Ligand selectivity between μ-opioid receptors (μ-OR) and δ-opioid receptors (δ-OR) is key for improving opioid analgesics. While the “message-address” hypothesis has been central to explaining this selectivity, we present evidence for an additional mechanism. Chimeric receptor and mutagenesis studies identify residue 2.63 in the orthosteric pocket as a key determinant of morphine's μ/δ-OR selectivity, while EM-1's selectivity involves a combination of residues at positions 2.63 and 3.29, the N-terminus, and extracellular loop 3 (ECL3). Approaches like voltage-clamp fluorometry, engineered zinc-bridge and microscale thermophoresis show that EM-1's Y¹P²W³F⁴ sequence confers over 1500-fold μ/δ-OR selectivity, driven by steric hindrance and a β-turn structure. β-Endorphin, with a more flexible sequence, binds non-selectively to both receptors. Morphine's rigid isoquinoline scaffold and broader geometry restrict it to binding through the wide ECL2-transmembrane (TM) 5 cleft, explaining its modest μ/δ-OR selectivity. These findings reveal that μ/δ-OR selectivity is driven by both “message-address” interactions and receptor-specific binding pathways, advancing opioid drug design.

Opioid receptor  /  Neuropharmacology  /  Binding pathway  /  Message–address concept  /  Analgesia  /  Receptor selectivity  /  Endomorphin-1  /  Morphine