The Experts below are selected from a list of 1023 Experts worldwide ranked by ideXlab platform
Paul Greengard - One of the best experts on this subject based on the ideXlab platform.
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Multiple actions of Spinophilin regulate mu opioid receptor function.
Neuron, 2008Co-Authors: Joanna J. Charlton, Patrick B. Allen, Paul Greengard, Kassi Psifogeorgou, Sumana Chakravarty, Ivone Gomes, Rachael L. Neve, Lakshmi A. Devi, Eric J. Nestler, Venetia ZachariouAbstract: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.
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Regulation of Spinophilin Ser94 phosphorylation in neostriatal neurons involves both DARPP-32-dependent and independent pathways.
Journal of neurochemistry, 2005Co-Authors: Ken Uematsu, Linda C. Hsieh-wilson, Angus C. Nairn, Paul Greengard, Marie Futter, Hideho Higashi, Hisao Maeda, Akinori NishiAbstract:Spinophilin is a protein phosphatase-1 (PP-1)- and actin-binding protein that is enriched in dendritic spines. Phosphorylation of the actin-binding domain of rat Spinophilin at one or more sites by protein kinase A (PKA) inhibits actin binding. Here, we investigated the regulation of mouse Spinophilin that contains only a single PKA-site (Ser94) within its actin-binding domain. In vitro phosphorylation of Ser94 resulted in the dissociation of Spinophilin from actin filaments. In mouse neostriatal slices, phospho-Ser94 (p-Ser94) was dephosphorylated mainly by PP-1 and also by PP-2A. Activation of dopamine D1 receptors in striatonigral medium spiny neurons, and of adenosine A2A receptors in striatopallidal medium spiny neurons increased, whereas activation of dopamine D2 receptors in striatopallidal neurons decreased, Spinophilin Ser94 phosphorylation. In neostriatal slices from DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) knockout mice, the effects of D1, D2 and A2A receptors were largely attenuated. Activation of NMDA receptors decreased Ser94 phosphorylation in a PP-2A-dependent, but DARPP-32-independent, manner. These results suggest that PKA-dependent phosphorylation of Spinophilin at Ser94 in both striatonigral and striatopallidal neurons requires synergistic contributions from the PKA and DARPP-32/PP-1 pathways. In addition, PP-2A plays a role in Ser94 dephosphorylation in response to activation of both D2 and NMDA receptors.
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The Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology
Neuron, 2005Co-Authors: Xiaozhou P. Ryan, Patrick B. Allen, Angus C. Nairn, Per Svenningsson, Jacqueline Alldritt, Paul GreengardAbstract:Neurabin and Spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or Spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or Spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/Spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.
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Phosphorylation of Spinophilin by ERK and cyclin-dependent PK 5 (Cdk5).
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Marie Futter, Paul Greengard, Ken Uematsu, Stewart A. Bullock, Yong Kim, Hugh C. Hemmings, Akinori Nishi, Angus C. NairnAbstract:Spinophilin is a protein that binds to protein phosphatase-1 and actin and modulates excitatory synaptic transmission and dendritic spine morphology. We have identified three sites phosphorylated by ERK2 (Ser-15 and Ser-205) and cyclin-dependent PK 5 (Cdk5) (Ser-17), within the actin-binding domain of Spinophilin. Cdk5 and ERK2 both phosphorylated Spinophilin in intact cells. However, in vitro, phosphorylation by ERK2, but not by Cdk5, was able to modulate the ability of Spinophilin to bind to and bundle actin filaments. In neurons and HEK293 cells expressing GFP-tagged variants of Spinophilin, imaging studies demonstrated that introduction of a phospho-site mimic (Ser-15 to glutamate) was associated with increased filopodial density. These results support a role for Spinophilin phosphorylation by ERK2 in the regulation of spine morphogenesis.
