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• W2108498532 abstract "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 μ 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. 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 μ 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. Repeated exposure to opiates leads to addiction, characterized by drug craving, compulsive drug use, dependence, and analgesic tolerance (Koob et al., 1998Koob G.F. Sanna P.P. Bloom F.E. Neuroscience of addiction.Neuron. 1998; 21: 467-476Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, Kreek, 2001Kreek M.J. Drug addiction: molecular and cellular endpoints.Ann. N Y Acad. Sci. 2001; 937: 27-49Crossref PubMed Scopus (74) Google Scholar, De Vries and Shippenberg, 2002De Vries T.J. Shippenberg T.S. Neural systems underlying opiate addiction.J. Neurosci. 2002; 22: 3321-3325Crossref PubMed Google Scholar, Inturrisi, 2002Inturrisi C.E. Clinical pharmacology of opioids for pain.Clin. J. Pain. 2002; 18: S3-S13Crossref PubMed Scopus (420) Google Scholar). At the cellular level, opiates exert their effects via G protein-coupled receptors, primarily the μ opioid receptor (MOR) (Contet et al., 2004Contet C. Kieffer B.L. Befort K. Mu opioid receptor: a gateway to drug addiction.Curr. Opin. Neurobiol. 2004; 14: 370-378Crossref PubMed Scopus (224) Google Scholar). Several proteins, including adenylyl cyclases, G protein receptor kinases (GRKs), β-arrestins, and regulators of G protein signaling (RGS proteins), have been shown to play a role in different aspects of opiate action by modulating the signaling duration and desensitization of MORs (Li et al., 2006Li S. Lee M.L. Bruchas M.R. Chan G.C. Storm D.R. Chavkin C. Calmodulin-stimulated adenylyl cyclase gene deletion affects morphine responses.Mol. Pharmacol. 2006; 70: 1742-1749Crossref PubMed Scopus (41) Google Scholar, Terman et al., 2004Terman G.W. Jin W. Cheong Y.P. Lowe J. Caron M.G. Lefkowitz R.J. Chavkin C. G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal.Br. J. Pharmacol. 2004; 141: 55-64Crossref PubMed Scopus (74) Google Scholar, Marker et al., 2004Marker C.L. Stoffel M. Wickman K. Spinal G-protein-gated K+ channels formed by GIRK1 and GIRK2 subunits modulate thermal nociception and contribute to morphine analgesia.J. Neurosci. 2004; 24: 2806-2812Crossref PubMed Scopus (106) Google Scholar, Bohn et al., 2000Bohn L.M. Gainetdinov R.R. Lin F.T. Lefkowitz R.J. Caron M.G. Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence.Nature. 2000; 408: 720-723Crossref PubMed Scopus (658) Google Scholar, Zachariou et al., 2003Zachariou V. Georgescu D. Sanchez N. Rahman Z. DiLeone R.J. Berton O. Neve R.L. Sim-Selley L.J. Selley D.E. Gold S.J. Nestler E.J. Essential role for RGS9 in opiate action.Proc. Natl. Acad. Sci. USA. 2003; 100: 13656-13661Crossref PubMed Scopus (195) Google Scholar, Kim et al., 2006Kim K.S. Lee K.W. Lee K.W. Im J.Y. Yoo J.Y. Kim S.-W. Lee J.K. Nestler E.J. Han P.L. Adenylyl cyclase-5 is an essential mediator of morphine action.Proc. Natl. Acad. Sci. USA. 2006; 103: 3908-3913Crossref PubMed Scopus (90) Google Scholar). Among the best characterized cellular adaptations to chronic opiate exposure are upregulation of cAMP signal transduction, changes in the rate of opioid receptor endocytosis, and altered ERK activation (Nestler and Aghajanian, 1997Nestler E.J. Aghajanian G.K. Molecular and cellular basis of addiction.Science. 1997; 278: 58-63Crossref PubMed Scopus (1109) Google Scholar, Law and Loh, 1999Law P.Y. Loh H.H. Regulation of opioid receptor activities.J. Pharmacol. Exp. Ther. 1999; 289: 607-612PubMed Google Scholar, von Zastrow et al., 2003von Zastrow M. Svingos A. Haberstock-Debik H. Evans C. Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs.Curr. Opin. Neurobiol. 2003; 13: 348-353Crossref PubMed Scopus (95) Google Scholar). However, the timing and cellular specificity of these responses in the brain have yet to be understood. Growing evidence indicates that the actions of proteins which modulate G protein-coupled receptor signaling are dramatically affected by their binding partners and the multiprotein complexes that they form under specific physiological conditions. Consequently, molecules such as β-arrestin-2 may promote or inhibit receptor signaling depending on other factors (Wang et al., 2004Wang Q. Zhao J. Brady A.E. Feng J. Allen P.B. Lefkowitz R.J. Greengard P. Limbird L.E. Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors.Science. 2004; 304: 1940-1944Crossref PubMed Scopus (125) Google Scholar). Our hypothesis is that repeated opiate exposure leads to adaptations that promote deleterious actions of opiate drugs (craving, dependence, tolerance) by preventing the formation of multiprotein complexes necessary for proper MOR signaling. Therefore, optimizing the action of proteins that are essential for the function of such complexes may delay the progress of addiction. Spinophilin, a ubiquitously expressed, dendritic spine-enriched protein (Allen et al., 1997Allen P.B. Ouimet C.C. Greengard P. Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines.Proc. Natl. Acad. Sci. USA. 1997; 94: 9956-9961Crossref PubMed Scopus (368) Google Scholar) interacts with several elements of the G protein-coupled receptor signaling network, including protein phosphatase 1 (PP1), RGS proteins, and dopamine D2 receptors (Hsieh-Wilson et al., 1999Hsieh-Wilson L.C. Allen P.B. Watanabe T. Nairn A.C. Greengard P. Characterization of the neuronal targeting protein spinophilin and its interactions with protein phosphatase-1.Biochemistry. 1999; 38: 4365-4373Crossref PubMed Scopus (104) Google Scholar, Wang et al., 2005Wang X. Zeng W. Soyombo A.A. Tang W. Ross E.M. Barnes A.P. Milgram S.L. Penninger J.M. Allen P.B. Greengard P. Muallem S. Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors.Nat. Cell Biol. 2005; 7: 405-411Crossref PubMed Scopus (124) Google Scholar, Smith et al., 1999Smith F.D. Oxford G.S. Milgram S.L. Association of the D2 dopamine receptor third cytoplasmic loop with Spinophilin, a protein phosphatase-1-interacting protein.J. Biol. Chem. 1999; 274: 19894-19900Crossref PubMed Scopus (170) Google Scholar). Recent studies revealed that spinophilin modulates α2-adrenergic responses by blocking association of GRK2 with the agonist-receptor-Gβγ complex and, therefore, by antagonizing β-arrestin-2 function (Richman et al., 2001Richman J.G. Brady A.E. Wang Q. Hensel J.L. Colbran R.J. Limbird L.E. Agonist-regulated interaction between alpha 2-adrenergic receptors and spinophilin.J. Biol. Chem. 2001; 276: 15003-15008Crossref PubMed Scopus (104) Google Scholar, Brady et al., 2003Brady A.E. Wang Q. Colbran R.J. Allen P.B. Greengard P. Limbird L.E. Spinophilin stabilizes cell surface expression of alpha 2B-adrenergic receptors.J. Biol. Chem. 2003; 278: 32405-32412Crossref PubMed Scopus (56) Google Scholar, Wang et al., 2004Wang Q. Zhao J. Brady A.E. Feng J. Allen P.B. Lefkowitz R.J. Greengard P. Limbird L.E. Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors.Science. 2004; 304: 1940-1944Crossref PubMed Scopus (125) Google Scholar). Since MORs share many of their signal transduction pathways with α2 receptors (i.e., both are Gi linked), it is likely that spinophilin plays a role in MOR signal transduction and desensitization and, consequently, in opiate addiction. Neurabin is a structural analog of spinophilin, with PPI and F-actin binding properties, and a similar pattern of distribution (Burnett et al., 1998Burnett P.E. Blackshaw S. Lai M.M. Qureshi I.A. Burnett A.F. Sabatin D.M. Snyder S.H. Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton.Proc. Natl. Acad. Sci. USA. 1998; 95: 8351-8356Crossref PubMed Scopus (106) Google Scholar, Terry-Lorenzo et al., 2002Terry-Lorenzo R.T. Carmody L.C. Voltz J.W. Connor J.H. Li S. Smith F.D. Milgram S.L. Colbran R.J. Shenolikar S. The neuronal actin-binding proteins, neurabin I and neurabin II, recruit specific isoforms of protein phosphatase-1 catalytic subunits.J. Biol. Chem. 2002; 277: 27716-27724Crossref PubMed Scopus (72) Google Scholar). A major difference between the two proteins is that spinophilin contains two PKA phosphorylation sites (Ser-94 and Ser-117) and a receptor binding/interacting domain (aa 169–255) that are absent from neurabin (Allen et al., 1997Allen P.B. Ouimet C.C. Greengard P. Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines.Proc. Natl. Acad. Sci. USA. 1997; 94: 9956-9961Crossref PubMed Scopus (368) Google Scholar, Richman et al., 2001Richman J.G. Brady A.E. Wang Q. Hensel J.L. Colbran R.J. Limbird L.E. Agonist-regulated interaction between alpha 2-adrenergic receptors and spinophilin.J. Biol. Chem. 2001; 276: 15003-15008Crossref PubMed Scopus (104) Google Scholar, Hsieh-Wilson et al., 1999Hsieh-Wilson L.C. Allen P.B. Watanabe T. Nairn A.C. Greengard P. Characterization of the neuronal targeting protein spinophilin and its interactions with protein phosphatase-1.Biochemistry. 1999; 38: 4365-4373Crossref PubMed Scopus (104) Google Scholar). The goal of the present study was to determine the influence of spinophilin on the acute and chronic actions of opiates. We relied on the analysis of spinophilin and neurabin knockout mice (Feng et al., 2000Feng J. Yan Z. Ferreira A. Tomizawa K. Liauw J.A. Zhuo M. Allen P.B. Ouimet C.C. Greengard P. Spinophilin regulates the formation and function of dendritic spines.Proc. Natl. Acad. Sci. USA. 2000; 97: 9287-9292Crossref PubMed Scopus (308) Google Scholar, Allen et al., 2006Allen P.B. Zachariou V. Svenningsson P. Centonze D. Costa C. Rossi S. Bender G. Chen G. Feng J. Snyder G. et al.Distinct roles for spinophilin and neurabin in dopamine mediated plasticity.Neuroscience. 2006; 140: 897-911Crossref PubMed Scopus (70) Google Scholar) in a series of behavioral paradigms. We show that spinophilin, but not neurabin, plays a prominent physiological role in regulating behavioral responses to opiates by opposing the development of dependence and tolerance and by decreasing sensitivity to the rewarding actions of the drugs. We also show that spinophilin affects MOR functional responses by promoting endocytosis and by inhibiting MOR signaling. Furthermore, our data indicate that opiate drugs induce association of spinophilin with MOR in striatum by promoting the formation of complexes between spinophilin and GRK2, RGS9-2, and Gβ subunits, signal transduction molecules that are essential for MOR signaling and desensitization. As a first step to investigate the role of spinophilin in physiological responses to opiates, we administered several MOR agonists to mice, at doses which produce maximal analgesic responses in behavioral tests, and monitored spinophilin levels in the nucleus accumbens, the ventral portion of the striatum which is important for many opiate actions. Table 1 shows regulation of spinophilin levels, measured by immunoblotting, in the nucleus accumbens two hrs after administration of different MOR agonists. Spinophilin is downregulated in this brain region by acute morphine (20 mg/kg, i.p.) administration. A similar effect was observed in response to acute administration of other opioid agonists, such as methadone (20 mg/kg, i.p) and fentanyl (0.25 mg/kg, s.c.). Our initial working hypothesis was that downregulation of spinophilin following acute opiate administration is one of the cellular adaptations that contribute to MOR desensitization. Interestingly, exposure to opiates has no effect on neurabin levels and administration of the α2-adrenergic agonist clonidine does not affect spinophilin levels in the nucleus accumbens at the 2 hr time point (Table 1). In contrast to the effects of acute morphine exposure, chronic morphine has no effect on spinophilin or neurabin levels in this brain region (not shown). Loss of spinophilin regulation by chronic morphine may be interpreted as one of the adaptive changes that follow continuous morphine use.Table 1Acute Opiate Administration Regulates Spinophilin Levels in Nucleus AccumbensAcute MorphineAcute MethadoneAcute FentanylAcute ClonidineSpinophilin levels (% control)62 ± 8.9∗80.8 ± 3.3∗73 ± 9.5∗109 ± 17Neurabin levels (% control)106 ± 2.8115 ± 9.4111 ± 5.6Decreased levels of spinophilin (but not of neurabin) in the nucleus accumbens of C57Bl/6 mice 2 hr after acute morphine (20 mg/kg), methadone (20 mg/kg), or fentanyl (0.250 mg/kg) administration. In contrast, clonidine administration had no significant effect on spinophilin levels in this brain region. Data represent % change from saline control and are expressed as means ± SEM, n = 4–5 per group, ∗p < 0.05 from control by t test. Open table in a new tab Decreased levels of spinophilin (but not of neurabin) in the nucleus accumbens of C57Bl/6 mice 2 hr after acute morphine (20 mg/kg), methadone (20 mg/kg), or fentanyl (0.250 mg/kg) administration. In