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.     

Phosphorylation of spinophilin modulates its interaction with actin filaments

Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission.     

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.     

Regulation of opioid receptor trafficking and morphine tolerance by receptor oligomerization

The utility of morphine for the treatment of chronic pain is hindered by the development of tolerance to the analgesic effects of the drug. Morphine is unique among opiates in its ability to activate the mu opioid receptor (MOR) without promoting its desensitization and endocytosis. Here we demonstrate that [D-Ala(2)-MePhe(4)-Gly(5)-ol] enkephalin (DAMGO) can facilitate the ability of morphine to stimulate MOR endocytosis. As a consequence, rats treated chronically with both drugs show reduced analgesic tolerance compared to rats treated with morphine alone. These results demonstrate that endocytosis of the MOR can reduce the development of tolerance, and hence suggest an approach for the development of opiate analogs with enhanced efficacy for the treatment of chronic pain.     

Congenic C57BL/6 mu opiate receptor (MOR) knockout mice: baseline and opiate effects

Homozygous µ-opioid receptor (MOR) knockout (KO) mice developed on a chimeric C57B6/129SV background lack morphine-induced antinociception, locomotion and reward. Therefore it appears that MOR largely mediates these morphine actions. However, one factor that could affect the extent of knockout deficits in morphine-induced behavior is the genetic background against which the gene deletion is expressed. To examine the effect of genetic background chimeric C57B6/129SV MOR knockout mice from the 15th generation of those developed in our laboratory were backcrossed for 10 successive generations with C57BL/6 mice, a strain which is more sensitive to many of the properties of morphine, to produce congenic MOR (conMOR) KO mice. Heterozygote conMOR KO mice display attenuated morphine locomotion and reduced morphine analgesia compared to wild-type mice. Homozygote conMOR KO mice display baseline hyperalgesia, no morphine place preference, no morphine analgesia and no morphine locomotion. These results are not qualitatively different from those observed in the MOR KO strain with a chimeric C57B6/129SV background, and suggest that although the strain has separate influences on these functions, it does not substantially interact with deletion of the µ opiate receptor gene.     

Gbetagamma that interacts with adenylyl cyclase in opioid tolerance originates from a Gs protein

We previously demonstrated that chronic morphine induces a change in G protein coupling by the mu opioid receptor (MOR) from Gi/o to Gs, concurrent with the instatement of an interaction between Gbetagamma and adenylyl cyclase types II and IV. These two signaling changes confer excitatory effects on the cell in place of the typical inhibition by opioids and are associated with morphine tolerance and dependence. Both signaling changes and these behavioral manifestations of chronic morphine are attenuated by cotreatment with ultra-low-dose naloxone. In the present work, using striatum from chronic morphine-treated rats, we isotyped the Gbeta within Gs and Go heterotrimers that coupled to MOR and compared these to the Gbeta isotype of the Gbetagamma that interacted with adenylyl cyclase II or IV after chronic morphine treatment. Isotyping results show that chronic morphine causes a Gs heterotrimer associated with MOR to release its Gbetagamma to interact with adenylyl cyclase. These data suggest that the switch to Gs coupling by MOR in response to chronic morphine, which is attenuated by ultra-low-dose opioid antagonist cotreatment, leads to a two-pronged stimulation of adenylyl cyclase utilizing both Galpha and Gbetagamma subunits of the Gs protein novel to this receptor.     

Counteraction by morphine of stress-induced inhibition of growth hormone release in the rat

The purpose of this study was to determine the effect of morphine (MOR) administration on pituitary growth hormone (GH) release during stress in the male rat. Circulating GH levels were significantly decreased following a brief (2 min) exposure to either, during repetitive etherization coupled with blood withdrawal, and during continuous immobilization. Under all three stress conditions, systemic administration of MOR resulted in a significant increase in plasma GH levels compared to the vehicle-treated group. These results indicate that the pathway for opiate-induced stimulation of GH release is functional during stress, and suggest that the suppressive effect of stress does not involve a blockade of opiate receptor stimulation of GH. Thus, the present findings, taken together with reports that the overall activity of central opioid neurons is enhanced during stress, support the view that the decline in GH is due to the overriding inhibitory influence of an independent nonopioid mechanism. However, MOR can apparently increase opiate receptor stimulation sufficiently to counteract this inhibitory signal, implying that stress and the opiates may influence GH release via separate mechanisms.     

Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of opiate withdrawal.

