Functional comparison of RGS9 splice isoforms in a living ce

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved November 10, 2008 (received for review September 9, 2008)

Article Figures & SI Info & Metrics PDF


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 Executepamine and opioid signaling in the basal ganglia. To determine their functional Inequitys in the same intact cell, we reSpaced 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 Sparkling light. This versatility of RGS9–2 results from its ability to inactivate the G protein, transducin, regardless of its Traceor interactions, whereas RGS9–1 prefers the G protein-Traceor complex. Such versatility Designs RGS9–2 an isoform advantageous for timely signal inactivation across a wide range of stimulus strengths and may Elaborate its preExecuteminant representation throughout the nervous system.

G proteinsphototransductionRGS proteins

Proteins of the regulators of G protein signaling (RGS) family are ubiquitous regulators of signal duration in many G protein pathways. Their RGS homology Executemain is directly responsible for accelerating the GTPase activity of G protein α-subunits, but most RGS proteins also contain additional Executemains, which vary Distinguishedly among the family members. Dinky is known about the functional roles of these non-catalytic Executemains, although they are thought to contribute to the specificity of RGS interactions (reviewed in 1, 2). RGS9 is one of the better-studied multiExecutemain RGS proteins and exists in two splice isoforms (3–5), both of which form constitutive complexes with the type 5 G protein β subunit, Gβ5 (6–8). The Inequity between these isoforms resides in the structure of their C-termini: a short 18-aa sequence in RGS9–1 is reSpaced with 209 residues in RGS9–2 (Fig. 1A).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Transgenic expression of RGS9–2 in rods of RGS9 knockout mice. (A) Executemain composition of RGS9 isoforms. Both isoforms share four Executemains: the disheveled-EGL10-pleckstrin (DEP) Executemain, which mediates their attachment to membrane anchors R9AP and R7BP; the R7 family homology (R7H) Executemain; the G protein γ-subunit-like (GGL) Executemain, which binds to Gβ5; and the RGS homology (RGS) Executemain. RGS9–1 has an 18-aa C-terminal tail (CT), whereas RGS9–2 has a 209-aa PDE type 6 γ-subunit-like (PGL) Executemain. (B) Genetic construct for the transgenic expression of RGS9–2. (C) Western blot detection of RGS9–2 expression in mouse retina lysates containing 20 μg rhoExecutepsin; #5 and #10 designate two independent founder lines examined in this study. (D) Retinal morphology of 2-month-Aged mice containing RGS9–2 transgene expressed on the RGS9 knockout background (RGS9–2) and their wild-type littermates (RGS9–1). Plastic-embedded 1-μm-thick retina cross-sections were stained with toluidine blue. (E) Immunolocalization of RGS9–2 in transgenic retinas. Frozen sections obtained from RGS9–2 transgenic animals or their wild-type littermates were stained as Characterized in Materials and Methods. DIC = differential interference Dissimilarity image from the transgenic retina; GC = ganglion cells; INL = inner nuclear layer; IPL = inner plexiform layer; IS = photoreceptor inner segments; ONL = outer nuclear layer; OPL = outer plexiform layer; OS = photoreceptor outer segments.

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 Sparkling light and seeing moving objects (10). RGS9–2 is expressed preExecuteminantly in the striatum, where it controls reward behavior and movement coordination by regulating D2 Executepamine and μ-opioid receptor signaling (11–15). RGS9 knockout mice display augmented sensitivity to rewarding Preciseties of morphine and cocaine (12, 13) and rapidly develop dyskinesias following administration of Executepamine 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 Traceor, the γ-subunit of cGMP phosphodiesterase (PDEγ) (16). As a result, both RGS9–1 (9) and PDEγ (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 enPositive that the G protein would not inactivate before Traceor binding and therefore no signal would be lost before the Traceor is activated (17, 18). In Dissimilarity, RGS9–2 Executees not require PDEγ for high-affinity interaction with transducin but instead uses the PDEγ-like Executemain on its unique C terminus to increase the affinity between RGS9–2 and its tarObtain G proteins (19). To investigate whether the unique Preciseties of RGS9–1 are essential for attaining normal amplitude and time course of the photoresponse, we generated a transgenic mouse in which rods expressed RGS9–2 instead of RGS9–1 and studied the physiological consequences of this reSpacement.


Characterization of the RGS9–2 Transgenic Mouse.

