Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation

Pharmacol Res. 2016 Sep;111:600-609. doi: 10.1016/j.phrs.2016.07.009. Epub 2016 Jul 18.


Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of 2-arachidonoylglycerol (2-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and 2-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3μM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3μM), transiently elevated [Ca(2+)]i. Incubation with a subeffective dose of OX-A (0.1μM)+ACEA (0.1μM) led to stronger and longer lasting elevation of [Ca(2+)]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM251, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.2μM), or OX-A (0.1μM)+ACEA (0.1μM), but not after ACEA (0.2μM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2μM) or OX-A (0.1μM)+ACEA (0.1μM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2(Thr202/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.2μM), was induced by incubation with OX-A (0.1μM)+ACEA (0.1μM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca(2+)]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.

Keywords: 2-Arachidonoylglycerol; ACEA (PubChem CID: 5311006); AM251 (PubChem CID: 2125); Calcium imaging; Cannabinoid receptor type 1; Fluorescence resonance energy transfer; Orexin-A (PubChem CID: 56842143); Orexin-A/hypocretin 1; SB-334867 (PubChem CID: 6604926).

MeSH terms

  • Animals
  • Arachidonic Acids / biosynthesis
  • Arachidonic Acids / pharmacology*
  • Calcium / metabolism
  • Cell Line
  • Endocannabinoids / biosynthesis
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Glycerides / biosynthesis
  • Hypothalamus / cytology*
  • Mice
  • Orexin Receptors / metabolism*
  • Orexins / pharmacology*
  • Phosphorylation / drug effects
  • Receptor, Cannabinoid, CB1 / metabolism*


  • Arachidonic Acids
  • Endocannabinoids
  • Glycerides
  • Hcrtr1 protein, mouse
  • Orexin Receptors
  • Orexins
  • Receptor, Cannabinoid, CB1
  • arachidonyl-2-chloroethylamide
  • glyceryl 2-arachidonate
  • Extracellular Signal-Regulated MAP Kinases
  • Calcium