Expression of the third intracellular loop of the delta-opioid receptor inhibits signaling by opioid receptors and other G protein-coupled receptors.

To explore the feasibility of developing inhibitors of signaling by opioid receptors and other G protein-coupled receptors (GPCRs) that use the same G protein pool, we investigated the capacity of a minigene encoding the third intracellular loop of the δ-opioid receptor (δ-i3L) to act as competitive antagonist of the receptor-G protein interface interaction. In δ-i3L-expressing cells, the peptide blocked high-affinity agonist binding to both the δ- and the μ-opioid (δ-OR and μ-OR) and attenuated opioid and α2-adrenergic receptor (α2AR)-dependent [35S]guanosine-5′- O -(3-thio)triphosphate binding. Furthermore, δ-i3L expression resulted in inhibition of δ-, μ-OR-, and α2AR-receptor-mediated cAMP accumulation, whereas the cAMP response produced by activation of the β2-adrenergic receptor was unaffected, suggesting that the inhibitory effects of δ-i3L expression were selective for Gi/Go proteins. Moreover, although δ-i3L expression also attenuated drastically phospholipase C accumulation and Ca2+ release following μ- and δ-OR stimulation, it failed to inhibit carbachol-mediated stimulation of inositol phosphate accumulation in M1-muscarinic receptor-expressing human embryonic kidney 293 cells. Finally, we also examined the effects of δ-i3L expression on the regulation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway. Our results demonstrate that, although ERK activation by μ- and δ-ORs is attenuated by the presence of δ-i3L, ERK activation mediated by α2AR remained unaffected. Collectively, our data demonstrate that the δ-i3L can be used as potent inhibitor of G protein signaling for various GPCRs that use a common pool of G proteins.