μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB1 Receptor: Electrophysiological and FRET Assay Analysis

Minoru Hojo; Yuka Sudo; Yuko Ando; Koichiro Minami; Masafumi Takada; Takehiro Matsubara; Masato Kanaide; Kohtaro Taniyama; Koji Sumikawa; Yasuhito Uezono

doi:10.1254/jphs.08244FP

Full Papers

μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB₁ Receptor: Electrophysiological and FRET Assay Analysis

Minoru Hojo, Yuka Sudo, Yuko Ando, Koichiro Minami, Masafumi Takada, Takehiro Matsubara, Masato Kanaide, Kohtaro Taniyama, Koji Sumikawa, Yasuhito Uezono

Author information

Minoru Hojo
Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Yuka Sudo
Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Department of Molecular and Cellular Biology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Yuko Ando
Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Koichiro Minami
Department of Anesthesiology and Critical Care Medicine, Jichi Medical University, Japan
Masafumi Takada
Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Takehiro Matsubara
Department of Molecular and Cellular Biology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Masato Kanaide
Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Kohtaro Taniyama
Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Koji Sumikawa
Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Yasuhito Uezono
Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Department of Molecular and Cellular Biology, Nagasaki University Graduate School of Biomedical Sciences, Japan

Keywords: μ-opioid receptor, cannabinoid CB₁ receptor, receptor heterdimerization, fluorescence resonance energy transfer (FRET), electrophysiology

JOURNAL FREE ACCESS

2008 Volume 108 Issue 3 Pages 308-319

DOI https://doi.org/10.1254/jphs.08244FP

View "Advance Publication" version (November 13, 2008).

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Abstract

Interactions between μ-opioid receptor (μOR) and cannabinoid CB₁ receptor (CB₁R) were examined by morphological and electrophysiological methods. In baby hamster kidney (BHK) cells coexpressing μOR fused to the yellow fluorescent protein Venus and CB₁R fused to the cyan fluorescent protein Cerulean, both colors were detected on the cell surface; and fluorescence resonance energy transfer (FRET) analysis revealed that μOR and CB₁R formed a heterodimer. Coimmunoprecipitation and Western blotting analyses also confirmed the heterodimers of μOR and CB₁R. [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin (DAMGO) or CP55,940 elicited K⁺ currents in Xenopus oocytes expressing μOR or CB₁R together with G protein activated-inwardly rectifying K⁺ channels (GIRKs), respectively. In oocytes coexpressing both receptors, either of which was fused to the chimeric Gα protein G_qi5 that activates the phospholipase C pathway, both DAMGO and CP55,940 elicited Ca²⁺-activated Cl⁻ currents, indicating that each agonist can induce responses through G_qi5 fused to either its own receptor or the other. Experiments with endogenous G_i/o protein inactivation by pertussis toxin (PTX) supported the functional heterodimerization of μOR/CB₁R through PTX-insensitive G_qi5(m) fused to each receptor. Thus, μOR and CB₁R form a heterodimer and transmit a signal through a common G protein. Our electrophysiological method could be useful for determination of signals mediated through heterodimerized G protein–coupled receptors.

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