Endocrine Abstracts

Orexin-A and orexin-B orchestrate their diverse central and peripheral effects via two G-protein coupled receptors, orexin-1 receptor (OX1R) and OX2R, which activate multiple G-proteins. In many tissues, orexins activate MAPKs, however, the mechanism by which OX2R alone mediates MAPK activation is not understood. We studied the intracellular signaling mechanisms involved in OX2R-mediated ERK_1/2 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 ERK_1/2 and p38 MAPK, with maximal activation at 10 min for ERK_1/2 and 30 min for p38 MAPK. We used dominant-negative G-proteins and selective inhibitors of intracellular signaling cascades and pertussis toxin, and determined that orexin-A and orexin-B induced ERK_1/2 and p38 MAPK activation through multiple G-proteins and different intracellular signaling pathways. ERK_1/2 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, as compared to orexin-A, the Gq/PLC/PKC pathway plays a predominant role in orexin-B-induced ERK_1/2 activation. In conclusion, this is the first comprehensive signalling study of the OX2R recombinant receptor, showing ERK_1/2 and p38 MAPK activation are regulated by differential signaling pathways in HEK-293 cells, and that the ERK_1/2 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 a transfected system. This distinct modulation of the MAPKs through OX2R may contribute to the regulation of diverse biological actions of orexins.