The signalling profile of recombinant human orexin-2 receptor

Orexin-A and orexin-B orchestrate their diverse central and peripheral effects via two G-protein coupled receptors, OX1R and OX2R, which activate multiple G-proteins. In many tissues, orexins activate extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase (MAPK); however, the mechanism by which OX2R alone mediates MAPK activation is not understood. This study describes the intracellular signalling pathways involved in OX2R-mediated ERK1/2 and p38 MAPK activation. In HEK-293 cells stably over-expressing recombinant human OX2R, orexin-A/B resulted in a rapid, dose and time dependent increase in activation of ERK1/2 and p38 MAPK, with maximal activation at 10 min for ERK1/2 and 30 min for p38 MAPK. Using dominant-negative G-proteins and selective inhibitors of intracellular signalling cascades, we determined that orexin-A and orexin-B induced ERK1/2 and p38 MAPK activation through multiple G-proteins and different intracellular signalling pathways. ERK1/2 activation involves Gq/phospholipase C (PLC)/protein kinase C (PKC), Gs/adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) and Gi cascades; however, the Gq/PLC/PKC pathway, as well as PKA is not required for OX2R-mediated p38 MAPK activation. Interestingly, orexin-B-induced ERK1/2 activation is predominantly mediated through the Gq/PLC/PKC pathway. In conclusion, this is the first comprehensive signalling study of the human OX2R recombinant receptor, showing ERK1/2 and p38 MAPK activation are regulated by differential signalling pathways in HEK-293 cells, and that the ERK1/2 activation is severely affected by naturally occurring mutants associated with narcolepsy. Moreover, it is evident that the human OX2R has ligand specific effects, with orexin-B being more potent in this transfected system and this distinct modulation of the MAPKs through OX2R, may translate to the regulation of diverse biological actions of orexins.