contrast, clonidine administration had no significant effect on spinophilin levels in this brain region. Data represent % change from saline control and are expressed as means ± SEM, n = 4–5 per group, ∗p < 0.05 from control by t test. To gain insight into the influence of spinophilin on opioid receptor function, we investigated morphine analgesia in the 52°C hot plate assay in mice lacking the spinophilin gene. As shown in Figure 1A, spinophilin mutants are less sensitive to low doses of morphine in this assay compared to their wild-type littermate controls. The spinophilin knockout mice are similarly less sensitive to the analgesic actions of methadone and fentanyl in this assay (Figures 1B and 1C). These findings suggest that spinophilin may be a positive regulator of analgesic responses to opiate drugs. We next tested the effect of spinophilin knockout on analgesic responses to a nonopiate drug, namely, the α2-adrenergic receptor agonist, clonidine. It has been shown previously that spinophilin is a negative modulator of α2-adrenergic receptor responses (Wang et al., 2004Wang Q. Zhao J. Brady A.E. Feng J. Allen P.B. Lefkowitz R.J. Greengard P. Limbird L.E. Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors.Science. 2004; 304: 1940-1944Crossref PubMed Scopus (125) Google Scholar). Consistent with these earlier data, we found that spinophilin mutant mice are more sensitive to the analgesic effects of clonidine (Figure 1D). These findings show that spinophilin modulates analgesic responses in a receptor selective manner: it is a positive modulator of MOR actions, but a negative modulator of α2-adrenergic actions. To assess the role of spinophilin in the development of analgesic tolerance, we used the 52°C hot plate assay and monitored morphine effects once a day for 4 consecutive days. Under these conditions, mice show a gradually reduced response to morphine due to development of tolerance and, by day 4, they show no analgesic response to a constant dose of the drug. Interestingly, spinophilin knockout mice develop maximal tolerance following a single exposure to morphine, as they show no response to the drug by day 2 of the hot plate assay (Figure 1E). To confirm that this is a morphine-related phenotype, we used the same paradigm and administered the α2 adrenergic agonist, clonidine. In contrast to what we observed with morphine, analgesic responses to clonidine (0.4 mg/kg) remained unchanged in both genotypes over 3 days of testing (% MPE for wild-type, day 1 = 25 ± 7, day 2 = 23 ± 3, day 3 = 39 ± 10; %MPE for knockout, day 1 = 58 ± 7, day 2 = 64 ± 3, day 3 = 70 ± 10). Notably, neurabin knockout mice develop tolerance to morphine at the same rate as their wild-type littermate controls (Figure 1F). These latter data show that rapid development of tolerance is not a general feature of depletion of proteins containing PP1 interacting/F-actin binding domains. Furthermore, the facilitated tolerance seen in spinophilin knockout mice does not reflect a general learning phenomenon, since initial analgesic responses are reduced and the mice show no abnormality in the Morris water maze (not shown). In addition to tolerance, chronic exposure to opiates causes severe physical dependence. The role of spinophilin in opiate physical dependence and withdrawal was evaluated using a standard morphine withdrawal paradigm. Mice were treated chronically with increasing doses of morphine, and withdrawal was precipitated by administration of the opioid receptor antagonist, naloxone (1 mg/kg, s.c.). Several signs of opiate withdrawal were monitored for 25 min after naloxone administration. We found that the intensity of withdrawal is ∼2-fold higher in spinophilin knockout mice compared to their wild-type controls for most of the signs monitored (Figure 2A). In contrast, no significant difference in opiate withdrawal behavior was observed between neurabin knockout and wild-type mice. In fact, for some of the withdrawal signs, there was a trend for milder withdrawal in the neurabin mutants. However, this effect was significant only for weight loss (e.g., jumps for wild-type = 76.2 ± 34, for knockout = 22 ± 14; wet dog shakes for wild-type = 6 ± 2.6, for knockout = 3.6 ± 1.1; diarrhea for wild-type = 9 ± 1, for knockout = 5 ± 1.8; weight loss for wild-type = 3.9 ± 0.54, for knockout = 1.78 ± 0.38∗, n = 7–8 per group, p < 0.05, two way ANOVA followed by Bonferroni test). These data suggest that spinophilin plays an important physiological role opposing the development of morphine dependence. We next examined the influence of spinophilin on the rewarding actions of morphine using the place preference paradigm, in which animals learn to prefer an environment paired with drug exposure. In this test, knockout of spinophilin increases the sensitivity of animals to morphine reward (Figure 2B). No differences were observed between neurabin knockout mice and their wild-type controls in this paradigm—place preference scores (in s) for wild-type were as follows: saline = −64 ± 57, morphine (5 mg/kg, i.p.) = 284 ± 67, morphine (10 mg/kg, i.p.) = 390 ± 69; knockout were as follows: saline = −68 ± 24, morphine (5 mg/kg, i.p.) = 240 ± 39, morphine (10mg/kg, i.p.) = 272 ± 37. The opposite phenotype was observed using a model of spinophilin overexpression in the nucleus accumbens. C57Bl/6 mice overexpressing spinophilin in the nucleus accumbens 3 weeks after bilateral stereotaxic injection of an AAV-spinophilin vector (which causes a 78% ± 23% increase in spinophilin protein levels selectively in this brain region determined by immunoblotting), show reduced sensitivity to morphine place conditioning (5 mg/kg, s.c.) compared to AAV-GFP injected control mice (Figure 2C). These data specifically relate the altered behavioral sensitivity to morphine seen in spinophilin knockout mice to the nucleus accumbens per se and argue against the involvement of a developmental abnormality in this phenomenon. Finally, we examined the influence of spinophilin in the locomotor activating effects of morphine (10 mg/kg, s.c.). In this test, deletion of the spinophilin gene results in increased locomotor responses to acute and repeated morphine administration (Figure 2F). In contrast, viral-mediated overexpression of spinophilin in the nucleus accumbens does not affect the acute locomotor activating effect of morphine but prevents the development of locomotor sensitization to repeated drug exposure (Figure 2E). Deletion of the spinophilin gene does not alter opioid receptor levels in nucleus accumbens, as determined by specific binding of [3H]-DAMGO, a highly selective MOR ligand, in striatal membranes of wild-type and knockout mice: wild-type = 109.15 ± 15 fmols/mg protein, knockout = 99.9 ± 13 fmols/mg protein. In addition, MOR mRNA levels (measured using real time PCR and expressed as fold change over control) in this brain region are unaffected by the loss of spinophilin: wild-type = 1 ± 0.06 and knockout = 0.85 ± 0.26. Likewise, striatal levels of proteins involved in MOR responses, such as RGS9-2, RGS4, β-arrestin-2, and Gβ5, determined by immunoblotting, are also not different in nucleus accumbens between wild-type and knockout mice (not shown). We used coimmunoprecipitation to investigate the effect of opiate drugs on the formation of complexes between spinophilin and MOR in mouse striatum. These studies revealed that under basal conditions MOR is associated with spinophilin and that this association is greatly enhanced 30 min after s.c. administration of the MOR agonists, fentanyl or morphine (Figure 3A). We also found that activation of MOR promotes the association of spinophilin with GRK2 (Figure 3B) and with Gβ5 (Figure 3C). In fact, exposure to opiates promotes the formation of spinophilin complexes with all Gβ subunits in striatum (not shown). As Gβ5 is a binding partner of the striatal-enriched protein RGS9-2, we examined whether RGS9-2 is also coprecipitated with spinophilin after stimulation of MOR. As expected, the spinophilin-RGS9-2 complex is greatly enhanced in striatum following fentanyl or morphine treatment (Figure 3D). We next investigated the effect of loss of spinophilin on signal transduction events that follow activation of MOR. For this purpose, we monitored how the loss of spinophilin affects MOR endocytosis, inhibition of adenylyl cyclase, and ERK phosphorylation, all three being well-characterized responses to acute MOR activation (Nestler and Aghajanian, 1997Nestler E.J. Aghajanian G.K. Molecular and cellular basis of addiction.Science. 