2026 ECNP Congress

PS03-3055

Altered beta-arrestin-dependent regulation of a human obesity-associated melanocortin 3 receptor variant

Ceren Akcasoy — T. Yanik · Middle East Technical University, Molecular Biology and Genetics, Ankara, Türkiye

2026 ECNP Congress

Introduction

The hypothalamic melanocortin system regulates energy homeostasis. A melanocortin receptor (MCR) family member located at the arcuate nucleus (ARC), MC3R, is fully activated by gamma-melanocyte-stimulating hormone (γ-MSH), derived from pro-opiomelanocortin (POMC). Peripheral administration of γ-MSH enhances food intake by activating hypothalamic MC3R after crossing blood-brain barrier[1]. Nevertheless, MC3R knockout studies in mice show that MC3R deficiency leads to increased fat mass, reduced lean mass, and enhanced feed efficiency, highlighting the crucial role of MC3R in metabolism and energy control[2].

MC3R is a G protein-coupled receptor (GPCR) that primarily couples to Gs and increases intracellular cAMP levels. Loss-of-function MC3R variants, including human obesity-associated Thr6Lys/Val81Ile variant, cause impaired cAMP signaling[3]. GPCR signaling is tightly regulated by β-arrestin-dependent desensitization, initiated following receptor phosphorylation by GPCR kinases (GRKs). β-arrestin binding disrupts GPCR-G protein coupling and promotes receptor internalization, thus hindering further cAMP generation[4]. To investigate β-arrestin-2 and GRK2 effects on MC3R-mediated cAMP signaling, cAMP production was analyzed in HEK293 cells using SNAP-tagged wild type (WT) and Thr6Lys/Val81Ile MC3R and H187 (cAMP biosensor) under basal and ligand-stimulated conditions.

Methods

The H187 cAMP biosensor, an EPAC-based FRET probe, consists of a modified EPAC protein positioned between a donor (mTurquoise) and two tandem acceptor (Venus) fluorophores at N- and C-terminals, respectively. When cAMP is absent, the fluorophores are in Förster distance, allowing efficient FRET. Binding of cAMP leads to a conformational change in EPAC and increases the distance between fluorophores, thus reducing FRET efficiency[5].

HEK293 cells co-expressing SNAP-MC3R, β-arrestin-2, GRK2 and H187 were stimulated with 1 µM γ-MSH. Emission spectra were detected using a Varioskan Lux plate reader (Thermo Scientific, Germany), following excitation at 435 nm. Fluorescence emission signals from mTurquoise and Venus were recorded across 455-625 nm spectral range at 5 nm intervals. For each well, total emission intensity across all measured wavelengths was summed and defined as 100%, and individual wavelength values were normalized accordingly. Using these normalized spectra, acceptor-to-donor emission ratios were calculated for each experimental condition. Statistical comparisons of these ratios were performed in GraphPad Prism 10 using Brown-Forsythe and Welch ANOVA tests.

Results

Under basal conditions, WT MC3R exhibited greater cAMP signaling (0.765±0.013) compared to Thr6Lys/Val81Ile MC3R (0.936±0.013), consistent with previous results. Upon stimulation with 1 µM γ-MSH, both WT MC3R (0.699±0.011; ***p≤0.001) and Thr6Lys/Val81Ile MC3R (0.897±0.012; *p≤0.05) showed significantly increased activity. Co-expression of β-arrestin-2 and GRK2 altered basal signaling, with WT MC3R displaying decreased cAMP levels (0.799±0.009), whereas Thr6Lys/Val81Ile MC3R exhibited higher activity (0.812±0.009). Following γ-MSH stimulation, WT MC3R showed increased activity (0.765±0.004; **p≤0.01), while Thr6Lys/Val81Ile MC3R exhibited no significant change (0.811±0.010; p>0.05) relative to basal conditions.

Conclusions

Thr6Lys/Val81Ile MC3R exhibited impaired cAMP signaling, consistent with a loss-of-function phenotype. This study showed that co-expression of β-arrestin-2 and GRK2 prominently altered receptor activity, partially restoring basal signaling in Thr6Lys/Val81Ile MC3R while disrupting its ligand-induced response. These results suggested a dysregulated desensitization mechanism and altered receptor trafficking for Thr6Lys/Val81Ile MC3R, providing new insights into the regulatory role of β-arrestin-2 and GRK2 in loss-of-function MC3R signaling.

References

  1. Marks, D. L., Hruby, V., Brookhart, G., & Cone, R. D. (2006). The regulation of food intake by selective stimulation of the type 3 melanocortin receptor (MC3R). Peptides, 27(2), 259–264. https://doi.org/10.1016/J.PEPTIDES.2005.01.025
  2. Chen, A. S., Marsh, D. J., Trumbauer, M. E., Frazier, E. G., Guan, X. M., Yu, H., Rosenblum, C. I., Vongs, A., Feng, Y., Cao, L., Metzger, J. M., Strack, A. M., Camacho, R. E., Mellin, T. N., Nunes, C. N., Min, W., Fisher, J., Gopal-Truter, S., MacIntyre, D. E., … van der Ploeg, L. H. T. (2000). Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass. Nature Genetics, 26(1), 97–102. https://doi.org/10.1038/79254
  3. Feng, N., Young, S. F., Aguillera, G., Puricelli, E., Adler-Wailes, D. C., Sebring, N. G., & Yanovski, J. A. (2005). Co-occurrence of Two Partially Inactivating Polymorphisms of MC3R Is Associated With Pediatric-Onset Obesity. Diabetes, 54(9), 2663–2667. https://doi.org/10.2337/DIABETES.54.9.2663
  4. DeWire, S. M., Ahn, S., Lefkowitz, R. J., & Shenoy, S. K. (2007). Beta-arrestins and cell signaling. Annual Review of Physiology, 69, 483–510. https://doi.org/10.1146/ANNUREV.PHYSIOL.69.022405.154749
  5. Klarenbeek, J., Goedhart, J., van Batenburg, A., Groenewald, D., & Jalink, K. (2015). Fourth-Generation Epac-Based FRET Sensors for cAMP Feature Exceptional Brightness, Photostability and Dynamic Range: Characterization of Dedicated Sensors for FLIM, for Ratiometry and with High Affinity. PLoS ONE, 10(4), e0122513. https://doi.org/10.1371/JOURNAL.PONE.0122513