The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein β subunit

Makino, Elina; Handy, Jason W.; Li, Tiansen; Arshavsky, Vadim Y.

doi:10.1073/pnas.96.5.1947

Cited by 200 publications

(142 citation statements)

References 32 publications

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“…Since most of the RGS residues in direct contact with G ␣ are well conserved across the entire family, these residues are unlikely to determine RGS-G protein specificity alone. Rather, additional regulatory proteins, such as effectors (11), G␤ proteins (9,(35)(36)(37)(38), or G proteincoupled receptors (7,39) may be involved. Since five classspecific residues (r77, r117, r121, r122, r124, and r125) cluster above the RGS-G ␣ interface to form an active site common to all members of the family, a reasonable hypothesis is that this is the binding site for additional proteins that mediate specificity (Fig.…”

Section: Discussion Evolutionary Analysis Can Provide Insight Into Rgmentioning

confidence: 99%

A regulator of G protein signaling interaction surface linked to effector specificity

Sowa

Wensel

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Proteins of the regulator of G protein signaling (RGS) family accelerate GTP hydrolysis by the ␣ subunits (G␣) of G proteins, leading to rapid recovery of signaling cascades. Many different RGS proteins can accelerate GTP hydrolysis by an individual G ␣, and GTP hydrolysis rates of different G ␣s can be enhanced by the same RGS protein. Consequently, the mechanisms for specificity in RGS regulation and the residues involved remain unclear. Using the evolutionary trace (ET) method, we have identified a cluster of residues in the RGS domain that includes the RGS-G ␣ binding interface and extends to include additional functionally important residues on the surface. One of these is within helix ␣3, two are in ␣5, and three are in the loop connecting ␣5 and ␣6. A cluster of surface residues on G␣ previously identified by ET, and composed predominantly of residues from the switch III region and helix ␣3, is spatially contiguous with the ET-identified residues in the RGS domain. This cluster includes residues proposed to interact with the ␥ subunit of Gt␣'s effector, cGMP phosphodiesterase (PDE␥). The proximity of these clusters suggests that they form part of an interface between the effector and the RGS-G ␣ complex. Sequence variations in these residues correlate with PDE␥ effects on GTPase acceleration. Because ET identifies residues important for all members of a protein family, these residues likely form a general site for regulation of G protein-coupled signaling cascades, possibly by means of effector interactions.H eterotrimeric G proteins (G ␣␤␥ ) mediate a ubiquitous eukaryotic pathway that converts extracellular signals received by transmembrane serpentine receptors into changes in the concentrations of intracellular ions and small molecule second messengers, thereby controlling vision, cardiac function, and many aspects of neuroendocrine signaling. Upon activation, a receptor catalyzes the exchange of GDP for GTP in the ␣ subunit of a specific G protein (G ␣ ), and either G ␣-GTP or its G ␤␥ partner can interact with a membrane-bound downstream effector protein, leading to amplification of the initial signal. Essential to G protein signaling is the intrinsic temporal regulation of the cascade imposed by G ␣ 's ability to switch back to its inactive form through hydrolysis of GTP. The regulator of G protein signaling (RGS) family of proteins plays a critical role in this process by increasing the intrinsic GTP hydrolysis rate of G ␣ (1-4) and accelerating recovery of the system. Nearly 50 different RGS family members have been identified in eukaryotes thus far, ranging from yeast to humans; and in mammals, individual RGS proteins display distinct expression patterns. However, in general, different types of RGS proteins coexist with a variety of G proteins, leading to the question of how RGS-G ␣ specificity is maintained (5). The crystal structure of the RGS4-G i␣1 complex (6) reveals that the contact residues between the RGS domain and G ␣ are highly conserved in both proteins, implying that in situ RGS-G pr...