1997; 278: 58-63Crossref PubMed Scopus (1109) Google Scholar, Law and Loh, 1999Law P.Y. Loh H.H. Regulation of opioid receptor activities.J. Pharmacol. Exp. Ther. 1999; 289: 607-612PubMed Google Scholar, von Zastrow et al., 2003von Zastrow M. Svingos A. Haberstock-Debik H. Evans C. Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs.Curr. Opin. Neurobiol. 2003; 13: 348-353Crossref PubMed Scopus (95) Google Scholar, Eitan et al., 2003Eitan S. Bryant C.D. Saliminejad N. Yang Y.C. Vojdani E. Keith Jr., D. Polakiewicz R. Evans C.J. Brain region-specific mechanisms for acute morphine-induced mitogen-activated protein kinase modulation and distinct patterns of activation during analgesic tolerance and locomotor sensitization.J. Neurosci. 2003; 23: 8360-8369Crossref PubMed Google Scholar, Muller and Unterwald, 2004Muller D.L. Unterwald E.M. In vivo regulation of extracellular signal-regulated protein kinase (ERK) and protein kinase B (Akt) phosphorylation by acute and chronic morphine.J. Pharmacol. Exp. Ther. 2004; 310: 774-782Crossref PubMed Scopus (87) Google Scholar). We first monitored the effect of morphine on ERK phosphorylation and inhibition of cAMP formation in striatum of spinophilin knockout mice and their wild-type littermates. As expected, systemic administration of morphine increases phosphoERK levels in wild-type striatum at 20 min but causes a significantly greater induction of phosphoERK in the absence of spinophilin (250% of saline in striatum of knockout mice versus 150% of saline observed in wild-type controls; Figure 4A). As a control, we monitored phosphoERK levels in striatum from spinophilin wild-type and knockout mice following saline or clonidine (0.4 mg/kg) administration. Contrary to observations with morphine, clonidine had no effect of on ERK phosphorylation in striatum in wild-type (123% ± 20%) or spinophilin knockout (86% ± 11%) mice. In striking contrast to regulation of ERK, spinophilin is required for opioid inhibition of cAMP formation in striatum: the MOR agonist DAMGO reduces cAMP formation in striatal homogenates of wild-type mice, an effect not observed in extracts of knockout mice (Figure 4C). To evaluate the role of spinophilin in MOR endocytosis, we first monitored the rate of MOR internalization following exposure to opiate drugs in the presence or absence of spinophilin in PC12 cells. MOR internalization was quantified using ELISA in PC12 cells transiently transfected with epitope-tagged MOR (HA-MOR), along with a spinophilin plasmid or empty vector. Under control conditions, application of the opioid peptide DAMGO leads to fast internalization of MOR, while morphine application leads to delayed MOR internalization. As shown in Figure 5A, overexpression of spinophilin promotes morphine-induced MOR internalization so that the receptor is internalized within 30 min. Similar results were observed using immunofluorescence to directly visualize HA-MOR in PC12 cells (Figure 5B). We next monitored MOR endocytosis in cultured mouse embryonic fibroblasts (MEFs) from spinophilin wild-type and knockout embryos using ELISA. We used MEF cultures and not cultured striatal neurons, because homozygous spinophilin knockout mice do not breed well and, while we were able to obtain sufficient quantities of MEF cells from embryos of heterozygous crosses, it was not possible to derive sufficient quantities of striatal neurons. MEF cells were infected with a herpes simplex virus (HSV) vector expressing HA-MOR (HSV-HA-MOR) (Haberstock-Debic et al., 2003Haberstock-Debic H. Wein M. Barrot M. Colago E.E. Rahman Z. Neve R.L. Pickel V.M. Nestler E.J. von Zastrow M. Svingos A.L. Morphine acutely regulates opioid receptor trafficking selectively in dendrites of nucleus accumbens neurons.J. Neurosci. 2003; 23: 4324-4332Crossref PubMed Google Scholar), and drug treatments were applied 24 hr later. Similar to what was observed previously in striatal cultures (Haberstock-Debic et al., 2005Haberstock-Debic H. Kim K. Joy Y.Y. von Zastrow M. Morphine promotes rapid, arrestin-dependent endocytosis of μ-opioid receptors in striatal neurons.J. Neurosci. 