining in the ipsi-AAV–injected side compared with the contralateral side. Data are represented as mean ± SEM; *P < 0.05, **P < 0.01 using 2 way ANOVA with Sidak’s multiple-comparison test (E) and 2-tailed Student’s t test (F).