Received for publication May 23, 2005.
Revised September 8, 2005.
Accepted for publication September 8, 2005.

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

Abstract

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, µ-OR) and attenuated opioid and 2-adrenergic receptor (2AR) dependent-[³⁵S]GTPS binding. Furthermore, -i3L expression resulted in inhibition of -, µ-OR and 2AR-receptor-mediated cAMP accumulation while leaving unaffected the cAMP response produced by activation of the 2-adrenergic receptor, suggesting that the inhibitory effects of -i3L expression were selective for Gi/Go proteins. Moreover, while -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 HEK293 cells. Finally, we also examined the effects of -i3L expression on the regulation of the ERK mitogen-activated protein kinase pathway. Our results demonstrate that while 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.

Key words: G protein coupled receptors, G proteins, minigene, mu and delta opioid receptors, receptor antagonism, third intracellular loop