| Summary: | The neuromedin U (NmU) and neuromedin S (NmS), neuropeptides, from various species share the greatest homology in their C-terminal regions. Both NmU and NmS share the seven residues located in the C terminus of their sequence, including the amidation of the C-terminal amino acids. Two family A G protein-coupled receptors have been identified for these peptides; NMU1 and NMU2. Both receptors have high affinity for both ligands but the differential expression of receptors and ligands may dictate what they do. Both receptors can signal through Gαq/11 and Gαi/o, leading to increases in intracellular [Ca2+]i and pertussis toxin-sensitive inhibition of adenylyl cyclase activity, respectively. Data presented here demonstrate that following desensitisation of NMU2, re-sensitisation is dependent on receptor internalisation, endosomal acidification and receptor recycling. Re-sensitisation is also dependent on endothelin converting enzyme-1 (ECE-1) activity, most likely through proteolysis of NmU in endosomes, which may facilitate disassociation of receptor-β-arrestin complexes resulting in NMU2 recycling and re-sensitisation. In addition to Ca2+ release, NMUs activate extracellular signal-regulated kinase (ERK) and it is possible that this may involve a number of different mechanisms. This study shows that human (h) NmU-25 and hNmS-33 evoke time- and concentration-dependent activation of ERK by NMU2. In experiments in which the free ligand was removed following a 5 minute stimulation, hNmS-33 provoked prolonged activation of ERK whereas ERK activation returned to basal level following hNmU-25. In these experiments, the ECE-1 inhibitor, SM19712, prolonged ERK activation following hNmU. Knockdown of either β-arrestin-1 or -2 with siRNA and, in particular, combined isoformic knockdown reduced re-sensitisation of Ca2+ signalling by NMU2 following desensitisation with either ligand. Knockdown of β-arrestins-1 and -2 individually or in combination significantly enhanced and extended the duration of NmU-mediated ERK activation, but had a much more limited impact on hNmS-33-mediated ERK activation. The current study indicates that ERK is likely regulated by G-protein-dependent mechanisms, mainly through Gαq, with little evidence for a role for G-protein-independent mechanisms.
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