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α2-Adrenergic Agonist Enrichment of Spinophilin at the Cell Surface Involves βγ Subunits of Gi Proteins and Is Preferentially Induced by the α2A-Subtype
Molecular pharmacology, 2005Co-Authors: Ashley E. Brady, Patrick B. Allen, Paul Greengard, Qin Wang, Mark Rizzo, Lee E. LimbirdAbstract:Agonist activation regulates reciprocal interactions of Spinophilin and arrestin with the α2A- and α2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the α2A- and α2B -AR subtypes also led to Spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged Spinophilin examined Spinophilin localization and its regulation by α2 -AR agonist. Agonist activation of α2A-AR preferentially, compared with the α2B-AR, led to Spinophilin enrichment at the cell surface in human embryonic kidney 293 cells and in mouse embryo fibroblasts derived from Spinophilin null mice. Activation of the ΔLEESSSSα2A-AR, which has enriched association with Spinophilin compared with the wild-type (WT) α2A-AR, does not show an enhanced redistribution of Spinophilin to the surface compared with WT α2A-AR, demonstrating that the ability or affinity of the receptor in binding Spinophilin may be independent of the ability of the receptor to effect Spinophilin redistribution to the surface. Agonist-evoked enrichment of Spinophilin at the cell surface seems to involve downstream signaling events, manifested both by the pertussis toxin sensitivity of the process and by the marked attenuation of Spinophilin redistribution in cells expressing the β-adrenergic receptor kinase-C tail, which sequesters βγ subunits of G proteins. Together, the data suggest that agonist-evoked Spinophilin enrichment at the cell surface is caused by receptor-evoked signaling pathways and is independent of the affinity of the receptor for the Spinophilin molecule.
Qin Wang - One of the best experts on this subject based on the ideXlab platform.
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Age-dependent differential regulation of anxiety- and depression-related behaviors by neurabin and Spinophilin
PloS one, 2017Co-Authors: Christopher Cottingham, Pulin Che, Liping Chen, Hongxia Wang, Kexiang Liu, Qin WangAbstract:Affective disorders impact nearly 10% of the adult population in the United States in a given year. Synaptic dysfunction has recently emerged as a key neurobiological mechanism underlying affective disorders such as anxiety and depression. In this study, we investigate the potential role of two synaptic scaffolding proteins, neurabin and Spinophilin, in regulating anxiety- and depression-related behaviors at different ages using genetically deficient mice. Loss of the neurabin gene reduces anxiety-like behavior in the elevated zero maze in young adult mice (3–5 months old), but not in middle aged mice (11–13 months old), whereas loss of Spinophilin decreases anxiety in middle-aged mice, but not in young adult mice. Neurabin knockout (KO) mice also show reduced immobility in the repeated force swim test (FST) at 3–5 months, but not 11–3 months, of age, compared to age- and strain-matched wild type (WT) controls. Conversely, Spinophilin KO mice display a lower level of this behavioral despair than matched WT controls after repeated FST trials at the middle age (11–13 months) but not the young age (3–5 months). Together, these data indicate that, despite their structural similarities and overlapping function in regulating synaptic cytoskeleton, the two homologs neurabin and Spinophilin play important yet distinct roles in the regulation of anxiety- and depression-like behaviors in an age-dependent manner. Our studies provide new insights into the complex neurobiology of affective disorders.
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Expression of neurabin and Spinophilin in the mouse brain at 4 and 12 months of age.
2017Co-Authors: Christopher Cottingham, Pulin Che, Liping Chen, Hongxia Wang, Kexiang Liu, Qin WangAbstract:Total brain lysates were subjected to western blot. Neurabin, Spinophilin, PP1γ and tubulin (loading control) were detected by respective antibodies. (A) representative westerns. (B) Quantification of expression levels of neurabin, Spinophilin and PP1γ in the WT mouse brain at different ages. Data are presented as mean ± SEM. n = 3 for each group.
