Expression of the Third Intracellular Loop of the δ-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 -opioid (␦-OR and -OR) and attenuated opioid and ␣ 2 -adrenergic receptor (␣2AR)-dependent [35 S]guanosine-5Ј-O-(3-thio)triphosphate binding. Furthermore, ␦-i3L expression resulted in inhibition of ␦-, -OR-, and ␣2AR-receptormediated 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 Ca 2ϩ release following -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 -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.

Impaired M₃ Muscarinic Acetylcholine Receptor Signal Transduction Through Blockade of Binding of Multiple Proteins to its Third Intracellular Loop

“…These observations are consistent with those of other groups using a similar experimental approach with other GPCRs, such as -opioid, µ-opioid and 2 -adrenergic receptors [29]. Upon co-expression of the minigene with the M 5 subtype, we also noted low affinity levels with agonist-stimulated […”

“…Coexpressing the minigene construct with the wild-type receptor decreased G protein activation, phosphatidylinositol production, and subsequent signaling, without effecting normal ligand binding and receptor membrane expression. Our results showed that co-expression of the 3ILoop of the M 3 receptor with the M 5 subtype are similar to previous studies of the 3ILoop of the µ-opioid receptor, which altered the functionality of the intact µ-opioid receptor as well as other classes of GPCR [29].…”

Muscarinic receptor family interacting proteins: Role in receptor function

“…Previous experiments evidenced that co-expression of GPCR intracellular loops (or peptides derived from these loops) could mimic the intact receptor, interacting with the same molecular partners (Abdulaev et al, 2000;Morou and Georgoussi, 2005). In order to define protein complexes within GPCRs, we modified a TAP method suitable for the purification of associated proteins of the muscarinic acetylcholine receptor family (mAChRs), chosen as a model.…”

RGS2 and RGS4 proteins: New modulators of the κ-opioid receptor signaling

“…and Zioudrou [14]. proteins was verified by immunoblotting using the appropriate anti-223 bodies as described previously [24]. Protein samples were subjected to SDS-PAGE and electroblotted to 226 PVDF membranes.…”

“…200 nM of U-255 50,488H was added to the cells and allowed to incubate at 37°C for 256 the indicated times. Measurement of MAPK phosphorylation was 257 performed as described by Morou and Gergoussi[24].…”