Antagonists Perturb the MC2R MRAP Complex and Reshape Receptor Conformations

Melanocortin 2 receptor (MC2R) is a G protein-coupled receptor (GPCR) for adrenocorticotropic hormone (ACTH), and its trafficking and signalling are associated with the melanocortin receptor accessory protein (MRAP). Mutations in either MC2R or MRAP disrupt this signalling and cause familial glucocorticoid deficiency. Here, we combine native mass spectrometry (MS) and hydrogen deuterium exchange mass spectrometry (HDX MS) to uncover how MRAP association and post-translational modification status shape the conformations of MC2R. Using native MS, we demonstrate that MC2R associates with MRAP or when MRAP is depleted the protein is extensively palmitoylated at the C-terminus. ACTH binding is restricted to the MC2R MRAP complex. By contrast antagonists shift the equilibrium toward MRAP-independent receptor populations. Our HDX MS analysis shows that ACTH binding induces global stabilisation of MC2R and the MRAP N terminus, consistent with reinforcement of the receptor accessory protein interface. Antagonist binding by contrast destabilises this interface and increases dynamics in transmembrane helix 2 (TM2). Notably, TM2 destabilisation is retained when the MRAP complex is depleted. Together, MRAP association and palmitoylation define distinct MC2R assemblies with ligand-dependent dynamics, suggesting new ways to influence MC2R pharmacology.