Ligand-dependent temporal patterns of signalling and receptor phosphorylation at NMU2

H. S. Qassam1,2, A. J. Butcher3, A. B. Tobin4, G. B. Willars1. 1Molecular and Cell biology, University of Leicester, Leicester, UNITED KINGDOM, 2Faculty of Medicine, University of Kufa, Najaf Governorate, IRAQ, 3MRC Toxicology Unit, University of Leicester, Leicester, UNITED KINGDOM, 4Institute of Molecular, Cell and Systems Biology, University of Glasgow, Glasgow, UNITED KINGDOM.

Introduction: Neuromedins U (NmU) and S (NmS) mediate effects via Family A, Gαq/11-coupled, G-protein-coupled receptors, NMU1 and NMU2. NMU2 is predominantly in the CNS suggesting it mediates central anorexigenic effects of NmU and/or NmS. We reported previously that NMU2 challenge with NmS for 5 min, followed by ligand removal, caused slower recovery of Ca2+ signalling and more sustained ERK signalling than activation with NmU, despite comparable initial Ca2+ responses (1). Here we examined p38 activation and, to consider potential differences in NMU2 phosphorylation and links to signalling differences, we employed C-terminally HA-tagged NMU2 (NMU2-HA) to determine temporal profiles of ligand-dependent receptor phosphorylation and de-phosphorylation.

Method: HEK-293 cell lines with stable expression of NMU2 or NMU2-HA were used to determine ERK activation, p38 activation and receptor phosphorylation using established methods (2).

Results: Initial experiments demonstrated NMU2-HA behaved similarly to NMU2. Thus, immunoblotting phosphorylated ERK (pERK) to indicate activation showed equivalent potency of the ligands at the two receptors (pEC50 5 min: 8.56+0.08 and 8.49+0.11 for NmU and NmS respectively at NMU2; 8.70+0.08 and 8.90+0.08 for NmU and NmS at NMU2-HA; mean+sem, n>3). Consistent with our previous data using NMU2 (1), challenge of NMU2 or NMU2-HA with NmU (30 nM, 5 min) followed by ligand removal evoked rapid activation of ERK that subsided over 3 h. In contrast, NmS (30 nM, 5 min) ERK activation was sustained until at least 3 h. Immunoblotting of phospho-p38 indicated equivalent potency of NmU and NmS on NMU2-HA-mediated p38 activation (pEC50 5 min: NmU, 8.77+0.23; NmS, 8.91+0.19). Following NmU (30 nM, 5 min) and ligand removal, p38 activity subsided to basal by 3 h, whereas following NmS (30 nM) activation was more sustained. Immunoprecipitating NMU2-HA from 32P-labelled-cells and subsequent autoradiography demonstrated significant increases in receptor phosphorylation by the two ligands (1 µM, 5 min, ~7-11-fold over basal) with NmS significantly greater. Challenge followed by ligand removal caused more prolonged receptor phosphorylation following NmS (63+8% maximum at 90 min) than NmU (basal at 90 min).

Conclusion: These data show ligand-dependent differences in MAPK signalling by NMU2 using a protocol that may be similar to transient exposure in vivo. Further, this protocol revealed ligand-dependent temporal patterns of NMU2 phosphorylation, suggesting this may contribute to ligand-dependent differences in receptor signalling and processing.

References:

1. Bahattab O et al. (2014). Proceedings of the British Pharmacological Society http://www.pa2online.org/abstracts/Vol12Issue1abst043P.pdf

2. Prihandoko R et al. (2015). Curr Protoc Pharmacol 69: 11-26.