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

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.</div

Ligand-Specific Signaling Profiles and Resensitization Mechanisms of the Neuromedin U2 Receptor

The structurally related, but distinct neuropeptides, neuromedin U (NmU) and neuromedin S (NmS) are ligands of two G protein-coupled NmU receptors (NMU1, NMU2). Hypothalamic NMU2 regulates feeding behavior and energy expenditure and has therapeutic potential as an anti-obesity target, making an understanding of its signaling and regulation of particular interest. NMU2 binds both NmU and NmS with high affinity, resulting in receptor-ligand co-internalization. We have investigated whether receptor trafficking events post-internalization are 'biased' by the ligand bound and can therefore influence signaling function. Using recombinant cell-lines expressing human NMU2, we demonstrate that acute Ca2+ signaling responses to NmU or NmS are indistinguishable and that restoration of responsiveness (resensitization) requires receptor internalization and endosomal acidification. The rate of NMU2 resensitization is faster following NmU compared to NmS exposure, but is similar if endothelin-converting enzyme-1 activity is inhibited or knocked-down. Although acute activation of extracellular signal-regulated kinase (ERK) is also similar, activation by NMU2 is longer-lasting if NmS is the ligand. Furthermore, when cells were briefly challenged before removal of free, but not receptor-bound ligand, activation of ERK and p38 mitogen-activated protein kinase by NmS is more sustained, but only NmU responses are potentiated and extended by ECE-1 inhibition. These data indicate that differential intracellular ligand processing produces different signaling and receptor resensitization profiles and add to the findings of other studies demonstrating that intracellular ligand processing can shape receptor behavior and signal transduction