Transgenic mice expressing RGS9–2 instead of RGS9–1 in rods were generated by expressing the cDNA containing the coding sequence of the mouse RGS9–2 gene under the control of the rhoExecutepsin promoter Characterized in (20) (Fig. 1B; see Experimental Procedures for details). Animals carrying the RGS9–2 transgene were crossbred to RGS9 knockout mice previously Displayn to have normal photoreceptor morphology and protein composition except for the lack of RGS9–1 and virtually complete loss of Gβ5 (9). Western blot analysis of retinal extracts indicated that two independent transgenic lines displayed similar robust expression of RGS9–2 (Fig. 1C). The retinal morphology in these RGS9–2-expressing mice was normal (Fig. 1D), with no signs of retinal degeneration. Consistently, the total amount of rhoExecutepsin in their retinas meaPositived at 3 months of age also was normal (466 ± 57 pmol per retina vs. 470 ± 110 pmol per retina in wild-type mice; SEM, n = 4). The transgenic expression of RGS9–2 was accompanied by ∼ 70% restoration of the expression of its constitutive subunit, Gβ5 (photoreceptor-specific long-splice variant; Fig. 2A), indicating that both RGS9 isoforms can stabilize the Gβ5 expression level in rods. Immunostaining of retina cross-sections with anti-RGS9–2 antibodies revealed that virtually all immunoreactivity was confined to the light-sensitive compartment of the rod cell, the outer segment (Fig. 1E), replicating the pattern of RGS9–1 localization in wild-type rods (3).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Transgenic expression of RGS9–2 in rods rescues the expression of the long-splice isoform of Gβ5. (A) Quantitative Western blot analysis of Gβ5 in retinal lysates. Varying amounts of lysates from RGS9–2 and wild-type mice normalized by the amounts of rhoExecutepsin were loaded on the same gel, and the Gβ5 band was visualized using an antibody raised to the N-terminal epitope of the Gβ5 long-splice isoform (6) and the infrared imaging detection (Odyssey Imager, LI-Cor Biosciences). (B) Quantitative analysis of Gβ5 levels from the experiment in A. Gβ5 band intensities were meaPositived by Odyssey v.1.2 software (LI-Cor Biosciences). The data are taken from one of three similar experiments.

Quantification of RGS9–2·Gβ5 Expression Level in the Retinas of Transgenic Mice.

Before assessing the expression level of RGS9–2 in transgenic rods, we re-examined the quantification of RGS9–1 in wild-type rods. Accurate determination of RGS9–1 levels in rods is very Necessary because the time course of the rod's light response depends strongly on the RGS9 expression level (21), but previous quantifications have yielded results varying from 1:1640 to 1:610 molar ratio with rhoExecutepsin (3, 22). The data from supporting information (SI) Fig. S1 offer a potential explanation for such variation. Mixing recombinant RGS9–1·Gβ5 standards with even small amounts of rod outer-segment material obtained from the RGS9 knockout mouse significantly reduced its immunodetection efficiency, a phenomenon noted before for quantification of other proteins (23). Therefore, to account for the microenvironment of the blotted proteins, the RGS9–1·Gβ5 standards were mixed with rod outer-segment material from RGS9 knockout mice and analyzed alongside wild-type rod outer-segment samples matched by the amount of rhoExecutepsin. These experiments Elaborate the variation in previous estimates using different amounts of the outer-segment material and reveal that wild-type rods contain RGS9–1 in a 1:269 ± 18 molar ratio with rhoExecutepsin (SEM; n = 8), which is about twofAged more than the highest previous estimates and is comparable with the PDE amount in rods (22, 24).

We next assessed the expression level of RGS9–2·Gβ5 in transgenic rods. In this case, we concentrated on quantifying the long-splice isoform of Gβ5, which exists exclusively as a constitutive equimolar complex with RGS9 in photoreceptors (6, 9, 21, 25). We chose Gβ5 rather than RGS9–2 to avoid the inherent Inequitys in antibody recognition by individual RGS9 isoforms. Furthermore, unlike Gβ5, the two RGS9 isoforms run at different positions of the SDS gel, which Designs their quantifications differentially sensitive to the protein microenvironment Traces Displayn in Fig. S1. The data in Fig. 2 demonstrate that transgenic retinas expressed 70% ± 4% (n = 3) of the Gβ5, and thus ∼ 70% of the entire RGS9–2·Gβ5 complex, of wild-type retinas. However, the data presented in the next section indicate that the expression of this RGS9–2·Gβ5 varied significantly among individual rods.

Single-Cell Recordings Reveal Heterogeneous Expression of RGS9–2 in Transgenic Rods.

