R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

Hu, Guang; Wensel, Theodore G.

doi:10.1073/pnas.152094799

Cited by 157 publications

(156 citation statements)

References 48 publications

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“…The function of this unique sequence is still unknown. However, some data suggested its importance in the interactions of RGS9-1 with targets (10,17,65). As highlighted in the sequence alignment (Fig.…”

Section: Discussionmentioning

confidence: 95%

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Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

Sokal

Liang

et al. 2003

Journal of Biological Chemistry

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Inactivation of the visual G-protein transducin by GTP hydrolysis is regulated by the GTPase-accelerating protein (GAP) RGS9-1. Regulation of RGS9-1 itself is poorly understood, but we found previously that it is subject to a light-and Ca 2؉ -sensitive phosphorylation on Ser 475 . Because there are much higher RGS9-1 levels in cones than in rods, we investigated whether Ser 475 is phosphorylated in rods using Coneless mice and found that both the phosphorylation and its regulation by light occur in rods. Therefore, we used rod outer segments as the starting material for the purification of RGS9-1 kinase activity. Two major peaks of activity corresponded to protein kinase C (PKC) isozymes, PKC␣ and PKC. A synthetic peptide corresponding to the Ser 475 RGS9-1 sequence and RGS9-1 were substrates for recombinant PKC␣ and PKC. This phosphorylation was removed efficiently by protein phosphatase 2A, an endogenous phosphatase in rod outer segments, but not by PP1 or PP2B. Phosphorylation of RGS9-1 by PKC had little effect on its activity in solution but significantly decreased its affinity for its membrane anchor protein and GAP enhancer, RGS9-1 anchor protein (R9AP). PKC immunostaining was at higher levels in cone outer segments than in rod outer segments, as was found for the components of the RGS9-1 GAP complex. Thus, PKCmediated phosphorylation of RGS9-1 represents a potential mechanism for feedback control of the kinetics of photoresponse recovery in both rods and cones, with this mechanism probably especially important in cones.Transducin (Gt), 1 the visual G-protein, is inactivated when its bound GTP is hydrolyzed to GDP, a free phosphate dissociates from the catalytic site, and the ␣ subunit reassociates with its partner ␤␥ subunits. The rate of hydrolysis is regulated by the protein's association with a GTPase-accelerating protein (GAP). This GAP has been identified to be RGS9-1 (1), a member of a family of regulatory proteins for G␣ GAPs termed the regulators of G-protein signaling (RGS) family (reviewed in Refs. 2 and 3). The GAP activity of RGS9-1 is further enhanced by the ␥-subunit of the phototransduction effector cGMP phosphodiesterase (PDE␥) (1, 4). The acceleration of GTPase activity is essential for timely recovery of dark conditions of photoreceptor cells (5) (for recent reviews, see Refs. 6 and 7).RGS9-1 contains a G-protein-␥-subunit-like domain (8) that is employed for functional association with the G␤5L subunit (9 -12). This subunit regulates GAP activity by the effector subunit, PDE␥, and induces and stabilizes RGS9-1 (5, 10, 13). ROS membranes from knockout mice lacking RGS9 hydrolyze GTP more slowly than ROS membranes from control mice. Moreover, in electrophysiological measurements, the flash responses of RGS9Ϫ/Ϫ rods recovered much more slowly than normal after illumination (5). Alternative splicing of the RGS9 gene yields two molecular forms of RGS9, RGS9-1 and RGS9-2, that vary only in the amino acid sequence of the C terminus. RGS9-1 is a retina-specific transcript, whereas RGS9-2 is ex...

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Section: Discussionmentioning

confidence: 95%

“…RGS9-1 also forms a complex with a 25-kDa photoreceptor-specific phosphoprotein, RGS9-1 anchor protein (R9AP). R9AP binds to the N-terminal domain of RGS9-1 and anchors RGS9-1 to the disk membrane (17).…”

mentioning

confidence: 99%

Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

Sokal

Liang

et al. 2003

Journal of Biological Chemistry

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“…On the other hand, binding of monomeric RGS7 in vitro to G␣ o -GDP has been detected in a pull-down assay, where it was as high as 50% that in the presence of aluminummagnesium-fluoride (5). The interaction between G␤5L⅐RGS9 and G␣t-GDP has also been shown (29). Earlier surface plasmon resonance studies of RGS16 and G␣t revealed that in the presence of ␥ subunit of cGMP phosphodiesterase the affinity of RGS16 to G␣t-GDP-aluminum-magnesium-fluoride is only 2-fold higher than to G␣t-GDP (30), also illustrating measurable interaction of some RGS proteins to G␣-GDP.…”

