2026 ECNP Congress

PS03-3054

Melanocortin peptides and metabolic control: γ-MSH vs α-MSH in MC3R–GHSR1A signaling

Seyda Tugce Durhan — Z.N. Koyuncu, S. Yiğit, T. Yanik · Middle East Technical University, Biochemistry, Ankara, Türkiye

2026 ECNP Congress

Introduction

The melanocortin 3 receptor (MC3R) is a G-protein coupled receptor that couples with Gαs protein and specifically expressed in hypothalamus; arcuate nucleus and paraventricular region. MC3R is known as a regulator of energy metabolism with cAMP regulated secondary messenger pathway. In addition to its role in weight gain and appetite regulation mechanisms, recent studies have shown that MC3R is important for growth, puberty, circadian rhythm, and liver autophagy pathways[1]. Research has indicated that wild type (WT) MC3R and growth hormone secretagogue receptor-1A (GHSR-1A) receptors dimerize and increase MC3R-induced cAMP signaling with general MC3R and MC4R agonist, α-MSH but not with GHSR-1A agonist ghrelin[2].

Aims

Recent studies showed that GPCRs, specifically MC3R, heterodimerization and signaling partners were ligand selectively alters [3]. In this study our aim was to investigate whether MC3R-GHSR-1A heterodimerization efficiency changes between MC3R specific γ-MSH and general MC3R/MC4R agonist α-MSH. Hence, MC3R-GHSR-1A heterodimerization and MC3R-Gαs protein coupling efficiencies were compared with α-MSH and γ-MSH by using spectral FRET technique.

Methods

Spectral FRET (Förster Resonance Energy Transfer) technique can be used to detect protein interactions depending on the energy transfer from donor fluorescent protein to acceptor fluorescent protein. When donor fluorophore excited with its specific wavelength, it excites ground state non-excited acceptor fluorophore by long-range dipol-dipol interaction between donor and acceptor fluorophore that are located in between 10-100 angstrom distance[4].

In this study, WT MC3R-GHSR-1A heterodimerization efficiency and Gαs protein couplings were compared with specific MC3R agonist γ-MSH and MC3R/MC4R agonist α-MSH by using spectral FRET. MC3R-EGFP with GHSP-1A-mCherry or Gαs-mCherry plasmids were transfected into HEK 293 cells. EGFP in pcDNA 3.1(-) and Gap43 mCherry in pcDNA3.1(-) was used as negative FRET control. After 24h, cells were inoculated into black 96-well plates, then they were stimulated with 1µM α-MSH or γ-MSH. Spectral FRET measurements were conducted by using Varioskan LUX (Thermo Scientific,USA), and the excitation signal was 450nm to excite donor EGFP and emission spectrum was collected with 5nm intervals between 470-650nm to measure both donor and the acceptor emission signals. Emission signals were normalized and the FRET/Donor emission signal ratio was calculated[5]. After the Shapiro-Wilk normality test, MC3R-GHSR-1A data analyzed with One-Way ANOVA and MC3R-Gαs data analyzed with Kruskal-Wallis test.

Co-localization images of the interactions were taken by FloID (Thermo Scientific,USA) and created by ImageJ.

Results

MC3R-GHSR-1A heterodimerization efficiency were found as statistically significantly higher in γ-MSH (0.062±0.004) than α-MSH (0.052±0.003) (*p≤0.05, n=3). Both groups were not found statististically different than PBS control group (0.060±0.006) and all groups were found statistically significantly different than negative FRET (0.043±0.001) group (PBS:***p≤0.0001,α-MSH:*p≤0.05,γ-MSH:****p≤0.00001). Alhough there was an increase trend, MC3R-Gαs γ-MSH (0.078±0.005) group, did not found as statistically significantly different than α-MSH (0.072±0.003). Both groups did not found statistically significantly different than PBS added heterodimer (0.056±0.012). All groups were found statistically significantly different than the negative FRET (0.029±0.002)(PBS:*p≤0.05,α-MSH:**p≤0.001, γ-MSH:***p≤0.0001). The co-localization images were showed that both MC3R-GHSR-1A and Gαs heterodimerization co-localization was on the plasma membrabe.

Conclusions

Results showed that MC3R-GHSR-1A heterodimerization and Gαs coupling occured more with the γ-MSH stimulation.

References

  1. Yanik T, Durhan ST. Specific Functions of Melanocortin 3 Receptor (MC3R). J Clin Res Pediatr Endocrinol 2022. https://doi.org/10.4274/JCRPE.GALENOS.2022.2022-5-21.
  2. Rediger A, Piechowski CL, Yi C-X, Tarnow P, Strotmann R, Grüters A, et al. Mutually Opposite Signal Modulation by Hypothalamic Heterodimerization of Ghrelin and Melanocortin-3 Receptors 2011. https://doi.org/10.1074/jbc.M111.287607.
  3. Yang LK, Tao YX. Biased Signaling at Neural Melanocortin Receptors in Regulation of Energy Homeostasis. Biochim Biophys Acta 2017;1863:2486. https://doi.org/10.1016/J.BBADIS.2017.04.010.
  4. Sekar RB, Periasamy A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 2003;160:629. https://doi.org/10.1083/JCB.200210140.
  5. Durhan ST, Nalli Sezer E, Son CD, Kucuk Baloglu F. Fast Screening of Protein-Protein Interactions Using Förster Resonance Energy Transfer (FRET) Based Fluorescence Plate Reader Assay in Live Cells. Appl Spectrosc 2022:000370282211409. https://doi.org/10.1177/00037028221140914.