D Scott Witherow

Howard Hughes Medical Institute, Ashburn, Virginia, United States

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Publications (10)37.75 Total impact

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    ABSTRACT: In addition to their roles in desensitization and signaling of seven-membrane-spanning receptors, beta-arrestins have been more recently implicated in regulating non-seven-membrane-spanning receptor pathways. By using a yeast two-hybrid screen, we identified the inhibitor of NF-kappaB, IkappaBalpha, as a binding partner of beta-arrestin 1. Both beta-arrestin 1 and 2 interact with IkappaBalpha in transfected cells as assessed by immunoprecipitation experiments. Additionally, upstream kinases known to regulate the function of IkappaBalpha, such as IkappaB kinase alpha and beta and NF-kappaB-inducing kinase, were also shown to interact with beta-arrestin. Overexpression of either beta-arrestin 1 or beta-arrestin 2 led to marked inhibition of NF-kappaB activity, as measured by reporter gene activity. Inhibition of NF-kappaB activity was independent of the type of stimulus used for NF-kappaB activation. Conversely, suppression of beta-arrestin 1, but not beta-arrestin 2, expression by using RNA interference led to a 3-fold increase in tumor necrosis factor-stimulated NF-kappaB activity as measured by NF-kappaB mobility-shift analysis. These data uncover a role of beta-arrestins in the regulation of NF-kappaB-mediated gene regulation.
    Proceedings of the National Academy of Sciences 07/2004; 101(23):8603-7. · 9.81 Impact Factor
  • D Scott Witherow, Vladlen Z Slepak
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    ABSTRACT: Regulator of G-protein signaling (RGS) proteins of the R7 subfamily (RGS6, 7, 9, and 11) contain a unique Ggamma-like (GGL) domain that enables their association with the G-protein beta subunit Gbeta5. The existence of these complexes was demonstrated by their purification from native tissues as well as by reconstitution in vitro. According to pulse-chase analysis, Gbeta5 and RGS7 monomers undergo rapid proteolytic degradation in cells, whereas the dimer is stable. Studies of the functional role of Gbeta5-RGS dimers using GTPase activity, ion channel, and calcium mobilization assays showed that, similarly to other RGS proteins, they can negatively regulate G-protein-mediated signal transduction. Protein-protein interactions involving the Gbeta5-RGS7 complex can be studied in cells using fluorescence resonance energy transfer utilizing Gbeta5, RGS, and Galpha subunits fused to the cyan and yellow versions of green fluorescent protein.
    Methods in Enzymology 02/2004; 390:149-62. · 2.00 Impact Factor
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    ABSTRACT: A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo. In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways. However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.
    Journal of Biological Chemistry 07/2003; 278(23):21307-13. · 4.65 Impact Factor
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    ABSTRACT: A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Gγ-like domain through which they form obligatory dimers with the G protein subunit Gβ5 in vivo. In Caenorhabditis elegans, orthologs of Gβ5·RGS dimers are implicated in regulating both Gαi and Gαq signaling, and in cell-based assays these dimers regulate Gαi/o- and Gαq/11-mediated pathways. However, initial studies with purified Gβ5·RGS6 or Gβ5·RGS7 showed that they only serve as GTPase activating proteins for Gαo. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Gαo or Gαq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7·Gβ5 complex binds to Gαq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gβ5, and Gα subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gβ5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7·Gβ5 complex and cyan fluorescence protein-tagged Gαq, indicating a direct interaction between these molecules.
    Journal of Biological Chemistry 06/2003; 278(23):21307-21313. · 4.65 Impact Factor
  • D. Scott Witherow, Vladlen Z. Slepak
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    ABSTRACT: The fifth member of the G protein g the subunit family, G g 5, has been shown to bind exclusively to a subfamily of regulators of G protein signaling (RGS) including RGS6, RGS7, RGS9, and RGS11. This interaction occurs through a G protein gamma-like (GGL) domain present in members of this RGS subfamily and is the only reported instance in which a G g subunit is not bound to a G n subunit. The G g 5-RGS interaction has been demonstrated both in vitro and in vivo and has been shown to stabilize the dimer against proteolytic degradation. GTPase activating protein (GAP) assays suggest that G g 5-RGS7 acts specifically on G f o, however in cell-based assays it also inhibited G f i- and G f q-mediated signaling. The role of the dimer in signaling and the function of G g 5 moiety within the complex are poorly understood. This review summarizes the information about the assembly and function of G g 5-RGS dimers, as well as their posttranslational modifications and localization.
    Receptors and Channels 05/2003; 9(3):205-212.
  • D Scott Witherow, Vladlen Z Slepak
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    ABSTRACT: The fifth member of the G protein beta the subunit family, G beta5, has been shown to bind exclusively to a subfamily of regulators of G protein signaling (RGS) including RGS6, RGS7, RGS9, and RGS11. This interaction occurs through a G protein gamma-like (GGL) domain present in members of this RGS subfamily and is the only reported instance in which a G beta subunit is not bound to a G gamma subunit. The G beta5-RGS interaction has been demonstrated both in vitro and in vivo and has been shown to stabilize the dimer against proteolytic degradation. GTPase activating protein (GAP) assays suggest that G beta5-RGS7 acts specifically on G alphao, however in cell-based assays it also inhibited G alphai- and G alphaq-mediated signaling. The role of the dimer in signaling and the function of G beta5 moiety within the complex are poorly understood. This review summarizes the information about the assembly and function of G beta5-RGS dimers, as well as their posttranslational modifications and localization.
    Receptors and Channels 02/2003; 9(3):205-12.
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    ABSTRACT: RGS proteins regulate G protein-mediated signalling pathways through direct interaction with the Galpha subunits and facilitation of GTP hydrolysis. An RGS subfamily consisting of RGS 6, 7, 9, and 11 also interacts with the G protein beta subunit Gbeta5 via a characteristic Ggamma-like domain. Thus far, these complexes were found only in neurons, with RGS7 being the most widely distributed in the brain. Here we confirm the expression of RGS7 in spinal neurons and show as a novel finding that following an experimental spinal cord injury in rats, expression of RGS7 is induced in a subpopulation of other cells. Immunofluorescent confocal microscopy using a series of cell specific antibodies identified these RGS7 positive cells as activated microglia and/or invading peripheral macrophages. To rule out interference from the adjacent neurons and confirm the presence of RGS7-Gbeta5 complex in inflammatory cells, we performed immunocytochemistry, RT-PCR, Western blot, and immunoprecipitation using microglial (BV2) and peripheral macrophage (RAW) cell lines. Expression of RGS7 mRNA and protein are nearly undetectable in non-stimulated BV2 and RAW cells, but remarkably increased after stimulation with LPS or TNF-alpha In addition, RGS7-positive cells were also found in the perinodular rim in the rat spleen. Our findings show that RGS7-Gbeta5 complex is expressed in immunocompetent cells such as resident microglia and peripheral macrophages following spinal cord injury. This expression might contribute to the post-traumatic inflammatory responses in the central nervous system.
    European Journal of Neuroscience 03/2002; 15(4):602-12. · 3.75 Impact Factor
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    ABSTRACT: Regulators of G protein signaling RGS4 and RGS7 accelerate the kinetics of K(+) channels (GIRKs) in the Xenopus oocyte system. Here, via quantitative analysis of RGS expression, we reveal biphasic effects of RGSs on GIRK regulation. At low concentrations, RGS4 inhibited basal GIRK activity, but stimulated it at high concentrations. RGS7, which is associated with the G protein subunit G beta 5, is regulated by G beta 5 by two distinct mechanisms. First, G beta 5 augments RGS7 activity, and second, it increases its expression. These dual effects resolve previous controversies regarding RGS4 and RGS7 function and indicate that they modulate signaling by mechanisms supplementary to their GTPase-activating protein activity.
    FEBS Letters 04/2001; 492(1-2):20-8. · 3.58 Impact Factor
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    ABSTRACT: A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Galpha subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Ggamma -like domain, also has the unique ability to interact with the G protein beta subunit Gbeta(5). Here, we examined the behavior of Gbeta(5), RGS7, RGS9, and Galpha in tissue extracts using immunoprecipitation and conventional chromatography. Native Gbeta(5) and RGS7 from brain, as well as photoreceptor-specific Gbeta(5)L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gbeta(5) nor Gbeta(5)-free RGS could be detected. Co-expression in COS-7 cells of Gbeta(5) dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gbeta(5):RGS stoichiometry in a manner similar to Gbetagamma complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gbeta(5)-RGS and their coupled expression argue that in vivo, Gbeta(5) and Ggamma-like domain-containing RGSs only exist as heterodimers. Native Gbeta(5)-RGS7 did not co-precipitate or co-purify with Galpha(o) or Galpha(q); nor did Gbeta(5)L-RGS9 with Galpha(t). However, in transfected cells, RGS7 and Gbeta(5)-RGS7 inhibited Galpha(q)-mediated Ca(2+) response to muscarinic M3 receptor activation. Thus, Gbeta(5)-RGS dimers differ from other RGS proteins in that they do not bind to Galpha with high affinity, but they can still inhibit G protein signaling.
    Journal of Biological Chemistry 09/2000; 275(32):24872-80. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A novel protein class, termed regulators of G protein signaling (RGS), negatively regulates G protein pathways through a direct interaction with Gα subunits and stimulation of GTP hydrolysis. An RGS subfamily including RGS6, -7, -9, and -11, which contain a characteristic Gγ -like domain, also has the unique ability to interact with the G protein β subunit Gβ5. Here, we examined the behavior of Gβ5, RGS7, RGS9, and Gα in tissue extracts using immunoprecipitation and conventional chromatography. Native Gβ5 and RGS7 from brain, as well as photoreceptor-specific Gβ5L and RGS9, always co-purified as tightly associated dimers, and neither RGS-free Gβ5 nor Gβ5-free RGS could be detected. Co-expression in COS-7 cells of Gβ5 dramatically increased the protein level of RGS7 and vice versa, indicating that cells maintain Gβ5:RGS stoichiometry in a manner similar to Gβγ complexes. This mechanism is non-transcriptional and is based on increased protein stability upon dimerization. Thus, analysis of native Gβ5-RGS and their coupled expression argue that in vivo, Gβ5and Gγ-like domain-containing RGSs only exist as heterodimers. Native Gβ5-RGS7 did not co-precipitate or co-purify with Gαo or Gαq; nor did Gβ5 L-RGS9 with Gαt. However, in transfected cells, RGS7 and Gβ5-RGS7 inhibited Gαq-mediated Ca2+ response to muscarinic M3 receptor activation. Thus, Gβ5-RGS dimers differ from other RGS proteins in that they do not bind to Gα with high affinity, but they can still inhibit G protein signaling.
    Journal of Biological Chemistry 08/2000; 275(32):24872-24880. · 4.65 Impact Factor

Publication Stats

254 Citations
37.75 Total Impact Points

Institutions

  • 2004
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2000–2003
    • University of Miami Miller School of Medicine
      • Department of Molecular and Cellular Pharmacology
      Miami, FL, United States