mu-Opioid receptor forms a functional heterodimer with cannabinoid CB1 receptor: electrophysiological and FRET assay analysis.
Interactions between mu-opioid receptor (muOR) and cannabinoid CB1 receptor (CB1R) were examined by morphological and electrophysiological methods. In baby hamster kidney (BHK) cells coexpressing muOR 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 muOR and CB1R formed a heterodimer. Coimmunoprecipitation and Western blotting analyses also confirmed the heterodimers of muOR and CB1R. [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) or CP55,940 elicited K+ currents in Xenopus oocytes expressing muOR 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 Galpha protein Gqi5 that activates the phospholipase C pathway, both DAMGO and CP55,940 elicited Ca2+-activated Cl(-) currents, indicating that each agonist can induce responses through Gqi5 fused to either its own receptor or the other. Experiments with endogenous Gi/o protein inactivation by pertussis toxin (PTX) supported the functional heterodimerization of muOR/CB1R through PTX-insensitive Gqi5(m) fused to each receptor. Thus, muOR 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.
Hojo
Minoru
M
Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
Sudo
Yuka
Y
Ando
Yuko
Y
Minami
Koichiro
K
Takada
Masafumi
M
Matsubara
Takehiro
T
Kanaide
Masato
M
Taniyama
Kohtaro
K
Sumikawa
Koji
K
Uezono
Yasuhito
Y
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism; D003511:Cyclohexanols / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin / Q000494:pharmacology; D055503:Protein Multimerization; D051381:Rats; D043884:Receptor, Cannabinoid, CB1 / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D014981:Xenopus
D018118:Chloride Channels; D003511:Cyclohexanols; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D043884:Receptor, Cannabinoid, CB1; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium