Having a Complex is a Good Thing? Spinophilin Mediates RGS4 Activity at the Delta Opioid Receptor via Scaffolding to a Multiprotein Signaling

ComplexJustin Ross, Department of Biology, York College of Pennsylvania

Introduction

Opiates elicit a host of effects in the body including analgesia, euphoria, and respiratory depression, amongst others, due to complex signaling pathways initiated by agonist binding at opioid receptors (µ, δ, and κ)

Figure 1. Signal transduction of a G-coupled protein receptor (GPCR). A receptor agonist binds the GPCR at the extracellular binding region, causing phosphorylation of Gα which then disociates from the C-terminus and Gβγ. A regulator of G-protein signaling terminates signaling by dephosphorylating Gα.

RGS4 and RGS9-2 are highly expressed in the striatum, a brain region associated with memory, learning, and reward pathway functionality

In some instances, RGS proteins require scaffolding proteins like spinophilin and β arrestin-2 to exert their GTPase activity, but the formation and configuration of these signaling complexes is poorly understood

Spinophilin was identified as a component of a signaling complex comprised of β arrestin-2, Gβ5, and RGS9-2, and though it was suggested, it is unknown whether it also acts as a scaffolding protein for RGS4 at the delta opioid receptor.

Hypotheses/Objectives

This research will identify the necessity of the scaffolding protein spinophilin to the GTPase activity of RGS4 at the delta opioid receptor, and whether these two are components of an even larger signaling complex comprised of β arrestin-2, Gβ5, and RGS9-2.

Review of Literature

RGS proteins are divided into 4 subfamilies based upon structural similarity: RZ, R4, R7, and R12; mRNA expression of each RGS is tissue specific (Larminie et al. 2004), while recruitment and binding is receptor and agonist dependent (Georgoussi et al. 2006).

Adult mice with RGS4 knockout in the nucleus accumbens displayed increased sensitivity to the rewarding and movement agitating effects of morphine, a potent mu-opioid agonist (Zachariou et al. 2009).

Several studies indicate that RGS4 participates in signaling complexes with mu and delta opioid receptors (Charlton et al. 2008; Georgoussi et al. 2006), but the strength of association at the DOR may be greater due to interaction at both the third intracellular loop and the C-tail .

Figure 2. RGS4 has been reported to act as a GTPase accelerating protein (GAP) for delta opioid receptor signaling in several different cell systems, and is most often measured via promotion or inhibition of DOR-mediated cAMP and MAPK (Wang et al. 2009).

RGS4 binds to spinophilin and neurablin, scaffolding proteins with individual functionality, and in concert with RGS2 have been shown to modulate calcium signaling (Wang et al. 2005).

Other scaffolding proteins, such as β-arrestin2 have been shown to play permissive roles in the GAP activity of RGS9-2 by maintaining it in the open conformation (Zheng et al. 2011).

Under basal conditions, immunoprecipitation assays showed associations of spinophilin with Gβ5, RGS9-2, and the mu-opioid receptor; the strength of the association was intensified upon administration of morphine (Charlton et al. 2008).

Wang et al. (2009) were only able to show indirect mediation of DOR signaling via RGS4 in SY5Y-SH human neuroblastoma cells, and suggested that spinophilin may act as a scaffolding intermediate.

Methods

Expected Results

Expected Conclusions

RGS4, RGS9-2, and delta opioid receptor (DOR) will co-precipitate with spinophilin in striatal tissue samples excised from wild type mice under basal conditions, and these associations will be strengthened following exposure to DOR agonists.

RGS9-2, but not RGS4, will co-precipitate with DOR in striatum specific spinophilin knockout (KO) mice given delta opioid agonists. This indicates that spinophilin is an integral component of RGS4 scaffolding at DOR.

pERK and cAMP levels will increase and decrease, respectively, in WT mice relative to KO littermates. This indicates that spinophilin mediates the GTPase activating properties of RGS4 at the DOR most likely via acting as a scaffolding protein.

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Literature CitedCharlton, Joanna J., Patrick B. Allen, Kassi Psifogeorgou, Sumana Chakravarty, Ivone Gomes, Rachael L. Neve, Lakshmi A. Devi, Paul Greengard, Eric J. Nestler, and Venetia Zachariou. 2008. "Multiple actions of spinophilin regulate mu opioid receptor function." Neuron 58, no. 2: 238-247.

Georgoussi Z, Leontiadis L, Mazarakou G, Merkouris M, Hyde K, Hamm H. 2006. “Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the mu- and delta-opioid receptors regulate opioid receptor signaling.” Cell Signaling 18(6): 771-82.

Larminie, Christopher, Paul Murdock, Jean-Philippe Walhin, Malcolm Duckworth, Kendall J. Blumer, Mark A. Scheideler, and Martine Garnier. 2004. "Selective expression of regulators of G-protein signaling (RGS) in the human central nervous system." Molecular Brain Research 122, no. 1: 24-34.

Leontiadis, Leonidas J., Maria P. Papakonstantinou, and Zafiroula Georgoussi. 2009. "Regulator of G protein signaling 4 confers selectivity to specific G proteins to modulate μ- and δ-opioid receptor signaling." Cellular Signalling 21, no. 7: 1218-1228.

Wang, Xinhua, Weizhong Zeng, Abigail A Soyombo, Wei Tang, Elliott M Ross, Anthony P Barnes, and Shmuel Muallem, et al. 2005. "Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors." Nature Cell Biology 7, no. 4: 405-411.

Wang, Qin, Lee-Yuan Liu-Chen, and John R Traynor. 2009. "Differential modulation of mu- and delta-opioid receptor agonists by endogenous RGS4 protein in SH-SY5Y cells." The Journal Of Biological Chemistry 284, no. 27: 18357-18367.

Zheng, Mei, Sang-Yoon Cheong, Chengchun Min, Mingli Jin, Dong-Im Cho, and Kyeong-Man Kim. 2011. "β-arrestin2 plays permissive roles in the inhibitory activities of RGS9-2 on G protein-coupled receptors by maintaining RGS9-2 in the open conformation." Molecular And Cellular Biology 31, no. 24: 4887-4901

Acknowledgements

Thanks to Jeffrey Thompson Ph.D and the York College of Pennsylvania Biology Dept.

SP KO – C57BI/6 Mice Conditional (Striatal) Spinophilin Knockout(n=5 for each assay)

SP WT – C57BI/6 Mice(n=5 for each assay)

Coimmunoprecipitation

with Spinophilin

Coimmunoprecipitation of RGS4 and RGS9-2 with DOR

cAMP assay only (untreated striatal cells): Treatment with 10 μM forskolin for 10 min. Administration of 0.001, 0.01, 0.1, 1.0, and

10.0 μM DPDPE. cAMP assays using competitive ELISA (performed in triplicate)

No treatement for to living mice for cAMP assays. For all assays beside cAMP: administration of 60mg/kg DPDPE,

10mg/kg SNC-80, or 1mL saline (I.V.).

Rapid decapitation

and extraction of striatum

RGS4

RGS9-2

DOR

Control

RGS4

RGS9-2

Control

Spinophilin

ERK Phosphorylation

assays using Immunoblotting (performed in

triplicate)

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