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Section: Discussion Evolutionary Analysis Can Provide Insight Into Rgmentioning

confidence: 99%

A regulator of G protein signaling interaction surface linked to effector specificity

Sowa

Wensel

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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“…With the exception of the G␤5L⅐RGS9 complex in photoreceptors, which has been demonstrated to be a GAP for transducin in vivo (9), the function of G␤5⅐RGS complexes is not clear. For the G␤5⅐RGS7 dimer, there are seemingly contradicting lines of evidence.…”

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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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“…It is a tightly bound peripheral membrane protein (7), but has no obvious features that target it to membranes and is largely soluble when expressed in vitro (8,9). RGS9-1 forms a constitutive complex with its obligate subunit, G ␤5 (5,8,10), and it also interacts with the inhibitory subunit of the effector for G ␣t , cGMP phosphodiesterase-␥ (PDE␥) (11)(12)(13)(14). Neither G ␤5 nor PDE␥ can serve as its membrane anchor, because selective removal of either of them does not affect RGS9-1 membrane binding (2,9,15).…”

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confidence: 99%

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

Wensel²

2002

Proc. Natl. Acad. Sci. U.S.A.

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150

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The regulator of G protein signaling (RGS)-9-1⅐G␤5 complex forms the GTPase accelerating protein for G␣t in vertebrate photoreceptors. Although the complex is soluble when expressed in vitro, extraction of the endogenous protein from membranes requires detergents. The detergent extracts contain a complex of RGS9-1, G␤5, G␣t, and a 25-kDa phosphoprotein, R9AP (RGS9-1-Anchor Protein). R9AP is encoded by one intronless gene in both human and mouse. Full or partial cDNA or genomic clones were obtained from mice, cattle, human, zebrafish, and Xenopus laevis. R9AP mRNA was detected only in the retina, and the protein only in photoreceptors. R9AP binds to the N-terminal domain of RGS9-1, and anchors it to the disk membrane via a C-terminal transmembrane helix.T o ensure high efficiency of phototransduction, the essential components of the phototransduction cascade are packed at a high density on photoreceptor outer segment disk membranes (1), where phototransduction occurs. One challenge in photoreceptor biology has been to determine the mechanisms by which these molecules and their modulators are localized to the disk membranes. This question has been particularly puzzling for regulator of G protein signaling (RGS)-9-1, which is the GTPase accelerating protein (GAP) for the visual G protein G ␣t (2-4).RGS9-1 plays an essential role in setting the timing of the recovery phase of light responses in vertebrate photoreceptors (5, 6). It is a tightly bound peripheral membrane protein (7), but has no obvious features that target it to membranes and is largely soluble when expressed in vitro (8, 9). RGS9-1 forms a constitutive complex with its obligate subunit, G ␤5 (5, 8, 10), and it also interacts with the inhibitory subunit of the effector for G ␣t , cGMP phosphodiesterase-␥ (PDE␥) (11)(12)(13)(14). Neither G ␤5 nor PDE␥ can serve as its membrane anchor, because selective removal of either of them does not affect RGS9-1 membrane binding (2, 9, 15). Closely related proteins like RGS7 and the RNA processing variant RGS9-2, are found in the cytoplasm or nucleus (16-18), as well as on plasma membranes. Other RGS proteins in general show highly varied distribution patterns (19)(20)(21)(22)(23)(24)(25)(26). Variable subcellular targeting of RGS proteins may be a mechanism for regulation of their functions (18,(27)(28)(29).We describe here the identification by coimmunoprecipitation of a heterotetrameric complex containing RGS9-1, G ␤5L , G ␣t , and a previously unknown photoreceptor-specific protein, R9AP. R9AP interacts with the N-terminal domain of RGS9-1 and has a transmembrane helix at its C terminus, allowing it to serve as the RGS9-1-anchor protein. Materials and MethodsBuffers. The following standard buffers were used: low-salt buffer (5 mM Tris⅐HCl͞0.5 mM MgCl 2 ), GAPN buffer (10 mM Hepes͞ 100 mM NaCl͞2 mM MgCl 2 ), high-salt buffer (10 mM Hepes͞1 M NH 4 Cl͞2 mM MgCl 2 ), urea buffer (4 M urea͞5 mM Tris⅐HCl). The pH of all buffers was adjusted to 7.4-7.5, and 1 mM DTT and Ϸ20 mg/liter solid PMSF were added before use.P...

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The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein β subunit

Cited by 200 publications

References 32 publications

A regulator of G protein signaling interaction surface linked to effector specificity

A regulator of G protein signaling interaction surface linked to effector specificity

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

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

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