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Title: Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB{sub 1} cannabinoid receptors

Highlights: • OX{sub 1} and OX{sub 2} orexin and CB{sub 1} cannabinoid receptor dimerization was investigated. • Bioluminescence resonance energy transfer method was used. • All receptors readily formed constitutive homo- and heteromeric complexes. - Abstract: Human OX{sub 1} orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB{sub 1} 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 OX{sub 1}, OX{sub 2} and CB{sub 1} receptors, C-terminally fused with either Renilla luciferase or GFP{sup 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. CB{sub 1} 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 maymore » place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP{sup 2} to CB{sub 1} produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX{sub 1}–OX{sub 2} 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 CB{sub 1} receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.« less
Authors:
 [1] ;  [2] ;  [1]
  1. Department of Veterinary Biosciences, POB 66, FIN-00014 University of Helsinki (Finland)
  2. Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, Riga LV 1067 (Latvia)
Publication Date:
OSTI Identifier:
22416310
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 445; Journal Issue: 2; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BIOLUMINESCENCE; CHO CELLS; COMPLEXES; CONCENTRATION RATIO; DIMERIZATION; DIMERS; ENERGY TRANSFER; FLUORESCENCE; GTP-ASES; HUMAN POPULATIONS; LUCIFERASE; MUTANTS; ORIENTATION; RECEPTORS; RESONANCE; SYMMETRY; TRANSIENTS