The Effector Enzyme Regulates the Duration of G Protein Signaling in Vertebrate Photoreceptors by Increasing the Affinity between Transducin and RGS Protein

Skiba, Nikolai P.; Hopp, Johnathan A.; Arshavsky, Vadim Y.

doi:10.1074/jbc.c000413200

Cited by 62 publications

(66 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As discussed in our previous studies (7,8), this approach allows an application of Michaelis analysis to studying RGS9-1⅐G␤5L activity with and without PDE␥. The experiment shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…GTPase Measurements-Transducin GTPase activity was determined by using either a multiple turnover ([GTP] Ͼ [transducin]) or single turnover ([GTP] Ͻ [transducin]) technique as described previously (8). The assays were conducted at room temperature (22-24°C).…”

Section: Methodsmentioning

confidence: 99%

“…An important property of RGS9-1⅐G␤5L is its selective interaction with activated G␣ t bound to its effector, the ␥-subunit of cGMP phosphodiesterase (PDE␥), rather than with free G␣ t . The specific RGS9-1⅐G␤5L interaction with the G␣ t ⅐PDE␥ complex is possible, because the RGS9-1⅐G␤5L affinity for G␣ t ⅐PDE␥ is much higher than the affinity for free activated G␣ t (8).…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Martemyanov

Arshavsky

2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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 ...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Martemyanov

Arshavsky

2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Because both native and recombinant RGS9-G␤5L interact much more efficiently with the complex of transducin with its effector, PDE␥, than with free activated transducin (13,18,20,21), we conducted all GTPase experiments in the presence of 50 M C-terminal PDE␥ peptide, PDE␥-(63-87). This peptide has been shown to completely replace the full-length PDE␥ in this assay (18,20). All data fitting and statistical analyses were performed with SigmaPlot software, version 6.…”

Section: Methodsmentioning

confidence: 99%

Specific Binding of RGS9-Gβ5L to Protein Anchor in Photoreceptor Membranes Greatly Enhances Its Catalytic Activity

Lishko¹,

Martemyanov²,

Hopp

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The complex between the short splice variant of the ninth member of the RGS protein family and the long splice variant of type 5 G protein ␤ subunit (RGS9-G␤5L) plays a critical role in regulating the duration of the light response in vertebrate photoreceptors by activating the GTPase activity of the photoreceptor-specific G protein, transducin. RGS9-G␤5L is tightly associated with the membranes of photoreceptor outer segments; however, the nature of this association remains unknown. Here we demonstrate that rod outer segment membranes contain a limited number of sites for high affinity RGS9-G␤5L binding, which are highly sensitive to proteolysis. In membranes isolated from bovine rod outer segments, all of these sites are occupied by the endogenous RGS9-G␤5L, which prevents the binding of exogenous recombinant RGS9-G␤5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-G␤5L, allowing for high affinity binding of recombinant RGS9-G␤5L to these sites. This binding results in a striking ϳ70-fold increase in the RGS9-G␤5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-G␤5L membrane attachment, as evident from the analysis of membrane-binding properties of deletion mutants lacking either N-or C-terminal parts of the RGS9 molecule. Our data indicate that specific association of RGS9-G␤5L with photoreceptor disc membranes serves not only as a means of targeting it to an appropriate subcellular compartment but also serves as an important determinant of its catalytic activity.Vertebrate photoreceptors produce rapid electrical responses to light and rapidly recover from excitation upon extinction of illumination (see Refs. 1-4 for recent reviews). Essential for the high speed of the photoresponse are the rates at which the G protein, transducin, is first activated by photoexcited rhodopsin and at which it is then inactivated by RGS9-G␤5L. 1 The rates of activation and inactivation were both documented to be at the high end of the range observed in the characterized G protein-based signaling pathways (Refs. 5 and 18; reviewed in Ref. 4). The rapid rates of both processes may be explained, at least in part, by the membrane association of most of the signaling proteins participating in phototransduction (reviewed in Ref. 1). Membrane association may increase the probability that these proteins encounter each other in orientations optimized for their productive interactions. The membrane association of most phototransduction proteins results from either their transmembrane position (e.g. rhodopsin) or their posttranslational modifications by fatty acids of isoprenoids (e.g. the subunits of transducin) (reviewed in Ref. 1). However, the nature of RGS9-G␤5L membrane attachment remains unknown. Neither RGS9 nor G␤5L has been shown to contain transmembrane domains or lipophilic posttranslational modifications. However, RGS9-G␤5L is bound to photoreceptor membranes so t...