Section: Function Of G␤5⅐rgsmentioning

confidence: 99%

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

Witherow

Tovey

Wang

et al. 2003

Journal of Biological Chemistry

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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 G␥-like domain through which they form obligatory dimers with the G protein subunit G␤5 in vivo. In Caenorhabditis elegans, orthologs of G␤5⅐RGS dimers are implicated in regulating both G␣ i and G␣ q signaling, and in cell-based assays these dimers regulate G␣ i/o -and G␣ q/11 -mediated pathways. However, initial studies with purified G␤5⅐RGS6 or G␤5⅐RGS7 showed that they only serve as GTPase activating proteins for G␣ o . Pull-down assays and coimmunoprecipitation with these dimers failed to detect their binding to either G␣ o or G␣ q , indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7⅐G␤5 complex binds to G␣ q using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, G␤5, and G␣ 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 G␤5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7⅐G␤5 complex and cyan fluorescence protein-tagged G␣ q , indicating a direct interaction between these molecules.Signal transduction from G protein-coupled receptors is regulated by the cycle of GTP binding and hydrolysis of heterotrimeric G proteins (G␣␤␥). Upon binding of an extracellular ligand, the activated receptor acts as a guanine nucleotide exchange factor, catalyzing the release of GDP and subsequent binding of GTP to the G␣ subunit, which promotes the heterotrimeric G protein to dissociate into G␣-GTP and a G␤␥ subunit complex. Both G␣-GTP and the tightly associated G␤␥ can modulate the activity of effector enzymes or ion channels until the GTP is hydrolyzed and the inactive G␣␤␥ heterotrimer is reformed. Control of the duration of the active state of the pathway takes place on several levels. One critical family of proteins involved in this regulation is the regulators of G protein signaling (RGS) 1 proteins, which act as GTPase-activating proteins (GAPs) to accelerate GTP hydrolysis on G␣ subunits. All RGS proteins have an ϳ120-amino acid RGS domain, which binds to G␣ subunits and stimulates their GTPase activity (1-3). Many RGS proteins contain additional domains involved in other proteins-protein interactions as well. One such subfamily of RGS proteins, consisting of RGS6, -7, -9, and -11, contains a unique G protein ␥-like domain and exists as an obligatory heterodimer with the G protein subunit G␤5 (4 -7). In the brain and retina, it has been shown that G␤5 exists only with RGS proteins and, surprisingly, does not exist as a traditional dimer with G␥ (8 -11).Although the majority of RGS proteins have been shown to be GAPs, some RGS proteins can a...

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“…The R7-Gb5 association is essential for the protein's stability and function (He et al, 2000;Chen et al, 2003). R7 proteins also share a DEP domain (Martemyanov et al, 2003;Ballon et al, 2006), which is required for their interaction with the adaptor molecules R9AP in the retina (Hu and Wensel, 2002) and R7 family binding protein (R7BP) in the brain (Martemyanov et al, 2005). The R7-R7BP interaction is essential for the cellular localization and function of R7 proteins, as it determines the localization of the protein at the cell membrane (Drenan et al, 2005;Anderson et al, 2009;Jayaraman et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

R7BP Modulates Opiate Analgesia and Tolerance but not Withdrawal

Terzi

Cao

Agrimaki

et al. 2011

Neuropsychopharmacol

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The adaptor protein R7 family binding protein (R7BP) modulates G protein coupled receptor (GPCR) signaling and desensitization by controlling the function of regulator of G protein signaling (RGS) proteins. R7BP is expressed throughout the brain and appears to modulate the membrane localization and stability of three proteins that belong to R7 RGS family: RGS6, RGS7, and RGS9-2. RGS9-2 is a potent negative modulator of opiate and psychostimulant addiction and promotes the development of analgesic tolerance to morphine, whereas the role of RGS6 and RGS7 in addiction remains unknown. Recent studies revealed that functional deletion of R7BP reduces R7 protein activity by preventing their anchoring to the cell membrane and enhances GPCR responsiveness in the basal ganglia. Here, we take advantage of R7BP knockout mice in order to examine the way interventions in R7 proteins function throughout the brain affect opiate actions. Our results suggest that R7BP is a negative modulator of the analgesic and locomotor activating actions of morphine. We also report that R7BP contributes to the development of morphine tolerance. Finally, our data suggest that although prevention of R7BP actions enhances the analgesic responses to morphine, it does not affect the severity of somatic withdrawal signs. Our data suggest that interventions in R7BP actions enhance the analgesic effect of morphine and prevent tolerance, without affecting withdrawal, pointing to R7BP complexes as potential new targets for analgesic drugs.

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R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

Cited by 157 publications

References 48 publications

Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

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

R7BP Modulates Opiate Analgesia and Tolerance but not Withdrawal

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