Biochemical purification and functional analysis of complexes between the G-protein subunit Gbeta5 and RGS proteins

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

A novel kind of G protein heterodimer: the G beta5-RGS complex

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

Gβ5·RGS7 Inhibits Gαq-mediated Signaling via a Direct Protein-Protein Interaction

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

G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction

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

Role for the Abi/Wave Protein Complex in T Cell Receptor-Mediated Proliferation and Cytoskeletal Remodeling

SummaryBackgroundThe molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation.ResultsHere we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex.ConclusionsThe Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation

Spinal cord injury induces expression of RGS7 in microglia/macrophages in rats

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