Rapid Kinetics of Regulator of G-protein Signaling (RGS)-mediated Gαi and Gαo Deactivation

The complex between the photoreceptor-specific regulator of G protein signaling (RGS) protein, RGS9-1, and type 5 G protein ␤-subunit, G␤5L, regulates the duration of the cellular response to light by stimulating the GTPase activity of G protein, transducin. An important property of RGS9-1⅐G␤5L is that it interacts specifically with transducin bound to its effector, cGMP phosphodiesterase, rather than with transducin alone. The minimal structure within the RGS9-1⅐G␤5L complex capable of activating transducin GTPase is the catalytic domain of RGS9. This domain itself is also able to discriminate between free and effector-bound transducin but to a lesser degree than RGS9-1⅐G␤5L. The goal of this study was to determine whether other, noncatalytic domains of RGS9-1⅐G␤5L enhance the intrinsic specificity of the catalytic domain or whether they set the specificity of RGS9-1⅐G␤5L regardless of the specificity of its catalytic domain. We found that a double L353E/R360P amino acid substitution reversed the specificity of the recombinant catalytic domain but did not reverse the specificity of RGS9-1⅐G␤5L. However, the degree of discrimination between free and effector-bound transducin was reduced. Therefore, noncatalytic domains of RGS9-1⅐G␤5L play a decisive role in establishing its substrate specificity, yet the high degree of this specificity observed under physiological conditions requires an additional contribution from the catalytic domain.RGS 1 proteins regulate the duration of signaling in many G protein pathways (1-3). They act by stimulating the GTP hydrolysis on G protein ␣-subunits, which results in termination of signaling events conferred by activated G proteins. All RGS proteins share a conservative catalytic domain of ϳ130 amino acid residues capable of accelerating the GTP hydrolysis on G protein ␣-subunits (2). Many RGS proteins are also equipped with other, noncatalytic domains (3). There are indications that these noncatalytic domains may participate in modulation of the RGS catalytic activity or may allow RGS proteins to interact with components of other intracellular pathways (reviewed in Ref. 4).Another putative role for the noncatalytic domains of RGS proteins is to contribute to the recognition of their specific G protein ␣-subunit targets. The idea that noncatalytic domains of RGS proteins may contribute to establishing the specificity in the RGS-G protein interactions is supported by several recent observations (5-7). Understanding the molecular mechanisms conferring this specificity has been complicated by many reports that RGS proteins and especially their catalytic domains can stimulate the GTPase activity of a broad spectrum of G protein ␣-subunits in vitro.The phototransduction cascade from vertebrate photoreceptors provides a useful model for studying substrate specificity in RGS proteins. In this cascade, the complex between the short splice isoform of RGS9 (RGS9-1) and the long splice isoform of type 5 G protein ␤-subunit (G␤5L) stimulates the GTPase activity of G protein, transducin (G ...

“…5 for additional examples). In contrast, some RGS proteins, like RGS4, seem to be rather nonspecific in interacting with their G␣ partners (5,26).…”

Section: Rgs9-1⅐g␤5l Is a Useful Model For Studying The Specificity Imentioning

confidence: 97%

Section: Rgs9-1⅐g␤5l Is a Useful Model For Studying The Specificity Imentioning

confidence: 99%

Noncatalytic Domains of RGS9-1·Gβ5L Play a Decisive Role in Establishing Its Substrate Specificity

Martemyanov

Arshavsky

2002

“…This idea is consistent with a model in which the receptor, G protein, and RGS do not physically dissociate but, rather, go through a series of conformational changes while progressing through the functional cycle. Such a model helps explain the rapid kinetics of many G protein cascades and the sub-second time constant of RGS-G␣ interactions (36). At this point it is not clear whether the dynamics of RGS-G␣ binding was not detected due the technical limitations of CFP-YFP FRET method or due to the true properties of G␣ q -G␤5⅐RGS7 complex in cells.…”

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

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...

“…The maximal stimulation of GTP hydrolysis by 10 M morphine at 2 min was 45 Ϯ 5% in the C6-G␣ o PTXi membranes and increased significantly (p Ͻ 0.05) to 94 Ϯ 17% in the presence of 1 M GST-RGS8, although relative to DAMGO, morphine was significantly less efficacious (p Ͻ 0.05) in the presence of GST-RGS8 (61 Ϯ 3%) than in its absence (75 Ϯ 1%). RGS8 was chosen for these studies because it is structurally a simpler RGS protein and is known to be a GAP for G␣ o (22).…”

Section: Methodsmentioning

confidence: 99%

Endogenous RGS Protein Action Modulates μ-Opioid Signaling through Gαo

Clark¹,

Harrison²,

Zhong³

et al. 2003

Self Cite

RGS (regulators ofGOpioid receptors are a typical seven-transmembrane domain receptor family that signal through inhibitory G proteins to a multitude of second messengers and cellular effectors, including adenylyl cyclase, voltage-operated calcium channels, and inwardly rectifying K ϩ channels (1); intracellular calcium stores (2); and the extracellular signal-regulated kinase (ERK) 1 mitogen-activated protein kinase (MAPK) pathway (3, 4).There are three principal types of opioid receptors, , ␦, and , with ϳ60% homology. However, the -opioid receptor has generated the most interest as the principal site of action for clinical analgesics and abused opiate drugs. The -opioid receptor can couple to all members of the G␣ i/o family, with little selectivity for particular G␣ subunits (5). Selectivity of intracellular -opioid signaling would therefore appear to depend on cell-specific expression of G protein subunits coupled with the selectivity of G proteins to interact with particular effectors. However, it has been suggested that other factors besides G protein and effectors may contribute to signaling specificity (6 -8).Agonist activation of G protein-coupled receptors leads to exchange of GDP for GTP on G␣ and dissociation of the G␣-GTP and G␤␥ subunits. Deactivation is brought about by the intrinsic GTPase activity of G␣, causing GTP to be hydrolyzed to GDP and the subsequent reassociation of the G␣-GDP and G␤␥ subunits. G protein signaling in this fashion is negatively controlled by a family of proteins known as RGS (regulators of G protein signaling) proteins (6). These proteins act as GTPaseactivating proteins (GAPs) for G␣ and speed up the hydrolysis of the G␣-bound GTP, thus reducing the steady-state levels of G␣-GTP and inhibiting signaling. Therefore, it has been suggested that, as with other G protein-coupled receptors, RGS proteins act to inhibit -opioid signaling and may play a controlling role in the effectiveness of opioid receptor ligands. In support of this idea, overexpression of RGS2 shifts the concentration effect curve for morphine-stimulated pigment aggregation to the right to a small (2-fold) degree in cultured dermal melanophores from Xenopus laevis transiently expressing a murine -opioid receptor (9). Furthermore, a reduction in RGS9 levels in mice using antisense oligonucleotide leads to an increase in the anti-nociceptive potency of morphine (10). Although these changes are small, they are suggestive of a role for RGS proteins in opioid coupling efficiency.An important question is whether RGS proteins alter the efficiency of all intracellular signaling pathways equally or whether there is a variable effect that would provide for selectivity. Selectivity for particular pathways may be obtained by several mechanisms. RGS-containing proteins have a wide variety of non-RGS domains (11-13) that, when RGS protein binds to G␣, can link other proteins and signaling pathways to provide for diversity of signaling. In addition, the interaction of RGS protein with receptors may contribute to selectiv...