POMC-specific modulation of metabolic and immune pathways via melanocortin-3 receptor signaling

This proteomic study provides a nuanced mechanistic understanding of the signaling processes upon agonist binding to the melanocortin-3 receptor (MC3R). Utilizing thermal proteome profiling (TPP) combined with LC-MS, we uncovered the distinct influences of the endogenous agonists adrenocorticotropic hormone (ACTH), α-melanocyte-stimulating hormone (α-MSH), and γ-melanocyte-stimulating hormone (γ-MSH) on protein thermal stability and pathway activation. In our 2D-TPP study, transfected HEK293 cells for expression of MC3R were exposed to the three endogenous MC3R-ligands across several concentrations followed by incubation at several temperatures, centrifugation and LC-MS analysis of the resulting supernatants. This enabled us to assess the effects of type of ligand and concentration on the thermal stability of proteins in these cells. We employed a combination of multivariate analysis, differential expression, TPP and pathway analysis to deeply characterize the impact of MC3R activation on molecular mechanisms. All three ligands affected signaling pathways related to the immune system and energy homeostasis. While α-MSH significantly modulated the IL-6 pathway via STAT3, and γ-MSH prominently activated interferon signaling, ACTH uniquely affected NADPH-related proteins. All ligands shared involvement in the cAMP-PKA-CREB and varied impacts on PI3K and ERK pathways, crucial for energy metabolism. All proteomic data are available under DOI: https://doi.org/10.6019/PXD039945.