Opioid-induced Down-Regulation of RGS4: role of ubiquitination and implications for receptor cross-talk

Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2011; 286(10):7854-64. DOI: 10.1074/jbc.M110.160911
Source: PubMed


Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate μ- and δ- 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 ∼60% 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 Gαi/o and Gαq proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the Gαi/o-coupled DOR and the Gαq-coupled M3 muscarinic receptor (M3R) was increased but not signaling of the α2 adrenergic receptor or bradykinin BK2 receptor, suggesting the development of cross-talk between the DOR and M3R involving RGS4.

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    • "In neuronal cell lines, expression of Rgs4 is reduced after treatment with nerve growth factor [13], cAMP [14] or camptothecin [15], whereas opioid receptor agonists lead to an increase in the expression levels of Rgs4 mRNA [16] and RGS4 protein [17]. Administration of corticosterone to adult rats decreases the level of Rgs4 mRNA in the paraventricular nucleus of the hypothalamus and increases the levels in locus coeruleus [18], but has no effect in the hippocampus [19], [20]. "
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    ABSTRACT: Regulator of G-protein Signaling 4 (RGS4) plays an important role in regulating smooth muscle contraction, cardiac development, neural plasticity and psychiatric disorder. However, the underlying regulatory mechanisms remain elusive. Our recent studies have shown that upregulation of Rgs4 by interleukin (IL)-1β is mediated by the activation of NFκB signaling and modulated by extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, and phosphoinositide-3 kinase. Here we investigate the effect of the c-Jun N-terminal kinase (JNK) pathway on Rgs4 expression in rabbit colonic smooth muscle cells. Cultured cells at first passage were treated with or without IL-1β (10 ng/ml) in the presence or absence of the selective JNK inhibitor (SP600125) or JNK small hairpin RNA (shRNA). The expression levels of Rgs4 mRNA and protein were determined by real-time RT-PCR and Western blot respectively. SP600125 or JNK shRNA increased Rgs4 expression in the absence or presence of IL-1β stimulation. Overexpression of MEKK1, the key upstream kinase of JNK, inhibited Rgs4 expression, which was reversed by co-expression of JNK shRNA or dominant-negative mutants for MKK4 or JNK. Both constitutive and inducible upregulation of Rgs4 expression by SP600125 was significantly inhibited by pretreatment with the transcription inhibitor, actinomycin D. Dual reporter assay showed that pretreatment with SP600125 sensitized the promoter activity of Rgs4 in response to IL-1β. Mutation of the AP1-binding site within Rgs4 promoter increased the promoter activity. Western blot analysis confirmed that IL-1β treatment increased the phosphorylation of JNK, ATF-2 and c-Jun. Gel shift and chromatin immunoprecipitation assays validated that IL-1β increased the in vitro and ex vivo binding activities of AP1 within rabbit Rgs4 promoter. Activation of MEKK1-MKK4-JNK-AP1 signal pathway plays a tonic inhibitory role in regulating Rgs4 transcription in rabbit colonic smooth muscle cells. This negative regulation may aid in maintaining the transient level of RGS4 expression.
    PLoS ONE 04/2012; 7(4):e35646. DOI:10.1371/journal.pone.0035646 · 3.23 Impact Factor
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    • "The changes in RGS proteins caused by MOR agonists not only serve to alter MOR signaling but have the potential to regulate signaling at receptors co-expressed with MOR; this could very well have implications for maintaining cellular homeostasis in the morphine-dependent state. Knockout of RGS9-2 or RGS4 protein activity in the NAc enhances morphine-induced conditioned place preference and locomotor sensitization, and consequently, compounds that promote RGS activity -for example by stabilization of the protein (Wang and Traynor, 2011) would be expected to reduce rewarding behaviors and presumably potential to cause addiction. However, since RGS9-2 also negatively modulates the analgesic effect of morphine (Zachariou et al., 2003) enhancement of RGS9-2 activity will decrease the analgesic effectiveness of morphine. "
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    ABSTRACT: Mu-opioid receptors (MOR) are the therapeutic target for opiate analgesic drugs and also mediate many of the side-effects and addiction liability of these compounds. MOR is a seven-transmembrane domain receptor that couples to intracellular signaling molecules by activating heterotrimeric G proteins. However, the receptor and G protein do not function in isolation but their activities are moderated by several accessory and scaffolding proteins. One important group of accessory proteins is the regulator of G protein signaling (RGS) protein family, a large family of more than thirty members which bind to the activated Gα subunit of the heterotrimeric G protein and serve to accelerate signal termination. This action negatively modulates receptor signaling and subsequent behavior. Several members of this family, in particular RGS4 and RGS9-2 have been demonstrated to influence MOR signaling and morphine-induced behaviors, including reward. Moreover, this interaction is not unidirectional since morphine has been demonstrated to modulate expression levels of RGS proteins, especially RGS4 and RGS9-2, in a tissue and time dependent manner. In this article, I will discuss our work on the regulation of MOR signaling by RGS protein activity in cultured cell systems in the context of other in vitro and behavioral studies. In addition I will consider implications of the bi-directional interaction between MOR receptor activation and RGS protein activity and whether RGS proteins might provide a suitable and novel target for medications to manage addictive behaviors.
    Drug and alcohol dependence 11/2011; 121(3):173-80. DOI:10.1016/j.drugalcdep.2011.10.027 · 3.42 Impact Factor
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    Journal of Medicinal Chemistry 09/2011; 54(21):7433-40. DOI:10.1021/jm101572n · 5.45 Impact Factor
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