title: Agonist potency differentiates G protein activation and Ca2+ signalling by the orexin receptor type 1.
abstract: The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with Gq/G11 proteins as well as with Gs and Gi proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate Gq/G11 proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+]i. No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the Galphas protein backbone with various Galpha donor sequences (Galphas/x) at the N and C termini, and measured cAMP as functional output. The Galphas/x chimeric proteins with Galpha11(Galphaq) and Galpha16 structure in the C terminus were stimulated by the OX1R. Concentration-response curves with Galphas/16 revealed an agonist potency correlation between G protein activation and the elevation of [Ca2+]i via discharge of intracellular Ca2+ stores, a feature also recognized for the muscarinic M3 receptor. However, in contrast to the M3 receptor, the OX1R elevated [Ca2+]i via influx from extracellular space at about 30-fold lower agonist concentration. The results suggest that the OX1R is linked to influx of Ca2+ through a signal pathway independent of Gq/G11 protein activation.
authors.affiliation: A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, FIN-70211 Kuopio, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Magga
authors.forename: Genevieve
authors.initials: G
authors.lastname: Bart
authors.forename: Christian
authors.initials: C
authors.lastname: Oker-Blom
authors.forename: Jyrki P
authors.initials: JP
authors.lastname: Kukkonen
authors.forename: Karl E O
authors.initials: KE
authors.lastname: Akerman
authors.forename: Johnny
authors.initials: J
authors.lastname: Näsman
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism; D009479:Neuropeptides / Q000494:pharmacology*; D018377:Neurotransmitter Agents / Q000494:pharmacology*; D064446:Orexin Receptors; D000068797:Orexins; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014161:Transduction, Genetic
chemical_list: C000596229:HCRT protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D064446:Orexin Receptors; D000068797:Orexins; D043587:Receptor, Muscarinic M3; D043562:Receptors, G-Protein-Coupled; D018013:Receptors, Neuropeptide; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D002118:Calcium