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Response
2016Co-Authors: Adrenergic Receptor-elicited Hypertensive, Yunjia Chen, Kai Jiao, Pulin Che, Ning Peng, Mary Gannon, Michael J. Wyss, Qin WangAbstract:The α2 adrenergic receptor (AR) subtypes are important for blood pressure control. When activated, the α2A subtype elicits a hypotensive response whereas the α2B subtype medi-ates a hypertensive effect that counteracts the hypotensive response by the α2A subtype. We have previously shown that Spinophilin attenuates the α2AAR-dependent hypotensive response; in Spinophilin null mice, this response is highly potentiated. In this study, we dem-onstrate that Spinophilin impedes arrestin-dependent phosphorylation and desensitization of the α2BAR subtype by competing against arrestin binding to this receptor subtype. The Del301-303 α2BAR, a human variation that shows impaired phosphorylation and desensiti-zation and is linked to hypertension in certain populations, exhibits preferential interaction with Spinophilin over arrestin. Furthermore, Del301-303 α2BAR-induced ERK signaling is quickly desensitized in cells without Spinophilin expression, showing a profile similar to that induced by the wild type receptor in these cells. Together, these data suggest a critical role of Spinophilin in sustaining α2BAR signaling. Consistent with this notion, our in vivo study reveals that the α2BAR-elicited hypertensive response is diminished in Spinophilin deficien
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Spinophilin Is Indispensable for the α2B Adrenergic Receptor-Elicited Hypertensive Response
PloS one, 2015Co-Authors: Pulin Che, Yunjia Chen, Kai Jiao, Ning Peng, Mary C. Gannon, J. Michael Wyss, Qin WangAbstract:The α2 adrenergic receptor (AR) subtypes are important for blood pressure control. When activated, the α2A subtype elicits a hypotensive response whereas the α2B subtype mediates a hypertensive effect that counteracts the hypotensive response by the α2A subtype. We have previously shown that Spinophilin attenuates the α2AAR-dependent hypotensive response; in Spinophilin null mice, this response is highly potentiated. In this study, we demonstrate that Spinophilin impedes arrestin-dependent phosphorylation and desensitization of the α2BAR subtype by competing against arrestin binding to this receptor subtype. The Del301-303 α2BAR, a human variation that shows impaired phosphorylation and desensitization and is linked to hypertension in certain populations, exhibits preferential interaction with Spinophilin over arrestin. Furthermore, Del301-303 α2BAR-induced ERK signaling is quickly desensitized in cells without Spinophilin expression, showing a profile similar to that induced by the wild type receptor in these cells. Together, these data suggest a critical role of Spinophilin in sustaining α2BAR signaling. Consistent with this notion, our in vivo study reveals that the α2BAR-elicited hypertensive response is diminished in Spinophilin deficient mice. In arrestin 3 deficient mice, where the receptor has a stronger binding to Spinophilin, the same hypertensive response is enhanced. These data suggest that interaction with Spinophilin is indispensable for the α2BAR to elicit the hypertensive response. This is opposite of the negative role of Spinophilin in regulating α2AAR-mediated hypotensive response, suggesting that Spinophilin regulation of these closely related receptor subtypes can result in distinct functional outcomes in vivo. Thus, Spinophilin may represent a useful therapeutic target for treatment of hypertension.
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Del301-303 α2BAR has a stronger interaction with Spinophilin than WT α2BAR.
2015Co-Authors: Pulin Che, Yunjia Chen, Kai Jiao, Ning Peng, Mary Gannon, Michael J. Wyss, Qin WangAbstract:(A) Cells co-expressing Myc-Spinophilin together with HA-tagged WT or Del301-303 α2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs). (B) Quantitation of the agonist-induced fold change of Myc-Spinophilin in the IP complex with the WT α2B or Del301-303 α2BAR. n = 3–5 for each condition. **, p
Anthony J. Baucum - One of the best experts on this subject based on the ideXlab platform.
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Spinophilin limits GluN2B-containing NMDAR activity and sequelae associated with excessive hippocampal NMDAR function
2021Co-Authors: Asma B. Salek, Ruchi Bansal, Nicolas F. Berbari, Anthony J. BaucumAbstract:ABSTRACTN-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that Spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how Spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of Spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from Spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which Spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that Spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which Spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of Spinophilin promotes pathological sequelae associated with GluN2B dysfunction.HIGHLIGHTSSpinophilin bidirectionally regulates GluN2B-containing NMDAR function.Loss of Spinophilin in primary hippocampal neurons increases a pro-apoptotic marker.Loss of Spinophilin in vivo decreases measures of spatial cognitive flexibility.Graphical AbstractSpinophilin increases the phosphorylation of Ser-1284 on GluN2B, thereby enhancing calcium influx through the GluN2B containing NMDARs. In contrast, Spinophilin limits GluN2B-containing surface expression putatively due to modulation of GluN2B interactions with endocytotic proteins. Since the second effect of Spinophilin occurs independent of the first, we observe an overall decrease in calcium influx through GluN2B containing NMDARs when Spinophilin is present. This low, basal calcium influx is less likely to be promote calcium-dependent activation of caspase and downstream apoptotic pathways and permits flexible search strategies and behaviors. In the absence of Spinophilin, the Spinophilin-driven internalization of the receptors is decreased, more receptors are expressed on the surface and calcium influx into the cell is increased. This high levels of intracellular calcium triggers apoptotic pathways leading to cell death. This impact may be more dramatic in cells with high expression of GluN2B-containing NMDA receptors. This loss of Spinophilin reduces cognitive flexibility in hippocampal dependent tasks.
