A unique role of RGS9-2 in the striatum as a positive or negative regulator of opiate analgesia.

The signaling molecule RGS9-2 is a potent modulator of G-protein-coupled receptor function in striatum. Our earlier work revealed a critical role for RGS9-2 in the actions of the µ-opioid receptor (MOR) agonist morphine. In this study, we demonstrate that RGS9-2 may act as a positive or negative modulator of MOR-mediated behavioral responses in mice depending on the agonist administered. Paralleling these findings we use coimmunoprecipitation assays to show that the signaling complexes formed between RGS9-2 and Gα subunits in striatum are determined by the MOR agonist, and we identify RGS9-2 containing complexes associated with analgesic tolerance. In striatum, MOR activation promotes the formation of complexes between RGS9-2 and several Gα subunits, but morphine uniquely promotes an association between RGS9-2 and Gαi3. In contrast, RGS9-2/Gαq complexes assemble after acute application of several MOR agonists but not after morphine application. Repeated morphine administration leads to the formation of distinct complexes, which contain RGS9-2, Gß5, and Gαq. Finally, we use simple pharmacological manipulations to disrupt RGS9-2 complexes formed during repeated MOR activation to delay the development of analgesic tolerance to morphine. Our data provide a better understanding of the brain-region-specific signaling events associated with opiate analgesia and tolerance and point to pharmacological approaches that can be readily tested for improving chronic analgesic responsiveness.

Brain region specific actions of regulator of G protein signaling 4 oppose morphine reward and dependence but promote analgesia.

BACKGROUND: Regulator of G protein signaling 4 (RGS4) is one of the smaller members of the RGS family of proteins, which are known to control signaling amplitude and duration via interactions with G protein alpha subunits or other signaling molecules. Earlier evidence suggests dynamic regulation of RGS4 levels in neuronal networks mediating actions of opiates and other drugs of abuse, but the consequences of RGS4 actions in vivo are largely unknown. METHODS: In this study, we use constitutive and nucleus accumbens-inducible RGS4 knockout mice as well as mice overexpressing RGS4 in the nucleus accumbens via viral mediated gene transfer, to examine the influence of RGS4 on behavioral responses to opiates. We also use electrophysiology and immunoprecipitation assays to further understand the mechanisms underlying the tissue-specific actions of RGS4. RESULTS: Inducible knockout or selective overexpression of RGS4 in the nucleus accumbens reveals that, in this brain region, RGS4 acts as a negative regulator of morphine reward, whereas in the locus coeruleus RGS4 opposes morphine physical dependence. In contrast, we show that RGS4 does not affect morphine analgesia or tolerance but is a positive modulator of certain opiate analgesics, such as methadone and fentanyl. CONCLUSIONS: These findings provide fundamentally novel information concerning the role of RGS4 in the cellular mechanisms underlying the diverse actions of opiate drugs in the nervous system.

Multiple actions of spinophilin regulate mu opioid receptor function.

Spinophilin, a dendritic spine-enriched scaffold protein, modulates synaptic transmission via multiple functions mediated by distinct domains of the protein. Here, we show that spinophilin is a key modulator of opiate action. Knockout of the spinophilin gene causes reduced sensitivity to the analgesic effects of morphine and early development of tolerance but a higher degree of physical dependence and increased sensitivity to the rewarding actions of the drug. At the cellular level, spinophilin associates with the mu opioid receptor (MOR) in striatum and modulates MOR signaling and endocytosis. Activation of MOR by opiate agonists such as fentanyl and morphine promotes these events, which feedback to suppress MOR responsiveness. Our findings support a potent physiological role of spinophilin in regulating MOR function and provide a potential new target for the treatment of opiate addiction.

RGS9-2 is a negative modulator of mu-opioid receptor function.

Regulators of G-protein signaling (RGS) 9-2 is a striatal enriched protein that controls G protein coupled receptor signaling duration by accelerating Galpha subunit guanosine triphosphate hydrolysis. We have previously demonstrated that mice lacking the RGS9 gene show enhanced morphine analgesia and delayed development of tolerance. Here we extend these studies to understand the mechanism via which RGS9-2 modulates opiate actions. Our data suggest that RGS9-2 prevents several events triggered by mu-opioid receptor (MOR) activation. In transiently transfected PC12 cells, RGS9-2 delays agonist induced internalization of epitope HA-tagged mu-opioid receptor. This action of RGS9-2 requires localization of the protein near the cell membrane. Co-immunoprecipitation studies reveal that RGS9-2 interacts with HA-tagged mu-opioid receptor, and that this interaction is enhanced by morphine treatment. In addition, morphine promotes the association of RGS9-2 with another essential component of MOR desensitization, beta-arrestin-2. We also show that over-expression of RGS9-2 prevents opiate-induced extracellular signal-regulated kinase phosphorylation. Our data indicate that RGS9-2 plays an essential role in opiate actions, by negatively modulating MOR downstream signaling as well as the rate of MOR endocytosis.