MON-661 Melanocortin-4 Receptor is Rapidly Ubiquitinated, Exits Primary Cilia and Undergoes Endocytosis After Activation

Abstract Disclosure: A.G. Diamant: None. I. Ojeda-Naharros: None. C. Vaisse: None. M.V. Nachury: None. MC4R is the most frequently implicated gene in obesity accounting for ∼6% of severe childhood obesity cases. This gene encodes melanocortin-4 receptor (MC4R), a G protein-coupled receptor (GPCR) expressed in a subset of hypothalamic neurons where it is essential for the regulation of food intake. MC4R responds to its endogenous ligands - the agonist α-MSH and the antagonist/inverse agonist AgRP - at the primary cilium, a signaling antenna of the cell. Surprisingly, MC4R is highly active in the absence of ligands and constitutively exits cilia in a ubiquitin- and β-arrestin-dependent manner; and suppressing this constitutive activity with AgRP causes accumulation of MC4R within cilia. Recent studies have revealed that GPCRs remain active and engage downstream signaling machinery even after endocytosis. Our aim was to characterize the fate of a MC4R molecule after activation and ciliary exit to further clarify MC4R’s role in regulating energy balance. To observe MC4R’s exit from cilia, we used a mouse kidney cell line modified to express MC4R fused C-terminally to three copies of mNeonGreen (MC4R-3NG). Ciliated cells were treated with AgRP to promote MC4R accumulation in cilia before replacing with plain medium or medium supplemented with α-MSH to contrast the effects of constitutive vs. ligand-induced activity. Cells were processed for immunofluorescence at various timepoints after removal of AgRP. We leveraged AI-based image segmentation to quantify MC4R levels in cilia, early endosomes and late endosomes in 3D. Ciliary ubiquitin was also quantified at each time point. Within 4 h of AgRP removal, ciliary ubiquitin in both groups approximately doubled from baseline levels. The half-life for ciliary depletion of MC4R-3NG after AgRP removal was 1 h with plain medium, but only 30 min with α-MSH. In both conditions, MC4R-3NG signal increased in early endosomes within 1 h and late endosomes within 2 h of AgRP removal. These data demonstrate that within a few hours of activation, ciliary MC4R is ubiquitinated, exits primary cilia and localizes to endosomes. This process occurs more rapidly when AgRP is replaced with α-MSH than plain medium, highlighting a difference in receptor trafficking dynamics between ligand-induced and constitutive activity. These experiments mimic MC4R trafficking during refeeding after a period of starvation and we speculate that ongoing MC4R activity after endocytosis may be important for the transition from fasting to a fed state. Presentation: Monday, July 14, 2025