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Spinophilin limits glun2b containing nmdar activity and sequelae associated with excessive hippocampal nmdar function
bioRxiv, 2021Co-Authors: Asma B. Salek, Ruchi Bansal, Nicolas F. Berbari, Anthony J. BaucumAbstract:N-methyl-D-Aspartate receptors (NMDARs) are calcium-permeable ion channels that are ubiquitously expressed within the glutamatergic postsynaptic density. Phosphorylation of NMDAR subunits defines receptor activity and surface localization. Modulation of NMDAR phosphorylation by kinases and phosphatases regulates calcium entering the cell and subsequent activation of calcium-dependent processes. Spinophilin is the major synaptic protein phosphatase 1 (PP1) targeting protein that controls phosphorylation of myriad substrates via targeting or inhibition of PP1. Spinophilin limits NMDAR function in a PP1-dependent manner and we have previously shown that Spinophilin sequesters PP1 away from the GluN2B subunit of the NMDAR, which results in increased phosphorylation of Ser-1284. However, how Spinophilin modifies NMDAR function is unclear. Herein, we detail that while Ser-1284 phosphorylation increases calcium influx via GluN2B-containing NMDARs, overexpression of Spinophilin decreases GluN2B-containing NMDAR activity by decreasing its surface expression. In hippocampal neurons isolated from Spinophilin knockout animals there is an increase in cleaved caspase-3 levels compared to wildtype mice; however, this effect is not exclusively due to NMDAR activation; suggesting multiple putative mechanisms by which Spinophilin may modulate caspase cleavage. Behaviorally, our data suggest that Spinophilin knockout mice have deficits in spatial cognitive flexibility, a behavior associated GluN2B function within the hippocampus. Taken together, our data demonstrate a unique mechanism by which Spinophilin modulates GluN2B containing NMDAR phosphorylation, channel function, and trafficking and that loss of Spinophilin promotes pathological sequelae associated with GluN2B dysfunction.
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Spinophilin regulates phosphorylation and interactions of the GluN2B subunit of the N-methyl-d-aspartate receptor.
Journal of neurochemistry, 2019Co-Authors: Asma B. Salek, Michael C. Edler, Jonathon P. Mcbride, Anthony J. BaucumAbstract:N-methyl-d-Aspartate receptors (NMDARs) are abundant postsynaptic proteins that are critical for normal synaptic communication. NMDAR channel function is regulated by multiple properties, including phosphorylation. Inhibition of protein phosphatase 1 (PP1) in hippocampal neurons increases NMDAR activity, an effect abrogated by loss of Spinophilin, the major PP1-targeting protein in the postsynaptic density. However, how Spinophilin regulates PP1-dependent NMDAR function is unclear. We hypothesize that Spinophilin regulates PP1 binding to the NMDAR to alter NMDAR phosphorylation. Our data demonstrate that Spinophilin interacts with the GluN2B subunit of the NMDAR. In human embryonic kidney 293 FT cells, activation and/or overexpression of protein kinase A increased the association between Spinophilin and the GluN2B subunit of the NMDAR. Functionally, we found that Spinophilin overexpression decreased PP1 binding to the GluN2B subunit of the NMDAR and attenuated the PP1-dependent dephosphorylation of GluN2B at Ser-1284. Moreover, in P28 hippocampal lysates isolated from Spinophilin KO compared to WT mice, there was increased binding of GluN2B to PP1, decreased phosphorylation of GluN2B at Ser-1284, and altered GluN2B protein interactions with postsynaptic density-enriched proteins. Together, our data demonstrate that Spinophilin decreases PP1 binding to GluN2B and concomitantly enhances the phosphorylation of GluN2B at Ser-1284. The putative consequences of these Spinophilin-dependent alterations in GluN2B phosphorylation and interactions on synaptic GluN2B localization and function are discussed. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.