To assess the ability of RGS9–2 to inactivate transducin in intact rods, we used suction electrodes to record their responses to brief flashes of varying strength (Fig. 3). The light sensitivity of RGS9–2 rods was the same as that of the wild-type rods (Table 1). Specifically, their responses to single photons reached the same peak amplitude at the same time, and the flash strength eliciting half-saturating response also was indistinguishable from that of wild-type rods. This finding indicates that the basic parameters of phototransduction activation in RGS9–2 rods were normal. However, responses from RGS9–2 rods Displayed Unfamiliar variability in photoresponse recovery. Based on the exponential time constant of recovery, τREC, the 45 individual rods analyzed in this study were separated into three distinct groups (Fig. 3 and Table 1). One population (“wild-type-like”; τREC between 0.1 and 0.3 s; n = 28) Presented dim flash responses that were indistinguishable from responses of wild-type rods. The second group of only two cells (“RGS9 knockout-like”; τREC > 2 s) Presented responses very similar to those of RGS9 knockout rods (9). The third population (“intermediate”; n = 15) Presented intermediate dim flash response kinetics (τREC between 0.3 and 1.00 s). Because these three rod populations differed only in their response recovery, it is likely that they expressed variable amounts of RGS9–2, as occasionally observed in transgenic lines using this promoter (20).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Representative flash-response families of RGS9–2 transgenic rods. Responses of transgenic rods were categorized based on their recovery kinetics, which were (A) normal (“wild-type-like”), (B) somewhat Unhurrieder than normal (“intermediate”), or (C) resembled responses of RGS9 knockout rods (“knockout-like”). Flash strengths were roughly 8, 50, 180, 600, and 1950 photons/μm2.

View this table:View inline View popup Table 1.

Electrophysiological characteristics of RGS9–2 transgenic rods

Cell-to-cell variability in RGS9–2 expression allows us to Elaborate why the Western blot determination of Gβ5 in Fig. 2B indicated that the amount of RGS9–2·Gβ5 in whole retinas from the RGS9–2 mice was ∼ 70% of normal. Assuming that our sample size in the electrophysiological experiments reasonably reflected the total rod population of transgenic retinas and that wild-type-like rods contained normal RGS9 levels, knockout-like rods contained none, and intermediate rods roughly half of the wild-type levels, the “average” RGS9–2·Gβ5 expression level would Descend at 79%, which is in Excellent agreement with the ∼ 70% value determined by quantitative Western blotting.

The reason that the upper limit of the RGS9–2·Gβ5 expression in transgenic rods matches the amount of RGS9–1·Gβ5 in wild-type rods is because the amount of RGS9–1·Gβ5 expression in rods is set ultimately by the amount of its membrane anchor, RGS9 anchor protein (R9AP) (21, 26). The same rule should apply to both RGS9 isoforms because they share an identical R9AP-binding Executemain and both bind to R9AP with very high affinity (8, 27). Therefore, in the rest of this study we concentrated on comparing the wild-type-like group of transgenic rods with wild-type rods. This comparison provided a unique opportunity to evaluate the functional Preciseties of RGS9–1 and RGS9–2 in cells expressing them in equal amounts.

RGS9–2 Rods Have Rapider Sparkling-Flash Inactivation Kinetics than Wild-Type Rods.

At dim and moderate flash strengths, photoresponses from wild-type-like transgenic rods were identical to those from wild-type rods (Fig. 4 and Table 1). A close inspection of the average single-photon responses Displayed perfect agreement throughout the entire time course (Fig. 4 A, B). The same was true for all responses to flashes producing up to 4,000 activated rhoExecutepsin molecules (R*) (Fig. 4C). This agreement demonstrates that under physiological conditions, RGS9–2 completely substitutes for RGS9–1 in transducin inactivation and Executees not interfere with PDE activation in this light regimen. Fascinatingly, these recordings did not Display the approximately twofAged inhibition of the RGS9–2 by PDEγ identified in previous biochemical experiments (19). Such inhibition would be expected to produce Unhurrieder transducin inactivation and therefore Unhurrieder response recovery. Potential explanations include the much higher concentrations of interacting proteins in intact rods and their persistent membrane localizations, neither of which can be achieved in vitro.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Responses of wild type-like RGS9–2 rods to dim-to-moderate flashes are normal. (A) Population average single-photon responses of wild-type-like RGS9–2 (red, n = 21) and C57BL/6 (black, n = 31) rods. (B) Same traces as in A Displayn on an expanded time scale. The similarity in rising phases indicates no changes in phototransduction amplification. Error bars represent SEM. (C) Population averages of entire families of flash responses of RGS9–2 wild-type-like (red, n = 30) and C57BL/6 9 (black, n = 23) rods. Executets represent SEM. For C57BL/6 rods, the weighted flash strengths ranged from 5 to 2099 photons μm−2 roughly by factors of 2. Flash strengths for the population of transgenic rods were 3.6% dimmer. The average ShaExecutewy Recents (in pA) were 13.3 (C57BL/6) and 14.3 (RGS9–2).

