A Setmelanotide-like Effect at MC4R Is Achieved by MC4R Dimer Separation

Abstract

Melanocortin 4 receptor (MC4R) is part of the leptin-melanocortin pathway and plays an essential role in mediating energy homeostasis. Mutations in the MC4R are the most frequent monogenic cause for obesity. Due to increasing numbers of people with excess body weight, the MC4R has become a target of interest in the search of treatment options. We have previously reported that the MC4R forms homodimers, affecting receptor G_s signaling properties. Recent studies introducing setmelanotide, a novel synthetic MC4R agonist, suggest a predominant role of the G_q/11 pathway regarding weight regulation. In this study, we analyzed effects of inhibiting homodimerization on G_q/11 signaling using previously reported MC4R/CB1R chimeras. NanoBRET^TM studies to determine protein-protein interaction were conducted, confirming decreased homodimerization capacities of chimeric receptors in HEK293 cells. G_q/11 signaling of chimeric receptors was analyzed using luciferase-based reporter gene (NFAT) assays. Results demonstrate an improvement of alpha-MSH-induced NFAT signaling of chimeras, reaching the level of setmelanotide signaling at wild-type MC4R (MC4R-WT). In summary, our study shows that inhibiting homodimerization has a setmelanotide-like effect on G_q/11 signaling, with chimeric receptors presenting increased potency compared to MC4R-WT. These findings indicate the potential of inhibiting MC4R homodimerization as a therapeutic target to treat obesity.

Keywords: GPCR; Gq/11; MC4R; homodimer; melanocortin 4 receptor; obesity; signaling.

Figure 1

Arrangement of MC4R/CB1R chimeric constructs. The table illustrates amino acid sequences and regions interchanged with the CB1R via overlap PCR. Chimera specification: Chim 1: substitution of TMH3; Chim 2: substitution of TMH4; Chim 3: substitution of TMH3 and TMH4 regions; Chim 4: substitution of ICL2 and intracellular parts of TMH4; Chim 5: subsitution of ICL2; Chim 6: substitution of intracellular parts of TMH3, ICL2, and intracellular parts of TMH4; Chim 7: substitution of intracellular parts of TMH3 and ICL2 [21]. Cloning of HaloTag (HT) and NanoLuc (NL) was performed using Sgfl and EcoICRI enzymes. The HT Tag is situated at the C-terminal end of the gene after a HT7 linker and a TEV protease recognition sequence. The NL is also situated at the C-terminal, NL protein coding region following after a Linker (region 14471461). The vectors were obtained from Promega, and cloning was conducted according to Flexi^® Vector Systems protocol.

Figure 2

Receptor dimerization of MC4R chimeras compared to MC4R-WT. Dimerization of chimeric MC4R/CB1R receptors and H158R gain of function mutation was analyzed by performing NanoBRET™ assays. HEK293 cells were co-transfected with either BRET partners, C-terminally tagged with the energy donor NanoLuc or the protein tag HaloTag, able to bind the energy acceptor, the NanoBRET^TM ligand 618. (A) CB1R is a non-interactive partner of MC4R and served as the negative control. Data are shown as BRET ratio in milliBRET units (mBU). (B) ΔBRET values were calculated as difference between BRET ratios of chimeras and MC4R-WT. Negative values represent a decrease in dimerization capacities compared to MC4R-WT dimerization. Data represent three independent experiments, each performed in triplicate. Values represent mean ± SEM of calculated BRET ratios. A one-way ANOVA with Kruskal–Wallis test was performed for statistical analysis, and the mean of the WT column was compared to the mean of all the other columns. Statistical significance was defined as ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 3

G_s signaling properties of MC4R-WT and chimeric receptors under alpha-MSH and setmelanotide stimulation. HEK293 cells were transfected with WT receptor or chimeric receptors and GloSensor™ reporter. Cells were stimulated with (A) alpha-MSH (1 µM) or (B) setmelanotide (1 µM) and assayed for increase in cAMP. The arrow indicates the start of ligand stimulation. cAMP accumulation was assessed over time and quantified in relative light units (rlu). (C) Chimeric receptors showed a higher basal G_s activity compared to MC4R-WT. The data are shown as area under the curve (AUC) of luminescence values from live cell cAMP accumulation. For statistical analysis, a one-way ANOVA with Kruskal–Wallis test was performed, comparing area under the curve of MC4R-WT to chimeric receptors stimulated with alpha-MSH or setmelanotide. The results turned out to be non-significant. Data represent four independent experiments, each performed in triplicate. Values represent mean ± SEM.

Figure 4

G_q/11 signaling of chimeric receptors and the H158R mutation compared to MC4R-WT. NFAT-reporter gene assay quantifying relative light units (rlu) to determine G_q/11 signaling capacities. HEK 293 cells were transfected with MC4R-WT, chimeric receptors, or H158R mutation. (A) Basal activity and 1 µM stimulated values of chimeric receptors and the H158R mutation compared to MC4R-WT. Basal activity of chimeric receptors is very similar to MC4R-WT. Chim 4, Chim 5, Chim 6, and Chim 7 displayed significant alpha-MSH induced increase in G_q/11 activity compared to alpha-MSH stimulated MC4R-WT. Values represent fold over 1 µM stimulated MC4R-WT. For statistical analysis, a one-way ANOVA with Kruskal–Wallis test was performed, comparing MC4R-WT to chimeric receptors and the H158R mutation. Values represent mean ± SEM. Statistical significance is indicated by * p < 0.05, *** p < 0.001, and **** p < 0.0001. Concentration response curves of Chim5, Chim 6, and Chim 7 cells were stimulated with different concentrations of (B) alpha-MSH or (C) setmelanotide. Concentration response curves of Chim 4 and H158R mutation were stimulated with different concentrations of (D) alpha-MSH or (E) setmelanotide. Data are given as raw rlu. Data represent WT (alphaMSH n = 10, setmelanotide n = 17), H158R (n = 4), Chim 4 (alpha-MSH n = 5, setmelanotide n = 7), Chim 5 (n = 4), Chim 6 (alpha-MSH n = 4, setmelanotide n = 3), and Chim 7 (alpha-MSH n = 9, setmelanotide = 10) experiments, each performed in triplicate. Values represent mean ± SEM. E_max and EC₅₀ values are summarized in Table 1.

Figure 5

NFAT reporter gene assay investigating signaling profile of PTX pretreated MC4R-WT-WT and Chim 7. Calcium mobilization was measured using NFAT reporter gene assay. HEK293 cells were pretreated with PTX or not, assessing possible increase in Ca²⁺ due to G_i signaling effects. Data are given in percentage of MC4R-WT-WT signaling. Cells were stimulated with (A) alpha-MSH (fold over MC4R-WT at 1 µM was set as 100%) or with (B) setmelanotide (fold over MC4R-WT at 1 µM was set as 100%). E_max and EC₅₀ values are summarized in Table 2. Data represent two to nine independent experiments, each performed in triplicate. Values represent mean ± SEM.

Figure 6

EC₅₀ of G_q/11 signaling of MC4R-WT stimulated with setmelanotide compared to EC₅₀ of Chim 7 stimulated with alpha-MSH. Concentration–response curves were used to determine potency. EC₅₀ of MC4R-WT after setmelanotide stimulation (4.04 ± 0.5 nM) appeared similar to EC₅₀ of Chim 7 after alpha-MSH stimulation (6.33 ± 1.1nM). Data are given as the result of 9 to 17 independent experiments performed in triplicate. Values represent mean ± SEM.