show abstract

“…High-affinity interaction of RGS9-1 with transducin requires that transducin first binds its effector, the ␥-subunit of cGMP phosphodiesterase (PDE␥) (16). As a result, both RGS9-1 (9) and PDE␥ (17) are needed for timely GTP hydrolysis by transducin and normal recovery of the rod from light excitation.…”

mentioning

confidence: 99%

Functional comparison of RGS9 splice isoforms in a living cell

Martemyanov

Krispel

Lishko

et al. 2008

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

Self Cite

View full text Add to dashboard Cite

Two isoforms of the GTPase-activating protein, regulator of G protein signaling 9 (RGS9), control such fundamental functions as vision and behavior. RGS9 -1 regulates phototransduction in rods and cones, and RGS9 -2 regulates dopamine and opioid signaling in the basal ganglia. To determine their functional differences in the same intact cell, we replaced RGS9 -1 with RGS9 -2 in mouse rods. Surprisingly, RGS9 -2 not only supported normal photoresponse recovery under moderate light conditions but also outperformed RGS9 -1 in bright light. This versatility of RGS9 -2 results from its ability to inactivate the G protein, transducin, regardless of its effector interactions, whereas RGS9 -1 prefers the G proteineffector complex. Such versatility makes RGS9 -2 an isoform advantageous for timely signal inactivation across a wide range of stimulus strengths and may explain its predominant representation throughout the nervous system. G proteins ͉ phototransduction ͉ RGS proteins P roteins of the regulators of G protein signaling (RGS) family are ubiquitous regulators of signal duration in many G protein pathways. Their RGS homology domain is directly responsible for accelerating the GTPase activity of G protein ␣-subunits, but most RGS proteins also contain additional domains, which vary greatly among the family members. Little is known about the functional roles of these non-catalytic domains, although they are thought to contribute to the specificity of RGS interactions (reviewed in 1, 2). RGS9 is one of the better-studied multidomain RGS proteins and exists in two splice isoforms (3-5), both of which form constitutive complexes with the type 5 G protein ␤ subunit, G␤5 (6-8). The difference between these isoforms resides in the structure of their C-termini: a short 18-aa sequence in RGS9-1 is replaced with 209 residues in RGS9-2 (Fig. 1A).RGS9-1 is expressed exclusively in rod and cone photoreceptors where it sets the duration of electrical responses to light by accelerating the GTPase activity of transducin (3). In mice, the lack of RGS9 causes a drastic delay in photoresponse recovery (9), and its mutation in humans leads to difficulties in adjusting to bright light and seeing moving objects (10). RGS9-2 is expressed predominantly in the striatum, where it controls reward behavior and movement coordination by regulating D2 dopamine and -opioid receptor signaling (11-15). RGS9 knockout mice display augmented sensitivity to rewarding properties of morphine and cocaine (12, 13) and rapidly develop dyskinesias following administration of dopamine receptor antagonists (15).The physiological significance of having two RGS9 isoforms is unknown. High-affinity interaction of RGS9-1 with transducin requires that transducin first binds its effector, the ␥-subunit of cGMP phosphodiesterase (PDE␥) (16). As a result, both RGS9-1 (9) and PDE␥ (17) are needed for timely GTP hydrolysis by transducin and normal recovery of the rod from light excitation. We hypothesized that the biological role for such a dual requirement is to ensure ...

show abstract

The Effector Enzyme Regulates the Duration of G Protein Signaling in Vertebrate Photoreceptors by Increasing the Affinity between Transducin and RGS Protein

Cited by 62 publications

References 36 publications

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

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

Specific Binding of RGS9-Gβ5L to Protein Anchor in Photoreceptor Membranes Greatly Enhances Its Catalytic Activity

Functional comparison of RGS9 splice isoforms in a living cell

Contact Info

Product

Resources

About