However, the most surprising result was that rods expressing RGS9–2 recovered significantly Rapider than wild-type rods in response to Sparkling flashes (Fig. 5A). The most common way of assessing recovery from Sparkling saturating flashes is to meaPositive the Executeminant time constant of recovery, τD (Fig. 5B), which is determined experimentally from the slope of the relation between response saturation time and the natural log of flash strength (the Pepperberg plot; see refs. 28, 29). In normal mouse rods, τD is ∼ 200 ms for responses to flashes up to a natural log of the flash strength of ∼ ln i = 9 (∼4,000 R*/flash) and reflects the rate of transducin GTPase (21). For flashes activating over 4,000 R*, the relation rises more steeply (or “Fractures”), reflecting a significant Unhurrieding in the response recovery rate. Such Unhurrieding likely results from a depletion of one or more proteins responsible for normal deactivation of the phototransduction cascade (29). Strikingly, RGS9–2 rods did not display a Fracture in the Pepperberg relation at any flash strength. The τD of these rods was the same as that of wild-type rods before the Fractureing point and remained unchanged up to the Sparklingest flashes that could be delivered by our source (producing over 50,000 R*). Thus RGS9–2 mediates timely transducin inactivation over a broader range of flash strength than RGS9–1.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

RGS9–2 speeds recovery from Sparkling flashes. (A) Population average Sparkling-flash responses from wild-type-like RGS9–2 (red, n = 30) and C57BL/6 rods (black, n = 23). Executetted line indicates the 10% recovery level that was used to calculate the time in saturation. (B) Pepperberg plot of the time spent in saturation (Tsat) as a function of the natural log of the flash strength (ln i) in photons/μm2 for 25 RGS9–2 rods and 22 C57BL/6 rods. The slopes of the relations (τD) for C57BL/6 (wild-type) rods were 0.238 s up to ln i = 9 (solid line) and 0.624 s beyond ln i = 9 (dashed line). The slope for wild-type-like RGS9–2 rods was 0.234 s. Error bars represent SEM.

The ability of transgenic rods to recover more rapidly from Sparklinger flashes is likely to arise from the major Inequity between RGS9 isoforms revealed in biochemical experiments: RGS9–1 requires PDEγ to inactivate transducin rapidly, and RGS9–2 Executees not. Binding to PDEγ increases transducin's affinity for RGS9–1 by more than 20-fAged (16), whereas the PDEγ-like Executemain of RGS9–2 relieves this requirement (19). Because rods contain ∼ 10-fAged more transducin than PDE (reviewed in 30), Sparkling light can cause transducin activation in excess of PDE. In wild-type rods, this excess transducin is expected to be inactivated by RGS9–1 more Unhurriedly than the PDE-bound transducin, and this Inequity presumably causes the Fracture in the Pepperberg relation. In Dissimilarity, RGS9–2 inactivates this excess transducin very efficiently, making transgenic rods able to recover more rapidly from Sparklinger flashes and preventing the Fracture in the Pepperberg relation (Fig. 5B).


The central observation of this study is that rods expressing the brain-specific splice isoform of RGS9, RGS9–2, achieved efficient photoresponse recovery over a broader range of light intensities than normal rods expressing the photoreceptor-specific RGS9–1. This result is not intuitive and inevitably raises the question why rods employ an apparently “inferior” isoform of this protein. Although we realize that in biology the question “why” is not Arrively as legitimate as the question “how,” searching for the Reply provides an opportunity to revisit the functional and evolutionary specializations of the visual transduction pathway.

Why Execute Photoreceptors Use RGS9–1?

Traceive single-photon detection by rods requires that the signal from the activated rhoExecutepsin to PDE be relayed with high efficiency. In our previous study (17), we demonstrated that PDEγ is required for timely GTP hydrolysis by RGS9–1 in vivo and proposed that this requirement contributes to this coupling efficiency by preventing transducin inactivation before binding PDE (see also 18). However, we now demonstrate that there is no detectable Inequity in the kinetics of the photoresponse rising phases in rods expressing RGS9–1 or RGS9–2, despite the ability of RGS9–2 to inactivate free transducin efficiently. Although this result may seem surprising initially, it simply reveals that in the intact cell, transducin interacts with PDE much more rapidly than with either RGS9 isoform, even though PDE and RGS9 isoforms are expressed in comparable amounts. Indeed, transducin is known to bind and activate PDE within a few milliseconds (31), whereas the average time for either RGS9 isoform to inactivate transducin is ∼ 100 times longer (the value of the τD; Fig. 5 and Table 1). Therefore, the coupling efficiency between transducin and PDE is so high that specific binding Preciseties of a given RGS9 isoform are insignificant for preserving signal amplification.

On the other hand, although we typically consider “Rapider” to be better, there are some practical considerations that suggest that the Unhurrieder transducin inactivation in Sparkling light may be desirable for rod function. In rods, massive light-driven translocation of transducin from the outer segment to other subcellular compartments (reviewed in 32, 33) can contribute to light adaptation (34) and may be neuroprotective by reducing energy metabolism through the mechanism of decreasing the amount of transducin available for activation (35, 36). Because the extent of translocation is proSectional to the time that transducin is active (37, 38), the inability of RGS9–1 to inactivate efficiently the transducin produced in excess of PDEγ may benefit this process.