2005; 25: 7847-7857Crossref PubMed Scopus (115) Google Scholar), both morphine and DAMGO induce MOR internalization within 30 min in MEF cells. Interestingly, endocytosis is observed within 30 min in wild-type MEFs, but not in MEFs from spinophilin knockout mice (Figure 5C). In addition, MEF cells from spinophilin wild-type and knockout mice were infected with HSV-HA-MOR, and MOR cellular localization was monitored 24 hr later using immunofluorescence. In wild-type MEFs, MOR is internalized 30 min following morphine treatment (10 μM), however, no internalization is observed in MEFs from spinophilin knockout embryos at this time point (Figure 5D). We also determined the action of spinophilin in MOR endocytosis in wild-type striatal neurons in primary culture. To knock down spinophilin expression in the neurons, we transfected striatal cultures with a small interfering RNA (siRNA) directed against spinophilin or a control siRNA as described by Wang et al., 2004Wang Q. Zhao J. Brady A.E. Feng J. Allen P.B. Lefkowitz R.J. Greengard P. Limbird L.E. Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors.Science. 2004; 304: 1940-1944Crossref PubMed Scopus (125) Google Scholar. We found that this treatment is effective in reducing spinophilin protein levels by >75% (24 ± 2 of control; Figure 6A) as determined by immunoblot analysis. The cells were then infected with HSV-HA-MOR and 24 hr later, HA-MOR localization was monitored using immunofluorescence. As shown in Figure 6B, under these conditions, administration of 10 μM morphine causes robust MOR internalization at 30 min in control, but internalization is significantly delayed in spinophilin siRNA transfected neurons (mean number of HA-MOR infected cells in which MOR showed intracellular localization at 30 min: control siRNA group = 65.9 ± 6.7, spinophilin siRNA group = 34 ± 1.8). Our study provides evidence for an important physiological role of spinophilin in the regulation of MOR signaling and behavioral responses to opiate drugs of abuse. Deletion of the spinophilin gene causes reduced analgesic effects of acute morphine but enhanced adaptations to repeated morphine exposure, including increased morphine dependence, place conditioning, locomotor sensitization, and analgesic toleranc" @default.
• W2108498532 created "2016-06-24" @default.
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• W2108498532 date "2008-04-01" @default.
• W2108498532 modified "2023-09-29" @default.
• W2108498532 title "Multiple Actions of Spinophilin Regulate Mu Opioid Receptor Function" @default.
• W2108498532 cites W1529268309 @default.
• W2108498532 cites W1565566868 @default.
• W2108498532 cites W1655589213 @default.
• W2108498532 cites W1858569662 @default.
• W2108498532 cites W1967072083 @default.
• W2108498532 cites W1967208942 @default.
• W2108498532 cites W1981891173 @default.
• W2108498532 cites W1990483753 @default.
• W2108498532 cites W1993614661 @default.
• W2108498532 cites W1994576663 @default.
• W2108498532 cites W2003104235 @default.
• W2108498532 cites W2004120139 @default.
• W2108498532 cites W2008935385 @default.
• W2108498532 cites W2015087988 @default.
• W2108498532 cites W2032568216 @default.
• W2108498532 cites W2033111233 @default.
• W2108498532 cites W2033670390 @default.
• W2108498532 cites W2040580510 @default.
• W2108498532 cites W2049796382 @default.
• W2108498532 cites W2055319462 @default.
• W2108498532 cites W2055363053 @default.
• W2108498532 cites W2061515123 @default.
• W2108498532 cites W2063372081 @default.
• W2108498532 cites W2063987862 @default.
• W2108498532 cites W2069726169 @default.
• W2108498532 cites W2072681884 @default.
• W2108498532 cites W2079708017 @default.
• W2108498532 cites W2083071923 @default.
• W2108498532 cites W2083409282 @default.
• W2108498532 cites W2089193522 @default.
• W2108498532 cites W2090232288 @default.
• W2108498532 cites W2096131954 @default.
• W2108498532 cites W2096140192 @default.
• W2108498532 cites W2102238490 @default.
• W2108498532 cites W2131699353 @default.
• W2108498532 cites W2135043861 @default.
• W2108498532 cites W2135326004 @default.
• W2108498532 cites W2139496188 @default.
• W2108498532 cites W2141611690 @default.
• W2108498532 cites W2149823377 @default.
• W2108498532 cites W2156480657 @default.
• W2108498532 cites W2158056917 @default.
• W2108498532 cites W2158636785 @default.
• W2108498532 cites W4291166395 @default.
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