title: Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors.
abstract: Human OX1 orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB1 cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX1, OX2 and CB1 receptors, C-terminally fused with either Renilla luciferase or GFP(2) green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB1 receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP(2) to CB1 produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX1-OX2 interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB1 receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.
authors: Department of Veterinary Biosciences, POB 66, FIN-00014 University of Helsinki, Finland. Electronic address: maria.jantti@helsinki.fi.
authors: Maria H
authors: MH
authors: Jäntti
authors: Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, Riga LV 1067, Latvia. Electronic address: ilona@biomed.lu.lv.
authors: Ilona
authors: I
authors: Mandrika
authors: Department of Veterinary Biosciences, POB 66, FIN-00014 University of Helsinki, Finland. Electronic address: jyrki.kukkonen@helsinki.fi.
authors: Jyrki P
authors: JP
authors: Kukkonen
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D008163:Luminescent Measurements; D064446:Orexin Receptors / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D043884:Receptor, Cannabinoid, CB1 / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D064446:Orexin Receptors; D043884:Receptor, Cannabinoid, CB1; D011993:Recombinant Fusion Proteins
keywords: Bioluminescence resonance energy transfer; Cannabinoid; G-protein-coupled receptor; Hypocretin; Orexin; dimerization