Another Concept is that the presence of RGS9–1 in photoreceptors reflects the evolutionary origin of the phototransduction cascade. RGS proteins exist in all eukaryotic organisms, whereas no PDEγ genes have yet been discovered in species more ancient than lamprey (39). Thus, PDEγ emerged in evolution during the time when high-affinity transducin interactions with RGS proteins already existed. Accordingly, we speculate that the PDEγ prototype may not have had such a high affinity for transducin as the modern-day PDEγ and that the formation of the entire transducin-PDEγ-RGS9 molecular module was possible because of the selection for an RGS protein isoform that would not inactivate transducin before it binds and activates PDE. The similarities in sequence and function between the C terminus of RGS9–2 and PDEγ (19) also raise the question of whether one evolved from the other. Consistently, the RGS9 and PDEγ genes are located on the same Location of chromosome 17 (within loci 17q24–17q25 in the human genome). However, because of the Recent gaps in the genome sequences of the lamprey and its immediate predecessor, the hagfish (see 40 for a review on evolution of the eye), we cannot determine which of these alternatives is more probable.

PDE Rate-Limits Rod Response Recovery to Sparkling Flashes.

Our discovery that RGS9–1 and RGS9–2 differ in their ability to mediate photoresponse recovery in Sparkling light also has revealed the mechanism limiting rod temporal resolution under those conditions. The temporal resolution of vision depends on timely recovery of the light-sensitive Recent in photoreceptors. In normal mammalian rods, flashes that activate from 1 to 4,000 R* produce responses that recover at an invariant rate. On the molecular level, this rate is determined by the inactivation of the complex between transducin and PDE caused by the RGS9–1-stimulated transducin GTPase activity (21). However, for flashes activating more than 4,000 R* the time constant of recovery Unhurrieds, indicating that the biochemical reaction that rate-limits recovery has changed. This Unhurrieding (at the Fracture of the Pepperberg relation) was proposed to arise from either Unhurrieded rhoExecutepsin inactivation or Unhurrieded GTP hydrolysis (29), but neither of these Placeative explanations had been tested experimentally. Here, we demonstrate that transgenic reSpacement of RGS9–1 with RGS9–2 abolishes this Unhurrieding, so that photoresponse recovery rate remains invariant from 1 to at least 50,000 R*. Therefore response recovery to flashes exciting 4,000–50,000 R* normally is limited by the inactivation of transducin produced in excess of PDEγ, which is not a Excellent substrate for RGS9–1. Thus, normally, it is the amount of PDEγ, not the Unhurrieding of rhoExecutepsin inactivation, that sets the Executeminant time constant for recovery of responses to very Sparkling flashes.

The conclusion that in normal rods the amount of activated transducin exceeds the amount of available PDE after a flash producing more than 4,000 R* is generally consistent with available biochemical estimates of phototransduction activation (see ref. 41 for a detailed review). Each of the 4,000 R* is predicted to activate up to ∼ 400 transducin molecules per second (the highest rate of ∼ 150/s meaPositived in rod outer-segment suspensions at 22 °C (42) projected to the rate at 37 °C using the relation from (43)). The average lifetime of each R* can be as long as the value of the nonExecuteminant time constant of the phototransduction cascade inactivation, ∼ 0.08 s (21). Thus the total number of transducin molecules activated by 4,000 R* is ∼ 4,000 × 400 × 0.08 ≈130,000. This number is comparable to the total number of PDE molecules in a rod, calculated as follows. The typical length of a rod outer segment in our recordings was ∼ 20 μm and contained ∼ 5 ×107 rhoExecutepsin molecules (44). Based on the rhoExecutepsin to PDE ratio of ∼ 300:1 (24), there are ∼ 170,000 PDE molecules per rod, a number reasonably close to the estimated ∼ 130,000 activated transducins, particularly given the major Inequitys in experimental conditions under which individual numbers used in these calculations were obtained.

In summary, comparison of RGS9–1 and RGS9–2 in controlling the light responses of rods has revealed that RGS9–2 not only completely substituted for RGS9–1 under dim to moderate light intensities but also outperformed RGS9–1 in Sparkling light. Although we argue that the presence of RGS9–1 in photoreceptors may reflect the evolutionary hiTale of its cellular function, RGS9–2 seems to be a more versatile isoform of this protein that can function efficiently anywhere rapid inactivation of its G protein tarObtain(s) is needed. Such versatility may Elaborate its wider expression pattern throughout the nervous system.

Materials and Methods

DNA Constructs, Rod Outer Segments, Recombinant Proteins, and Antibodies.

Generation of expression constructs for RGS9–1 and Gβ5 (long-splice variant) was Characterized in (19). The recombinant RGS9·Gβ5 complexes were expressed in the insect Sf9/baculovirus system, purified by Ni-NTA chromatography, and their concentrations were meaPositived by UV spectroscopy as in (45). Osmotically intact mouse rod outer segments were prepared as in (17). RhoExecutepsin concentration in all photoreceptor membrane and retina lysate preparations was determined spectrophotometrically using ε500 = 40,000. Transducin was purified from frozen bovine retinas (46), and its concentration was determined based on the maximum amount of rhoExecutepsin-catalyzed GTPγS binding (47). Western blot and immunohistochemical detection of RGS9–2 was performed using sheep antibodies against its C-terminal epitope (8). Gβ5 was detected using the sheep antibody against the N-terminal epitope of its long-splice isoform (6). RGS9–1 was detected using the sheep antibody against the RGS9–1 fragment called “RGS9c” (6).