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Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization
Proteomes, 2018Co-Authors: Darryl S. Watkins, Jason D. True, Amber L. Mosley, Anthony J. BaucumAbstract:Glutamatergic projections from the cortex and dopaminergic projections from the substantia nigra or ventral tegmental area synapse on dendritic spines of specific GABAergic medium spiny neurons (MSNs) in the striatum. Direct pathway MSNs (dMSNs) are positively coupled to protein kinase A (PKA) signaling and activation of these neurons enhance specific motor programs whereas indirect pathway MSNs (iMSNs) are negatively coupled to PKA and inhibit competing motor programs. An imbalance in the activity of these two programs is observed following increased dopamine signaling associated with exposure to psychostimulant drugs of abuse. Alterations in MSN signaling are mediated by changes in MSN protein post-translational modifications, including phosphorylation. Whereas direct changes in specific kinases, such as PKA, regulate different effects observed in the two MSN populations, alterations in the specific activity of serine/threonine phosphatases, such as protein phosphatase 1 (PP1) are less well known. This lack of knowledge is due, in part, to unknown, cell-specific changes in PP1 targeting proteins. Spinophilin is the major PP1-targeting protein in striatal postsynaptic densities. Using proteomics and immunoblotting approaches along with a novel transgenic mouse expressing hemagglutainin (HA)-tagged Spinophilin in dMSNs and iMSNs, we have uncovered cell-specific regulation of the Spinophilin interactome following a sensitizing regimen of amphetamine. These data suggest regulation of Spinophilin interactions in specific MSN cell types and may give novel insight into putative cell-specific, phosphatase-dependent signaling pathways associated with psychostimulants.
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The association of Spinophilin with disks large-associated protein 3 (SAPAP3) is regulated by metabotropic glutamate receptor (mGluR) 5.
Molecular and cellular neurosciences, 2018Co-Authors: Cameron W. Morris, Asma B. Salek, Darryl S. Watkins, Michael C. Edler, Anthony J. BaucumAbstract:Abstract Spinophilin is the most abundant protein phosphatase 1 targeting protein in the postsynaptic density of dendritic spines. Spinophilin associates with myriad synaptic proteins to regulate normal synaptic communication; however, the full complement of Spinophilin interacting proteins and mechanisms regulating Spinophilin interactions are unclear. Here we validate an association between Spinophilin and the scaffolding protein, disks large-associated protein 3 (SAP90/PSD-95 associated protein 3; SAPAP3). Loss of SAPAP3 leads to obsessive-compulsive disorder (OCD)-like behaviors due to alterations in metabotropic glutamate receptor (mGluR) signaling. Here we report that Spinophilin associates with SAPAP3 in the brain and in a heterologous cell system. Moreover, we have found that expression or activation of group I mGluRs along with activation of the mGluR-dependent kinase, protein kinase C β, enhances this interaction. Functionally, global loss of Spinophilin attenuates amphetamine-induced hyperlocomotion, a striatal behavior associated with dopamine dysregulation and OCD. Together, these data delineate a novel link between mGluR signaling, Spinophilin, and SAPAP3 in striatal pathophysiology.
Patrick B. Allen - One of the best experts on this subject based on the ideXlab platform.
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Multiple actions of Spinophilin regulate mu opioid receptor function.
Neuron, 2008Co-Authors: Joanna J. Charlton, Patrick B. Allen, Paul Greengard, Kassi Psifogeorgou, Sumana Chakravarty, Ivone Gomes, Rachael L. Neve, Lakshmi A. Devi, Eric J. Nestler, Venetia ZachariouAbstract: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.
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Arrestins and Spinophilin competitively regulate Na+,K+-ATPase trafficking through association with a large cytoplasmic loop of the Na+,K+-ATPase.
Molecular biology of the cell, 2007Co-Authors: Tohru Kimura, Patrick B. Allen, Angus C. Nairn, Michael J. CaplanAbstract:The activity and trafficking of the Na(+),K(+)-ATPase are regulated by several hormones, including dopamine, vasopressin, and adrenergic hormones through the action of G-protein-coupled receptors (GPCRs). Arrestins, GPCR kinases (GRKs), 14-3-3 proteins, and Spinophilin interact with GPCRs and modulate the duration and magnitude of receptor signaling. We have found that arrestin 2 and 3, GRK 2 and 3, 14-3-3 epsilon, and Spinophilin directly associate with the Na(+),K(+)-ATPase and that the associations with arrestins, GRKs, or 14-3-3 epsilon are blocked in the presence of Spinophilin. In COS cells that overexpressed arrestin, the Na(+),K(+)-ATPase was redistributed to intracellular compartments. This effect was not seen in mock-transfected cells or in cells expressing Spinophilin. Furthermore, expression of Spinophilin appeared to slow, whereas overexpression of beta-arrestins accelerated internalization of the Na(+),K(+)-ATPase endocytosis. We also find that GRKs phosphorylate the Na(+),K(+)-ATPase in vitro on its large cytoplasmic loop. Taken together, it appears that association with arrestins, GRKs, 14-3-3 epsilon, and Spinophilin may be important modulators of Na(+),K(+)-ATPase trafficking.
