Opioid-induced Down-Regulation of RGS4

Abstract

Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate -and ␦-opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ϳ60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both G␣ i/o and G␣ q proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the G␣ i/o -coupled DOR and the G␣ q -coupled M 3 muscarinic receptor (M 3 R) was increased but not signaling of the ␣ 2 adrenergic receptor or bradykinin BK 2 receptor, suggesting the development of cross-talk between the DOR and M 3 R involving RGS4. Regulator of G-protein signaling (RGS)2 proteins are a family of more than 30 molecules that control the duration of G protein-coupled receptor (GPCR)-mediated G protein signaling by acting as GTPase accelerating proteins (GAPs) (1-3). RGS4 is a small RGS protein (ϳ28 kDa) and a member of the R4 subfamily of RGS proteins with a structure consisting of the RGS homology domain (RH domain) and a small N terminus that may be important for defining specificity of action at GPCRs (1). However, RGS4 is a promiscuous GAP and shares activity at both G␣ q (4, 5) and G␣ i/o proteins (6, 7). The protein is unstable and subject to degradation by the N-end rule pathway (8 -12). RGS4 is also regulated by transcription and RNA stabilization (13). This means that levels of this protein can be readily regulated. Indeed, stress and several drugs of abuse have been shown to alter RGS4 levels (13-15).RGS4 has a wide distribution in brain, including regions important for the action of analgesic drugs acting at -opioid receptors (MOR) such as the cortex, thalamus, caudate putamen, nucleus accumbens, amygdala, locus ceruleus, periaqueductal gray, and dorsal horn of the spinal cord (16). Consequently, RGS4 has been linked to the regulation of MOR signaling in several in vitro systems (17-19), and a RGS4 knockout mouse shows phenotypic behaviors in response to MOR opioid agonist (20). In support of a role for RGS4 in the pharmacology of M...

μ-Opioid receptors and regulators of G protein signaling (RGS) proteins: From a symposium on new concepts in mu-opioid pharmacology

“…Consequently, to study MOR agonist regulation of RGS proteins we again turned to the SHSY5Y cell system. Unlike the PC12 cell system we observed no up-regulation of RGS4 following acute MOR agonist treatment, but did find that chronic treatment with the MOR agonists morphine or DAMGO led to a marked down regulation of RGS4 at the protein level, with no change in mRNA (Wang and Traynor, 2011), again indicating a disconnect between message and protein. This reduction in RGS4 was surprising, given that RGS4 does not modulate MOR signaling in these cells, but could support our hypothesis that there is redundancy of GAP activity at MOR (Wang et al, 2009).…”

“…In the presence of MOR agonist there is increased ubiquitination and degradation. The reduction in RGS4 protein was accompanied by marked MOR down-regulation, possible suggesting that RGS4 is stabilized in a complex with receptor and Gα protein, especially since the N-teminus is the site for both ubiquitination (Davydov et al, 2000) and for receptor interaction (Zeng et al, 1998; Wang et al, 2009) This reduction in cellular RGS4 levels causes enhanced signaling at co-expressed DOR and M3 receptors which are negatively modulated by RGS4, but not α2 adrenoceptors or bradykinin BK2 receptors which are not sensitive to RGS4 modulation (Figure 4; Wang and Traynor, 2011). The findings indicate that RGS4 modulation provides a mechanism for cross-talk between co-expressed receptors and may contribute to homeostatic changes in response to chronic opioid exposure.…”

The delta opioid receptor tool box

“…Like its parent line, SH-SY5Y cells express delta opioid receptors to a lesser extent than mu (Kazmi and Mishra, 1987; Yu and Sadee, 1988). SH-SY5Y cells also express ORL-1 receptors (Cheng et al, 1995), and signaling proteins like the Regulator of G protein signaling 4 (RGS4) (Lambert et al, 1989; McDonald et al, 1994; Wang et al, 2009; Levitt et al, 2011; Wang and Traynor, 2011). Given its repertoire of signaling proteins, SH-SY5Y cells have been widely used for the study of mu and delta opioid receptor activity, and receptor desensitization (Elliott et al, 1997; Nowoczyn et al, 2013).…”

Small Molecule Inhibitors of Regulators of G Protein Signaling (RGS) Proteins

“…It is broadly, but heterogeneously, expressed in the central nervous system (CNS), 6,7 less so in peripheral tissues, and has been shown to modulate activity of the mu opioid (MOP), delta opioid (DOP) and M3 muscarinic receptors amongst others. 8,9 The selectivity in regional distribution, coupled with the finding that RGS4 can selectively suppress signalling through DOP receptors compared to MOP receptors, 10 suggests a level of specificity for RGS4 inhibitory action will be possible.…”