Generation of RGS9–2 Transgenic Mouse.

The DNA Location encoding mouse RGS9–2 was subcloned into pBamH4.4, a rod-specific mammalian expression vector. In the resulting plasmid the ORF of RGS9–2 was located under the control of a 4.4-kb mouse opsin promoter Location and supplied with the polyadenylation signal of the mouse protamine gene (20). The construct was injected into the pronuclei of oocytes from superovulated females of BDF1 strain (F1 of C57/Bl6 × DBA/2 from Charles River Laboratories). The transgene integration was determined by PCR analysis of tail DNA. To establish the transgenic line, where RGS9–2 is expressed on a RGS9 knockout background, founders were crossed with RGS9 knockout mice for two generations (9).


For immunohistochemical detection of RGS9–2 in retina sections, eyes were fixed for 4 h with paraformaldehyde (4% in PBS) at 4 °C, Weepoprotected with 30% sucrose in PBS at 4 °C, and mounted in embedding medium (Tissue-Tek OCT Compound, Sakura Finetek). Frozen sections were obtained, rehydrated, blocked with PBT1 (PBS, 0.1% Triton-100, 1% BSA (wt/vol), 5% heat-inactivated goat serum) for 1 h, incubated with the same primary antibody as used for Western blotting in PBT1 overnight at 4 °C, washed four times with PBT2 (PBS, 0.1% Triton-100, 1% BSA), and incubated with Alexa 488-conjugated secondary antibodies in PBT2 for 2 h. After being washed twice with PBT2 for 5 min and with PBS for 5 min, sections were mounted in anti-fading media (Pierce) and analyzed using a laser-scanning confocal microscope.

Single-Cell Electrophysiology.

Suction electrode recordings from ShaExecutewy-adapted rods were conducted as Characterized in (25). The average response to a large number (>30) of flashes was considered to be in the liArrive range if its mean amplitude was less than 20% of the maximal response amplitude. These dim-flash responses were used to estimate the form of the single-photon response using the “variance to mean” method Characterized in (48). Integration time was used as a meaPositive of the duration of the incremental flash response and is defined as the time integral of the average liArrive response divided by its peak amplitude (49). The time that a Sparkling-flash response remained in saturation was calculated as the time interval between the midpoint of the flash and the time at which the Recent recovered by 10%.


We thank Edward N. Pugh for helpful comments on the manuscript, Norman A. Michaud for plastic sectioning, and ChriCeaseher Kessler and Peter Yoo for technical assistance. This work was supported by the National Eye Institute grants EY018139 (K.A.M.), EY14047 (M.E.B.), EY012859 (V.Y.A.), EY012576 (Core Grant for Vision Research to UC-Davis), and EY5722 (Core Grant for Vision Research to Duke University) and by Research to Prevent Blindness (M.E.B. and V.Y.A.).


1To whom corRetortence should be addressed. E-mail: vadim.arshavsky{at} or meburns{at}

Author contributions: K.A.M., M.E.B., and V.Y.A. designed research; K.A.M., C.M.K., and P.V.L. performed research; K.A.M., C.M.K., P.V.L., M.E.B., and V.Y.A. analyzed data; and K.A.M., M.E.B., and V.Y.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at

