Signalling, Desensitization and Resensitization of Neuromedin U Receptors

The neuropeptides neuromedin U (NmU) and neuromedin S (NmS) show a high degree of conservation across species, primarily at the amidated C-terminus. NmU in particular is widely distributed in both the central nervous system and periphery and is involved in a plethora of physiological and pathological events. NmU and NmS mediate their actions via two family A, G protein-coupled receptors, NMU1 and NMU2, which share ~50% homology. The present study confirmed receptor coupling to Gαq/11, leading to increases in intracellular [Ca2+] and activation of extracellular signal-regulated protein kinase (ERK), as well as coupling to Gαi/o, leading to pertussis toxin-sensitive inhibition of adenylyl cyclase activity. This study also confirmed that different NmU analogues bind pseudo-irreversibly to recombinantly expressed NMUs and has shown receptor-dependent internalization of a fluorescently-tagged version of NmU. C-terminal eGFP-tagged NMUs showed co-internalization of ligand and receptor within ~2.5 min of ligand exposure. Cell-surface, receptor-bound ligand could be removed by a rapid (20 s), pH 2.0 washing without detrimental effect on signal transduction or cell viability, allowing examination of desensitization and resensitization in the absence of cell-surface, ligand-bound receptors. Desensitization of NMU2-mediated Ca2+ responses (that was independent of continued ligand binding) occurred within minutes of exposure to human (h) NmU-25. Acute exposure (5 min) to a maximum concentration of hNmU-25 followed by recovery in the absence (pH 2.0 wash) or the presence (pH 7.4 wash) of cell-surface, receptor-bound hNmU-25 showed that the continued presence of ligand markedly delayed receptor resensitization. Receptor internalization via a dynamin-dependent pathway was crucial for resensitization of NMU1 and NMU2. Further, resensitization was dependent on endosomal acidification, recycling and endothelin-converting enzyme-1 (ECE-1) activity, but not de novo protein synthesis. This suggests that processing of hNmU-25 by ECE-1 in acidified endosomes is critical for resensitization. Inhibition of ECE-1 also prolonged NMU-mediated ERK activation, suggesting G protein-independent signalling by a ligand-receptor dependent complex within endosomes. Although no significant differences were demonstrated in potency and signalling between the NMU subtypes or their ligands, resensitization (and potentially therefore G protein-dependent/independent signalling) was influenced by both the ligand (nature and length of the N-terminus) and the receptor (NMU1 versus NMU2).