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

Interactions between mu-opioid receptor (mu OR) and cannabinoid CB1 receptor (CB1R) were examined by morphological and electrophysiological methods. In baby hamster kidney (BHK) cells coexpressing mu OR 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 mu OR and CB1R formed a heterodimer. Coimmunoprecipitation and Western blotting analyses also confirmed the heterodimers of mu OR and CB1R. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) or CP55,940 elicited K+ currents in Xenopus oocytes expressing mu OR 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 G alpha 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 Gqi5 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 mu OR/CB1R through PTX-insensitive G(qi5(m)) fused to each receptor. Thus, mu OR 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.