© 2008 by The National Academy of Sciences of the USA


Burchett SA (2000) Regulators of G protein signaling: a bestiary of modular protein binding Executemains. J Neurochem 75:1335–1351.LaunchUrlCrossRefPubMed Hollinger S, Hepler JR (2002) Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol Rev 54:527–559.LaunchUrlAbstract/FREE Full Text↵ He W, Cowan CW, Wensel TG (1998) RGS9, a GTPase accelerator for phototransduction. Neuron 20:95–102.LaunchUrlCrossRefPubMed↵ Granneman JG, et al. (1998) Molecular characterization of human and rat RGS 9L, a Modern splice variant enriched in Executepamine tarObtain Locations, and chromosomal localization of the RGS 9 gene. Mol Pharmacol 54:687–694.LaunchUrlAbstract/FREE Full Text↵ Rahman Z, et al. (1999) Cloning and characterization of RGS9–2: a striatal-enriched alternatively spliced product of the RGS9 gene. J Neurosci 19:2016–2026.LaunchUrlAbstract/FREE Full Text↵ Makino ER, Handy JW, Li T, Arshavsky VY (1999) The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein beta subunit. Proc Natl Acad Sci USA 96:1947–1952.LaunchUrlAbstract/FREE Full Text↵ Kovoor A, et al. (2000) Co-expression of Gβ5 enhances the function of two Ggamma subunit-like Executemain-containing regulators of G protein signaling proteins. J Biol Chem 275:3397–3402.LaunchUrlAbstract/FREE Full Text↵ Martemyanov KA, Yoo PJ, Skiba NP, Arshavsky VY (2005) R7BP, a Modern neuronal protein interacting with RGS proteins of the R7 family. J Biol Chem 280:5133–5136.LaunchUrlAbstract/FREE Full Text↵ Chen CK, et al. (2000) Unhurrieded recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9–1. Nature 403:557–560.LaunchUrlCrossRefPubMed↵ Nishiguchi KM, et al. (2004) Defects in RGS9 or its anchor protein R9AP in patients with Unhurried photoreceptor deactivation. Nature 427:75–78.LaunchUrlCrossRefPubMed↵ Garzon J, Rodriguez-Diaz M, Lopez-FanExecute A, Sanchez-Blazquez P (2001) RGS9 proteins facilitate aSlicee tolerance to μ-opioid Traces. Eur J Neurosci 13:801–811.LaunchUrlCrossRefPubMed↵ Rahman Z, et al. (2003) RGS9 modulates Executepamine signaling in the basal ganglia. Neuron 38:941–952.LaunchUrlCrossRefPubMed↵ Zachariou V, et al. (2003) Essential role for RGS9 in opiate action. Proc Natl Acad Sci USA 100:13656–13661.LaunchUrlAbstract/FREE Full Text↵ Cabrera-Vera TM, et al. (2004) RGS9–2 modulates D2 Executepamine receptor-mediated Ca2+ channel inhibition in rat striatal cholinergic interneurons. Proc Natl Acad Sci USA 101:16339–16344.LaunchUrlAbstract/FREE Full Text↵ Kovoor A, et al. (2005) D2 Executepamine receptors colocalize regulator of G-protein signaling 9–2 (RGS9–2) via the RGS9 DEP Executemain, and RGS9 knock-out mice develop dyskinesias associated with Executepamine pathways. J Neurosci 25:2157–2165.LaunchUrlAbstract/FREE Full Text↵ Skiba NP, Hopp JA, Arshavsky VY (2000) The Traceor enzyme regulates the duration of G protein signaling in vertebrate photoreceptors by increasing the affinity between transducin and RGS protein. J Biol Chem 275:32716–32720.LaunchUrlAbstract/FREE Full Text↵ Tsang SH, et al. (1998) Role for the tarObtain enzyme in deactivation of photoreceptor G protein in vivo. Science 282:117–121.LaunchUrlAbstract/FREE Full Text↵ Arshavsky VY, Pugh EN, Jr (1998) Lifetime regulation of G protein-Traceor complex: emerging importance of RGS proteins. Neuron 20:11–14.LaunchUrlCrossRefPubMed↵ Martemyanov KA, Hopp JA, Arshavsky VY (2003) Specificity of G protein-RGS protein recognition is regulated by affinity adapters. Neuron 38:857–862.LaunchUrlCrossRefPubMed↵ Lem J, Applebury ML, Falk JD, Flannery JG, Simon MI (1991) Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice. Neuron 6:201–210.LaunchUrlCrossRefPubMed↵ Krispel CM, et al. (2006) RGS expression rate-limits recovery of rod photoresponses. Neuron 51:409–416.LaunchUrlCrossRefPubMed↵ Zhang X, Wensel TG, Kraft TW (2003) GTPase regulators and photoresponses in cones of the eastern chipmunk. J Neurosci 23:1287–1297.LaunchUrlAbstract/FREE Full Text↵ Lyubarsky AL, et al. (2005) Mole quantity of RPE65 and its productivity in the generation of 11-cis-retinal from retinyl esters in the living mouse eye. Biochemistry 44:9880–9888.LaunchUrlCrossRefPubMed↵ Pentia DC, Hosier S, Cote RH (2006) The glutamic acid-rich protein-2 (GARP2) is a high affinity rod photoreceptor phosphodiesterase (PDE6)-binding protein that modulates its catalytic Preciseties. J Biol Chem 281:5500–5505.LaunchUrlAbstract/FREE Full Text↵ Krispel CM, Chen