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Distinct roles for Spinophilin and neurabin in dopamine-mediated plasticity.
Neuroscience, 2006Co-Authors: Patrick B. Allen, Venetia Zachariou, Per Svenningsson, A.c. Lepore, Diego Centonze, Cinzia Costa, Silvia Rossi, G. Bender, Guojun Chen, Jian FengAbstract:Protein phosphatase 1 plays a major role in the governance of excitatory synaptic activity, and is subject to control via the neuromodulatory actions of dopamine. Mechanisms involved in regulating protein phosphatase 1 activity include interactions with the structurally related cytoskeletal elements Spinophilin and neurabin, synaptic scaffolding proteins that are highly enriched in dendritic spines. The requirement for these proteins in dopamine-related neuromodulation was tested using knockout mice. Dopamine D1-mediated regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor activity was deficient in both striatal and prefrontal cortical neurons from neurabin knockout mice; in Spinophilin knockout mice this deficit was manifest only in striatal neurons. At corticostriatal synapses long-term potentiation was deficient in neurabin knockout mice, but not in Spinophilin knockout mice, and was rescued by a D1 receptor agonist. In contrast, long-term depression was deficient in Spinophilin knockout mice but not in neurabin knockout mice, and was rescued by D2 receptor activation. Spontaneous excitatory post-synaptic current frequency was increased in neurabin knockout mice, but not in Spinophilin knockout mice, and this effect was normalized by D2 receptor agonist application. Both knockout strains displayed increased induction of GluR1 Ser(845) phosphorylation in response to D1 receptor stimulation in slices, and also displayed enhanced locomotor activation in response to cocaine administration. These effects could be dissociated from cocaine reward, which was enhanced only in Spinophilin knockout mice, and was accompanied by increased immediate early gene induction. These data establish a requirement for synaptic scaffolding in dopamine-mediated responses, and further indicate that Spinophilin and neurabin play distinct roles in dopaminergic signal transduction and psychostimulant response.
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The Rho-Specific GEF Lfc Interacts with Neurabin and Spinophilin to Regulate Dendritic Spine Morphology
Neuron, 2005Co-Authors: Xiaozhou P. Ryan, Patrick B. Allen, Angus C. Nairn, Per Svenningsson, Jacqueline Alldritt, Paul GreengardAbstract:Neurabin and Spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or Spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or Spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/Spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.
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α2-Adrenergic Agonist Enrichment of Spinophilin at the Cell Surface Involves βγ Subunits of Gi Proteins and Is Preferentially Induced by the α2A-Subtype
Molecular pharmacology, 2005Co-Authors: Ashley E. Brady, Patrick B. Allen, Paul Greengard, Qin Wang, Mark Rizzo, Lee E. LimbirdAbstract:Agonist activation regulates reciprocal interactions of Spinophilin and arrestin with the α2A- and α2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the α2A- and α2B -AR subtypes also led to Spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged Spinophilin examined Spinophilin localization and its regulation by α2 -AR agonist. Agonist activation of α2A-AR preferentially, compared with the α2B-AR, led to Spinophilin enrichment at the cell surface in human embryonic kidney 293 cells and in mouse embryo fibroblasts derived from Spinophilin null mice. Activation of the ΔLEESSSSα2A-AR, which has enriched association with Spinophilin compared with the wild-type (WT) α2A-AR, does not show an enhanced redistribution of Spinophilin to the surface compared with WT α2A-AR, demonstrating that the ability or affinity of the receptor in binding Spinophilin may be independent of the ability of the receptor to effect Spinophilin redistribution to the surface. Agonist-evoked enrichment of Spinophilin at the cell surface seems to involve downstream signaling events, manifested both by the pertussis toxin sensitivity of the process and by the marked attenuation of Spinophilin redistribution in cells expressing the β-adrenergic receptor kinase-C tail, which sequesters βγ subunits of G proteins. Together, the data suggest that agonist-evoked Spinophilin enrichment at the cell surface is caused by receptor-evoked signaling pathways and is independent of the affinity of the receptor for the Spinophilin molecule.
