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NEUROPHARMACOLOGY

Expression of the Third Intracellular Loop of the -Opioid Receptor Inhibits Signaling by Opioid Receptors and Other G Protein-Coupled Receptors

Laboratory of Cellular Signaling and Molecular Pharmacology, Institute of Biology, National Center for Scientific Research "Demokritos", Athens, Greece

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 -opioid receptor (-i3L) to act as competitive antagonist of the receptor-G protein interface interaction. In -i3L-expressing cells, the peptide blocked high-affinity agonist binding to both the - and the µ-opioid (-OR and µ-OR) and attenuated opioid and ₂-adrenergic receptor (2AR)-dependent [³⁵S]guanosine-5'-O-(3-thio)triphosphate binding. Furthermore, -i3L expression resulted in inhibition of -, µ-OR-, and 2AR-receptor-mediated cAMP accumulation, whereas the cAMP response produced by activation of the ₂-adrenergic receptor was unaffected, suggesting that the inhibitory effects of -i3L expression were selective for G_i/G_o proteins. Moreover, although -i3L expression also attenuated drastically phospholipase C accumulation and Ca²⁺ release following µ- and -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 -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 -ORs is attenuated by the presence of -i3L, ERK activation mediated by 2AR remained unaffected. Collectively, our data demonstrate that the -i3L can be used as potent inhibitor of G protein signaling for various GPCRs that use a common pool of G proteins.

Address correspondence to: Dr. Zafiroula Georgoussi, Laboratory of Cellular Signaling and Molecular Pharmacology, Institute of Biology, National Center for Scientific Research "Demokritos", 15310 Ag. Paraskevi, Athens, Greece. E-mail: iro{at}bio.demokritos.gr