title: G-protein-dependency of orexin/hypocretin receptor signalling in recombinant Chinese hamster ovary cells.
abstract: Multiple signalling pathways for orexin receptors have been discovered, and most thoroughly mapped in Chinese hamster ovary K1 (CHO-K1) cells. It is also known that orexin receptors can couple to the G-protein families Gi, Gs and Gq. However, the connection between the G-proteins and the downstream signals is only vaguely established, and we now set out to resolve this for human orexin receptors expressed in CHO-K1 cells. Adenylyl cyclase (AC), phospholipase A2, C and D, and diacylglycerol lipase activities were assessed by precursor radiolabelling and chromatographic separation, and calcium by fluorescent methods. Pertussis toxin, cholera toxin and the cyclic depsipeptide, UBO-QIC a.k.a. FR900359, were used to assess the involvement of Gi-, Gs- and Gq-family G-proteins, respectively. Calcium elevations as well as activation of the phospholipases and diacylglycerol lipase were dependent on Gq, as they were fully blocked by UBO-QIC. The low-potency AC activation fully depended on Gs. Surprisingly, the assumed Gi-dependent inhibition of AC was (fully or partially) inhibited by UBO-QIC, in opposition to the previous findings of no sensitivity of Gi proteins to UBO-QIC. Orexin receptor signalling is indeed mostly Gq-driven in CHO-K1 cells, even with respect to the less clearly mapped cascades such as phospholipase A2 and C and calcium influx, underlining the importance of Gq even under physiological conditions. AC regulation warrants more studies.
authors.affiliation: Biochemistry and Cell Biology, Department of Veterinary Biosciences, POB 66, FIN-00014, University of Helsinki, Helsinki, Finland. Electronic address: jyrki.kukkonen@helsinki.fi.
authors.forename: Jyrki P
authors.initials: JP
authors.lastname: Kukkonen
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D003412:Cricetulus; D047630:Depsipeptides / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000378:metabolism; D064446:Orexin Receptors / Q000235:genetics / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D047630:Depsipeptides; C000607068:FR900359; C578630:HCRTR1 protein, human; C578626:HCRTR2 protein, human; D064446:Orexin Receptors; D011994:Recombinant Proteins; D002772:Cholera Toxin; D037342:Pertussis Toxin; D010740:Phospholipases; D008071:Lipoprotein Lipase; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000262:Adenylyl Cyclases; D002118:Calcium
keywords: Adenylyl cyclase; Calcium; Heterotrimeric G-protein; Hypocretin; Orexin; Phospholipase