Morphine is unusual in its failure to promote robust desensitization and endocytosis of the mu opioid receptor (MOR), processes that for many receptors contribute directly to tolerance. This apparent paradox has led us to revise the idea that receptor desensitization and endocytosis are solely responsible for tolerance and withdrawal to morphine, and instead test the hypothesis that these side effects occur due to abnormally prolonged MOR signaling. We report here that MOR mutations that facilitate endocytosis reduce the development of cellular tolerance and cAMP superactivation, a cellular hallmark of withdrawal. Moreover, mutant receptors with reduced endocytosis produce exacerbated superactivation. These data demonstrate a critical role for receptor endocytosis in the development of adverse side effects associated with prolonged opiate use.     

Morphine activation of c-fos expression in rat brain

The post-receptor mechanism of opiate action has been studied by examining the activation by morphine of the proto-oncogene c-fos and its encoded nucleoprotein pp55c-fos (FOS) in rat caudate-putamen, which is rich in the mu-type opiate receptor. Following an acute morphine treatment, c-fos mRNA levels in rat caudate-putamen were increased to maximum (420% of control level) at 45 minutes and returned to control levels at 90 minutes. This induction was completely abolished by naloxone, a morphine antagonist. Fos protein, detected by immunocytochemistry, was also increased 3 hours after morphine injection, in the caudate-putamen, but not in the olfactory tubercle, which does not have the mu-type opiate receptor. Upon activation of opiate receptors by morphine, the c-fos gene is activated and Fos protein may act as a signal transducer uniquely involved in the mechanism of opiate addiction at the level of gene regulation.     

Gβγ that interacts with adenylyl cyclase in opioid tolerance originates from a Gs protein

We previously demonstrated that chronic morphine induces a change in G protein coupling by the mu opioid receptor (MOR) from Gi/o to Gs, concurrent with the instatement of an interaction between Gβγ and adenylyl cyclase types II and IV. These two signaling changes confer excitatory effects on the cell in place of the typical inhibition by opioids and are associated with morphine tolerance and dependence. Both signaling changes and these behavioral manifestations of chronic morphine are attenuated by cotreatment with ultra‐low‐dose naloxone. In the present work, using striatum from chronic morphine‐treated rats, we isotyped the Gβ within Gs and Go heterotrimers that coupled to MOR and compared these to the Gβ isotype of the Gβγ that interacted with adenylyl cyclase II or IV after chronic morphine treatment. Isotyping results show that chronic morphine causes a Gs heterotrimer associated with MOR to release its Gβγ to interact with adenylyl cyclase. These data suggest that the switch to Gs coupling by MOR in response to chronic morphine, which is attenuated by ultra‐low‐dose opioid antagonist cotreatment, leads to a two‐pronged stimulation of adenylyl cyclase utilizing both Gα and Gβγ subunits of the Gs protein novel to this receptor. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Mu opioid receptor activation of ERK1/2 is GRK3 and arrestin dependent in striatal neurons

In this study we investigated the mechanisms responsible for MAP kinase ERK1/2 activation following agonist activation of endogenous mu opioid receptors (MOR) normally expressed in cultured striatal neurons. Treatment with the MOR agonist fentanyl caused significant activation of ERK1/2 in neurons derived from wild type mice. Fentanyl effects were blocked by the opioid antagonist naloxone and were not evident in neurons derived from MOR knock-out (-/-) mice. In contrast, ERK1/2 activation by fentanyl was not evident in neurons from GRK3-/- mice or neurons pretreated with small inhibitory RNA for arrestin3. Consistent with this observation, treatment with the opiate morphine (which is less able to activate arrestin) did not elicit ERK1/2 activation in wild type neurons; however, transfection of arrestin3-(R170E) (a dominant positive form of arrestin that does not require receptor phosphorylation for activation) enabled morphine activation of ERK1/2. In addition, activation of ERK1/2 by fentanyl and morphine was rescued in GRK3-/- neurons following transfection with dominant positive arrestin3-(R170E). The activation of ERK1/2 appeared to be selective as p38 MAP kinase activation was not increased by either fentanyl or morphine treatment in neurons from wild type, MOR-/-, or GRK3-/- mice. In addition, U0126 (a selective inhibitor of MEK kinase responsible for ERK phosphorylation) blocked ERK1/2 activation by fentanyl. These results support the hypothesis that MOR activation of ERK1/2 requires opioid receptor phosphorylation by GRK3 and association of arrestin3 to initiate the cascade resulting in ERK1/2 phosphorylation in striatal neurons.     

