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 (ERK(1/2)) 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 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. Using dominant-negative G-proteins and selective inhibitors of intracellular signalling cascades, we determined that orexin-A and orexin-B induced ERK(1/2) and p38 MAPK activation through multiple G-proteins and different intracellular signalling 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, orexin-B-induced ERK(1/2) 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 ERK(1/2) and p38 MAPK activation are regulated by differential signalling 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 this transfected system and this distinct modulation of the MAPKs through OX2R, may translate to the regulation of diverse biological actions of orexins.
Tang
Jiyou
J
Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.
Chen
Jing
J
Ramanjaneya
Manjunath
M
Punn
Anu
A
Conner
Alex C
AC
Randeva
Harpal S
HS
D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009479:Neuropeptides / Q000494:pharmacology; D064446:Orexin Receptors; D000068797:Orexins; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D010738:Type C Phospholipases / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D044385:GTP-Binding Protein alpha Subunits; C000596229:HCRT protein, human; C578626:HCRTR2 protein, human; D007295:Inositol Phosphates; D047908:Intracellular Signaling Peptides and Proteins; D050505:Mutant Proteins; D009479:Neuropeptides; D064446:Orexin Receptors; D000068797:Orexins; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases