Ligand-specific regulation and signalling by the neuromedin U 2 receptor

Abstract

Neuromedin U receptor 2 (NMU2) is a Family A, G protein-coupled receptor (GPCR) for both neuromedin U (NmU) and neuromedin S (NmS), particularly within the CNS where effects include the suppression of feeding behaviour and increased energy expenditure suggesting an anti-obesity target. Although NMU2 preferentially activates Gαq/11, it is unclear which signalling underlies physiological outcomes or if the ligands generate different responses. Here, NMU2 signalling and regulation have been explored, including using a protocol of brief ligand exposure to match exposure patterns of peptidergic receptors in vivo.In HEK293 cells stably expressing human (h) NMU2, hNmU-25 and hNmS-33 evoked similar Ca2+ signalling, although resensitisation required only 6 h following brief (5 min) exposure to hNmU-25 but more than 6 h following hNmS-33. Activation of the mitogen-activated protein kinases (MAPKs) ERK, P38 and JNK, was more sustained following brief exposure to hNmS-33 compared to hNmU-25. NMU2 phosphorylation was increased by both ligands and inhibition of protein kinase C (PKC) reduced this by ~50%. Phosphorylation was more sustained following brief exposure to hNmS-33 than hNmU-25. Sustained challenge with either ligand sustained the recruitment of arrestin 2 and 3 to NMU2. Ligand removal resulted in loss of arrestin interaction that was slower for arrestin 2 but similar for the two ligands. This suggests that following brief exposure to hNmS-33, sustained NMU2 phosphorylation does not sustain arrestin recruitment and arrestin recruitment is not responsible for sustained MAPK activation. Five phosphorylated serine or threonine residues were identified in the C-terminus of NMU2 by mass spectrometry. Mutation of these reduced phosphorylation by both ligands and phosphorylation was abolished by PKC inhibition. Mutation of seven other serine/threonine residues reduced phosphorylation by hNmU-25 more than hNmS-33, suggesting different phosphorylation patterns. Mutation of all fourteen C-terminal serine/threonine residues abolished agonist-dependent phosphorylation. These data highlight ligand-dependent NMU2 signalling and regulation, particularly following brief ligand exposure. Such ligand dependence may be relevant to other GPCRs but their consequence to physiology and implications for drug discovery require further study.