Evidence for a μ-δ opioid receptor complex in CHO cells co-expressing μ and δ opioid peptide receptors

Based on non-competitive binding interactions we suggested that μ and δ receptors associate as a μ/δ receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express μ and δ receptors, but not in cells that express just μ or δ receptors. We used CHO cells expressing the cloned human μ receptor, cloned human δ receptor, or cloned mouse δ/human μ (“dimer cell”). Cell membranes were prepared from intact cells pretreated with 100 nM SUPERFIT. [3H][d-Ala2,d-Leu5]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [3H][d-Ala2,d-Leu5]enkephalin binding to δ receptors by ∼75% and to μ receptors by ∼50% in dimer cells. SUPERFIT treatment did not decrease [3H][d-Ala2,d-Leu5]enkephalin binding to μ cells. The IC50 values observed in SUPERFIT-treated dimer cells were: [d-Pen2,d-Pen5]enkephalin (1820 nM) and morphine (171 nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen2,d-Pen5]enkephalin (5000 nM) was a competitive inhibitor. In contrast, morphine (1000 nM) lowered the Bmax from 1944 fmol/mg to 1276 fmol/mg protein (35% decrease). Both [d-Pen2,d-Pen5]enkephalin and morphine competitively inhibited [3H][d-Ala2,d-Leu5]enkephalin binding to SUPERFIT-treated μ cells. The results indicate that the μ-δ opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of μ-δ heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of μ-δ heterodimers.