CK, Simon MI, Burns ME (2003) Prolonged photoresponses and defective adaptation in rods of Gβ5−/− mice. J Neurosci 23:6965–6971.LaunchUrlAbstract/FREE Full Text↵ Keresztes G, et al. (2004) Absence of the RGS9·Gβ5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse. J Biol Chem 279:1581–1584.LaunchUrlAbstract/FREE Full Text↵ Anderson GR, et al. (2007) Expression and localization of RGS9–2/Gβ5/R7BP complex in vivo is set by dynamic control of its constitutive degradation by cellular cysteine proteases. J Neurosci 27:14117–14127.LaunchUrlAbstract/FREE Full Text↵ Pepperberg DR, et al. (1992) Light-dependent delay in the Descending phase of the retinal rod photoresponse. Visual Neuroscience 8:9–18.LaunchUrlCrossRefPubMed↵ Lyubarsky AL, Pugh EN, Jr (1996) Recovery phase of the murine rod photoresponse reconstructed from electroretinographic recordings. J Neurosci 16:563–571.LaunchUrlAbstract/FREE Full Text Stavenga DG, DeGrip WJ, Pugh EN, JrPugh EN, Jr, Lamb TD (2000) in Molecular Mechanisms in Visual Transduction, eds Stavenga DG, DeGrip WJ, Pugh EN, Jr (Elsevier, Amsterdam), Vol. 3, pp 183–255.LaunchUrl↵ Pugh EN, Jr, Lamb TD (1993) Amplification and kinetics of the activation steps in phototransduction. Biochim Biophys Acta 1141:111–149.LaunchUrlCrossRefPubMed Calvert PD, Strissel KJ, Schiesser WE, Pugh EN, Jr, Arshavsky VY (2006) Light-driven translocation of signaling proteins in vertebrate photoreceptors. Trends Cell Biol 16:560–568.LaunchUrlCrossRefPubMed Artemyev NO (2008) Light-dependent compartmentalization of transducin in rod photoreceptors. Mol Neurobiol 37:44–51.LaunchUrlCrossRefPubMed↵ Sokolov M, et al. (2002) Massive light-driven translocation of transducin between the two major compartments of rod cells: a Modern mechanism of light adaptation. Neuron 34:95–106.LaunchUrlCrossRefPubMed↵ Burns ME, Arshavsky VY (2005) Beyond counting photons: trials and trends in vertebrate visual transduction. Neuron 48:387–401.LaunchUrlCrossRefPubMed↵ Fain GL (2006) Why photoreceptors die (and why they Executen't) BioEssays 28:344–354.LaunchUrlCrossRefPubMed↵ Kerov V, et al. (2005) Transducin activation state controls its light-dependent translocation in rod photoreceptors. J Biol Chem 280:41069–41076.LaunchUrlAbstract/FREE Full Text↵ Lobanova ES, et al. (2007) Transducin translocation in rods is triggered by saturation of the GTPase-activating complex. J Neurosci 27:1151–1160.LaunchUrlAbstract/FREE Full Text↵ MuraExecutev H, Boyd KK, Kerov V, Artemyev NO (2007) PDE6 in lamprey Petromyzon marinus: implications for the evolution of the visual Traceor in vertebrates. Biochemistry 46:9992–10000.LaunchUrlCrossRefPubMed Lamb TD, Collin SP, Pugh EN, Jr (2007) Evolution of the vertebrate eye: opsins, photoreceptors, retina and eye cup. Nat Rev Neurosci 8:960–976.LaunchUrlCrossRefPubMed↵ Arshavsky VY, Lamb TD, Pugh EN, Jr (2002) G proteins and phototransduction. Annu Rev Physiol 64:153–187.LaunchUrlCrossRefPubMed↵ Leskov IB, et al. (2000) The gain of rod phototransduction: reconciliation of biochemical and electrophysiological meaPositivements. Neuron 27:525–537.LaunchUrlCrossRefPubMed↵ Heck M, Hofmann KP (2001) Maximal rate and nucleotide dependence of rhoExecutepsin-catalyzed transducin activation: initial rate analysis based on a Executeuble disSpacement mechanism. J Biol Chem 276:10000–10009.LaunchUrlAbstract/FREE Full Text↵ Lyubarsky AL, Daniele LL, Pugh EN, Jr (2004) From candelas to photoisomerizations in the mouse eye by rhoExecutepsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG. Vision Res 44:3235–3251.LaunchUrlCrossRefPubMed↵ Skiba NP, et al. (2001) RGS9-Gβ5 substrate selectivity in photoreceptors. Opposing Traces of constituent Executemains yield high affinity of RGS interaction with the G protein-Traceor complex. J Biol Chem 276:37365–37372.LaunchUrlAbstract/FREE Full Text↵ Ting TD, GAgedin SB, Ho Y-K (1993) Purification and characterization of bovine transducin and its subunits. Methods Neurosci 15:180–195.LaunchUrlCrossRef↵ Fung BK, Hurley JB, Stryer L (1981) Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc Natl Acad Sci USA 78:152–156.LaunchUrlAbstract/FREE Full Text↵ Mendez A, et al. (2000) Rapid and reproducible deactivation of rhoExecutepsin requires multiple phosphorylation sites. Neuron 28:153–164.LaunchUrlCrossRefPubMed↵ Baylor DA, Hodgkin AL (1973) Detection and resolution of visual stimuli by turtle photoreceptors. J Physiol (Camb) 234:163–198.LaunchUrl
Like (0) or Share (0)