Protein kinase A phosphorylation of spinophilin modulates its interaction with the alpha 2A-adrenergic receptor (AR) and alters temporal properties of alpha 2AAR internalization

Spinophilin plays critical roles in regulating trafficking and signaling of the alpha(2)-adrenergic receptor (AR) both in vitro and in vivo (Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940-1944). In the present study, we demonstrate that protein kinase A (PKA) phosphorylation of spinophilin modulates the spinophilin-alpha(2A)AR interaction to regulate alpha(2A)AR internalization. Activation of PKA by forskolin abolishes the agonist-enhanced interaction between spinophilin and the alpha(2A)AR, and this event can be blocked by Ser --> Ala mutations at the PKA phosphorylation sites of spinophilin. In addition, a Ser --> Asp mutation that mimics the phosphorylated state at the PKA phosphorylation site Ser-177, which is located within the alpha(2A)AR binding region of spinophilin, is sufficient to block the spinophilin-alpha(2A)AR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist-induced internalization of the alpha(2A)AR is accelerated and enhanced, as revealed by both intact cell enzyme-linked immunosorbent assay and quantitative immunofluorescent studies. Furthermore, activation of PKA by forskolin enhances agonist-induced internalization of the alpha(2A)AR in cells expressing wild type spinophilin, but not in cells lacking spinophilin or expressing the spinophilin mutant Sp177D. These results strongly support that PKA phosphorylation of spinophilin is functionally relevant in regulating alpha(2A)AR trafficking. Therefore, modulation of spinophilin-receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates G protein-coupled receptor trafficking.     

Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.     

C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization

The main analgesic effects of the opioid alkaloid morphine are mediated by the mu-opioid receptor. In contrast to endogenous opioid peptides, morphine activates the mu-opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse mu-opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four mu-receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell- and tissue-specific C-terminal splicing of the mu-opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.     

Thalidomide Promotes Morphine Efficacy and Prevents Morphine-Induced Tolerance in Rats with Diabetic Neuropathy

Opioid analgesics have less efficacy in diabetic neuropathy treatment, and tolerance often occurs after chronic usage. Given that thalidomide can potentiate the morphine efficacy in diabetic neuropathy treatment, we investigated the effects of intrathecal administrations of thalidomide on morphine tolerance during the treatment of diabetic neuropathy. We found that intrathecal administrations of thalidomide (25 mg/kg/ml) potentiated the analgesic effects of morphine on mechanical hyperalgesia and prevented the development of morphine tolerance. While this treatment regimen did not alter the protein levels of μ-opioid receptor (MOR) in the spinal cord of diabetic rats, chronic morphine treatment robustly increased MOR binding density in the synaptic plasma membranes fraction, but decreased it in the microsomal fraction. Furthermore, thalidomide was able to reverse the distribution of MOR altered by chronic morphine treatment. Finally, STZ-induced diabetes promoted PKC activation and enhanced TNFα level in the spinal cord, which were attenuated by intrathecal administrations of thalidomide. Taken together, these results suggested that thalidomide may potentiate morphine efficacy on diabetic neuropathy and prevent the development of morphine tolerance by suppressing PKC activation and TNFα level in the spinal cord.     

Morphine induces desensitization of insulin receptor signaling

Morphine analgesia is mediated principally by the micro -opioid receptor (MOR). Since morphine and other opiates have been shown to influence glucose homeostasis, we investigated the hypothesis of direct cross talk between the MOR and the insulin receptor (IR) signaling cascades. We show that prolonged morphine exposure of cell lines expressing endogenous or transfected MOR, IR, and the insulin substrate 1 (IRS-1) protein specifically desensitizes IR signaling to Akt and ERK cascades. Morphine caused serine phosphorylation of the IR and impaired the formation of the signaling complex among the IR, Shc, and Grb2. Morphine also resulted in IRS-1 phosphorylation at serine 612 and reduced tyrosine phosphorylation at the YMXM p85-binding motifs, weakening the association of the IRS-1/p85 phosphatidylinositol 3-kinase complex. However, the IRS-1/Grb2 complex was unaffected by chronic morphine treatment. These results suggest that morphine attenuates IR signaling to Akt by disrupting the IRS-1-p85 interaction but inhibits signaling to ERK by disruption of the complex among the IR, Shc, and Grb2. Finally, we show that systemic morphine induced IRS-1 phosphorylation at Ser612 in the hypothalamus and hippocampus of wild type, but not MOR knockout, mice. Our results demonstrate that opiates can inhibit insulin signaling through direct cross talk between the downstream signaling pathways of the MOR and the IR.     

Increased response to morphine in mice lacking protein kinase C epsilon

The protein kinase C (PKC) family of serine-threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCepsilon have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCepsilon null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCepsilon null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of mu-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCepsilon as a key regulator of opiate sensitivity in mice.