G-protein-dependency of orexin/hypocretin receptor signalling in recombinant Chinese hamster ovary cells.

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.