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Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods
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Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent

James Ellis1, John D Pediani, Meritxell Canals

  • 1Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.

The Journal of Biological Chemistry
|October 4, 2006
PubMed

Insights

Ligands can alter the function of unrelated receptors by targeting receptor heterodimers. This novel mechanism was observed with orexin-1 and cannabinoid CB1 receptors, impacting cellular signaling pathways.

Area of Science:

  • Neuropharmacology
  • Cell Biology
  • Receptor Signaling

Background:

  • The human orexin-1 receptor (OX1R) and cannabinoid CB1 receptor (CB1R) are G protein-coupled receptors involved in various physiological processes.
  • Receptor trafficking and internalization are critical for regulating cellular responses to stimuli.
  • Heterodimerization between different receptor types can influence their individual signaling properties.

Purpose of the Study:

  • To investigate the interaction and functional consequences of co-expressing OX1R and CB1R in HEK293 cells.
  • To explore the role of receptor heterodimerization in ligand-induced and spontaneous receptor trafficking.
  • To determine if ligands targeting one receptor can modulate the function of a co-expressed, unrelated receptor.

Main Methods:

  • Inducible and constitutive expression of OX1R and CB1R in HEK293 cells.
  • Confocal microscopy and single-cell fluorescence resonance energy transfer (FRET) imaging to assess receptor localization and heterodimerization.
  • Pharmacological treatments with receptor agonists and antagonists (SR-141716A for CB1R, SB-674042 for OX1R).
  • Measurement of mitogen-activated protein kinase (MAPK) pathway activation (ERK1/2 phosphorylation) to assess receptor function.

Main Results:

  • Constitutive CB1R expression led to spontaneous internalization, while OX1R showed agonist-induced internalization.
  • Co-expression of OX1R and CB1R resulted in both receptors exhibiting spontaneous internalization, forming intracellular heterodimers/oligomers.
  • Antagonists for one receptor (SR-141716A or SB-674042) induced cell surface re-localization of both co-expressed receptors.
  • Ligands targeting one receptor altered the potency of the other receptor's agonist in activating ERK1/2 signaling specifically in co-expressing cells.

Conclusions:

  • OX1R and CB1R form functional heterodimers that influence each other's trafficking and signaling.
  • Ligands can indirectly modulate the function of unrelated receptors through their action on receptor heterodimers.
  • This study reveals a novel pharmacological paradigm where receptor heterodimerization mediates cross-talk between distinct receptor systems.

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