Lee E. Limbird - One of the best experts on this subject based on the ideXlab platform.
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enhanced hypotensive bradycardic and hypnotic responses to α2 adrenergic agonists in Spinophilin null mice are accompanied by increased g protein coupling to the α2a adrenergic receptor
Molecular Pharmacology, 2010Co-Authors: Yunjia Chen, Kai Jiao, Lee E. Limbird, Ning Peng, C Cottingham, J M Wyss, Qin WangAbstract:We previously identified Spinophilin as a regulator of α2 adrenergic receptor (α2AR) trafficking and signaling in vitro and in vivo (Science 304:1940–1944, 2004). To assess the generalized role of Spinophilin in regulating α2AR functions in vivo, the present study examined the impact of eliminating Spinophilin on α2AR-evoked cardiovascular and hypnotic responses, previously demonstrated to be mediated by the α2AAR subtype, after systemic administration of the α2-agonists 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304) and clonidine in Spinophilin-null mice. Mice lacking Spinophilin expression display dramatically enhanced and prolonged hypotensive, bradycardic, and sedative-hypnotic responses to α2AR stimulation. Whereas these changes in sensitivity to α2AR agonists occur independent of any changes in α2AAR density or intrinsic affinity for agonist in the brains of Spinophilin-null mice compared with wild-type control mice, the coupling of the α2AAR to cognate G proteins is enhanced in Spinophilin-null mice. Thus, brain preparations from Spinophilin-null mice demonstrate enhanced guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of α2AAR in the guanine-nucleotide-sensitive higher affinity state compared with those from wild-type mice. These findings suggest that eliminating Spinophilin expression in native tissues leads to an enhanced receptor/G protein coupling efficiency that contributes to sensitization of receptor mediated responses in vivo.
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Enhanced Hypotensive, Bradycardia and Hypnotic Responses to α2-adrenergic Agonists in Spinophilin Null Mice Are Accompanied by Increased G Protein Coupling to the α2AAR
Molecular pharmacology, 2010Co-Authors: Yunjia Chen, Christopher Cottingham, Kai Jiao, Lee E. Limbird, Ning Peng, J. Michael Wyss, Qin WangAbstract:We previously identified Spinophilin as a regulator of α 2 adrenergic receptor (α 2 AR) trafficking and signaling 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). To assess the generalized role of Spinophilin in regulating α 2 AR functions in vivo , the present study examined the impact of eliminating Spinophilin on α 2 AR-evoked cardiovascular and hypnotic responses, previously demonstrated to be mediated by the α 2A AR subtype, following systemic administration of the α 2 -agonists, UK14,304 and clonidine, in Spinophilin null mice. Mice lacking Spinophilin expression display dramatically enhanced and prolonged hypotensive, bradycardic as well as sedative-hypnotic responses to α 2 AR stimulation. Whereas these changes in sensitivity to α 2 AR agonists occur independent of any changes in α 2A AR density or intrinsic affinity for agonist in the brain of Spinophilin null mice when compared to wild type controls, the coupling of the α 2A AR to cognate G proteins is enhanced in Spinophilin null mice. Thus, brain preparations from Spinophilin null mice demonstrate enhanced guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of α 2A AR in the guanine-nucleotide-sensitive higher affinity state when compared to those from wild type mice. These findings suggest that eliminating Spinophilin expression in native tissues leads to an enhanced receptor/G protein coupling efficiency that contributes to sensitization of receptor mediated responses in vivo .
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Knockout of Spinophilin, an endogenous antagonist of arrestin-dependent α2-adrenoceptor functions, enhances receptor-mediated antinociception yet does not eliminate sex-related differences
Behavioural brain research, 2008Co-Authors: Subodh Nag, Qin Wang, Lee E. Limbird, S. S. MokhaAbstract:We have previously shown gonadal steroid-dependent, gender specific modulation of nociception by α2-adrenoceptors. Agonist activation of the receptor enhances its association with Spinophilin that antagonizes arrestin functions both by diminishing receptor phosphorylation by G-protein-coupled receptor kinase 2 (GRK2) and by competing for receptor interactions with arrestin. Since Spinophilin is highly enriched in dendritic spines, we investigated whether α2-adrenoceptor-induced antinociception as well as sex-related differences are modified in Spinophilin knockout mice. We evaluated α2-adrenoceptor antinociception in a heat-evoked tail flick test in Spinophilin wild type (Sp+/+) and knockout (Sp−/−) mice. Baseline tail flick latencies (TFLs) did not change between any groups. Interestingly, the α2-adrenoceptor agonist, clonidine, increased TFL in male and diestrous (low estrogen) Sp−/− as well as Sp+/+ mice; in fact, this increase in TFL was significantly higher in Sp−/− male and diestrous groups than in their Sp+/+ counterparts. This unexpected finding is consistent with enhanced α2-adrenoceptor–mediated sedation observed previously in Sp−/− mice, presumably due to accelerated endocytosis of desensitized receptors and recycling of refreshed receptors when arrestin is not competed for by Spinophilin in Sp−/− mice. Despite modulation of α2-adrenoceptor effects in Sp−/− mice, sex-related differences were retained; thus, clonidine was ineffective in proestrous females (highest estrogen levels), in both Sp−/− and Sp+/+ mice, reaffirming that estrogen suppresses α2-adrenoceptor-evoked antinociception. These findings show that elimination of Spinophilin enhances α2-adrenoceptor-evoked antinociception in estrogen-deprived physiological settings, suggesting a role for Spinophilin to suppress these effects, and yet this enhanced response cannot overcome the absence of antinociception with elevated estrogen levels.
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α2-Adrenergic Agonist Enrichment of Spinophilin at the Cell Surface Involves βγ Subunits of Gi Proteins and Is Preferentially Induced by the α2A-Subtype
Molecular pharmacology, 2005Co-Authors: Ashley E. Brady, Patrick B. Allen, Paul Greengard, Qin Wang, Mark Rizzo, Lee E. LimbirdAbstract:Agonist activation regulates reciprocal interactions of Spinophilin and arrestin with the α2A- and α2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the α2A- and α2B -AR subtypes also led to Spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged Spinophilin examined Spinophilin localization and its regulation by α2 -AR agonist. Agonist activation of α2A-AR preferentially, compared with the α2B-AR, led to Spinophilin enrichment at the cell surface in human embryonic kidney 293 cells and in mouse embryo fibroblasts derived from Spinophilin null mice. Activation of the ΔLEESSSSα2A-AR, which has enriched association with Spinophilin compared with the wild-type (WT) α2A-AR, does not show an enhanced redistribution of Spinophilin to the surface compared with WT α2A-AR, demonstrating that the ability or affinity of the receptor in binding Spinophilin may be independent of the ability of the receptor to effect Spinophilin redistribution to the surface. Agonist-evoked enrichment of Spinophilin at the cell surface seems to involve downstream signaling events, manifested both by the pertussis toxin sensitivity of the process and by the marked attenuation of Spinophilin redistribution in cells expressing the β-adrenergic receptor kinase-C tail, which sequesters βγ subunits of G proteins. Together, the data suggest that agonist-evoked Spinophilin enrichment at the cell surface is caused by receptor-evoked signaling pathways and is independent of the affinity of the receptor for the Spinophilin molecule.
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Spinophilin blocks arrestin actions in vitro and in vivo at G protein-coupled receptors.
Science (New York N.Y.), 2004Co-Authors: Qin Wang, Patrick B. Allen, Paul Greengard, Ashley E. Brady, Jian Feng, Jiali Zhao, Robert J. Lefkowitz, Lee E. LimbirdAbstract:Arrestin regulates almost all G protein–coupled receptor (GPCR)–mediated signaling and trafficking. We report that the multidomain protein, Spinophilin, antagonizes these multiple arrestin functions. Through blocking G protein receptor kinase 2 (GRK2) association with receptor-Gβγ complexes, Spinophilin reduces arrestin-stabilized receptor phosphorylation, receptor endocytosis, and the acceleration of mitogen-activated protein kinase (MAPK) activity following endocytosis. Spinophilin knockout mice were more sensitive than wild-type mice to sedation elicited by stimulation of α2 adrenergic receptors, whereas arrestin 3 knockout mice were more resistant, indicating that the signal-promoting, rather than the signal-terminating, roles of arrestin are more important for certain response pathways. The reciprocal interactions of GPCRs with Spinophilin and arrestin represent a regulatory mechanism for fine-tuning complex receptor-orchestrated cell signaling and responses.