Transducin β-Subunit Can Interact with Multiple G-Protein γ-Subunits to Enable Light Detection by Rod Photoreceptors

Visual Overview


Introduction
The heterotrimeric G-protein transducin mediates visual signal transduction in the outer segments of vertebrate photoreceptor cells. The visual signal, or photoresponse, is initiated when photoexcited rhodopsin activates the transducin heterotrimer by catalyzing GDPϪGTP exchange on its ␣-subunit (G␣ t ). G␣ t subsequently dissociates from the ␤␥-subunit complex (consisting of G␤ 1 and G␥ 1 ) to stimulate the downstream effector type 6 cGMP phosphodiesterase. The resulting reduction in cytosolic cGMP causes cGMP-gated ion channel closure, photoreceptor hyperpolarization, and decreased glutamate release from the synaptic terminal (for review, see Arshavsky and Burns, 2012). This signal is processed further by downstream retinal neurons before being transmitted to the brain.
Previous studies have demonstrated that knocking out individual transducin subunits produces quite different phenotypes. For example, the rods of G␣ t knock-out mice are completely insensitive to light (Calvert et al., 2000), while G␥ 1 knock-out rods retain distinct light sensitivity despite the absence of this critical component of the transducin heterotrimer (Lobanova et al., 2008;Kolesnikov et al., 2011). Next, the absence of G␣ t does not affect the expression levels of its cognate G␤ 1 ␥ 1 complex (Calvert et al., 2000;Lobanova et al., 2008), whereas the expression levels of both G␣ t and G␤ 1 are drastically reduced in the G␥ 1 knockout (Lobanova et al., 2008;Kolesnikov et al., 2011). Finally, the knockout of G␣ t does not affect photoreceptor viability (Calvert et al., 2000), whereas G␥ 1 knock-out mice undergo progressive photoreceptor degeneration (Lobanova et al., 2008;Kolesnikov et al., 2011). The latter originates from proteostatic stress arising from the requirement to degrade vast amounts of G␤ 1 , unable to fold without its G␥ 1 partner (Lobanova et al., 2013).
In a previous attempt to understand the nature of light responses in G␥ 1 Ϫ/Ϫ rods, the reduction in transducin subunits in their outer segments was correlated with the reduction in their light sensitivity (Lobanova et al., 2008). G␥ 1 Ϫ/Ϫ outer segments were shown to contain ϳ50-fold less G␣ t and G␤ 1 than WT rods, a reduction very close to the ϳ67-fold loss in their light sensitivity also reported in that study. Because photoreceptor light sensitivity is directly proportional to the rate of transducin activation, which in turn is proportional to its concentration on outer segment discs (Pugh and Lamb, 1993;Heck and Hofmann, 2001;Sokolov et al., 2002;Arshavsky and Burns, 2014), it was suggested that G␥ 1 Ϫ/Ϫ light responses are conveyed by transducin heterotrimer using an alternate G-protein ␥-subunit to enable its efficient activation by rhodopsin. However, attempts to identify this replacement G␥ were unsuccessful due to the low transducin content in these retinas.
More recently, an alternative G␥ 1 Ϫ/Ϫ mouse with a transgenic design different from that used by Lobanova et al. (2008) was characterized by Kolesnikov et al. (2011). Notably, rod photoresponses recorded from these mice had characteristics very similar to those described by the first study, including a 90-fold reduction in light sensitivity from the WT level. However, the outer segment content of G␣ t was estimated to be only sixfold lower than normal. Based on previously published evidence that rhodopsin can activate G␣ t in vitro without G␤ 1 ␥ 1 , although significantly slower than normally (Fung, 1983;Kisselev et al., 1999;Herrmann et al., 2006), the authors argued that G␥ 1 Ϫ/Ϫ rod photoresponses are conveyed by G␣ t acting alone. They also described a slower progression of photoreceptor degeneration in G␥ 1 Ϫ/Ϫ retinas than observed by Lobanova et al. (2008), which was attributed to differences in the genetic backgrounds of these strains.
To settle these discrepancies and elucidate the mechanism driving visual signaling in G␥ 1 Ϫ/Ϫ rods, we performed a side-by-side analysis of both G␥ 1 Ϫ/Ϫ mouse strains. First, we compared the amounts of G␣ t and G␤ 1 in retinal lysates of these strains and found that the decrease in expression levels of both subunits was identical and corresponded to the decrease initially reported by Lobanova et al. (2008). Most importantly, this decrease was comparable to the decrease in rod photoresponse sensitivity reported for both strains. We next demonstrated that the remaining G␤ 1 is present in a complex with endogenous G␥ 2 and G␥ 3 subunits. These results indicate that G␤ 1 ␥ 1 is not the sole G␤␥ complex able to facilitate phototransduction and that G␤ 1 associated with alternative, noncanonical G␥ subunits supports transducin activation in G␥ 1 Ϫ/Ϫ rods at an efficiency comparable to that of the canonical G␤ 1 ␥ 1 complex.
Given the significant difference reported in the progression of photoreceptor degeneration for the two G␥ 1 The authors declare no competing financial interests. strains, we also performed a systematic analysis of retinal degeneration in these animals. We have found that photoreceptors of these mice degenerate at nearly the same rate, and both strains display phenotypes consistent with abnormal proteostasis in their rods.

Animals
Mouse care and experiments were performed in accordance with procedures approved by the Institutional Animal Care and Use Committee of Duke University. The Deltagen G␥ 1 knockout was licensed from Deltagen Inc. (San Mateo, CA) (Target ID 408) and was previously characterized in the study by Lobanova et al. (2008Lobanova et al. ( , 2013. In this mouse, regions of the G␥ 1 coding sequence (amino acids 17-44 and intron 2) were replaced with a 6.9 kb IRES-lacZ reporter and neomycin resistance cassette. The StL G␥ 1 knockout, previously characterized in the study by Kolesnikov et al. (2011), was provided by Dr. O.G. Kisselev (Saint Louis University, St. Louis, MO). In this mouse, the targeting construct replaced all three exons with a Neo cassette to eliminate the coding region of G␥ 1 . Transgenic mice heterozygously expressing the Ub G76V -GFP reporter are described in the study by Lindsten et al. (2003). WT mice used in this study were C57BL/6J from The Jackson Laboratory. None of the mouse lines contained the Rd8 mutation. Mice of either sex were used for all experiments.

Antibodies
Rabbit anti-G␣ t (sc-389), anti-G␤ 1 (sc-379), anti-G␥ 2 (sc-374), and anti-G␥ 3 (sc-375) antibodies, and mouse anti-G␥ 2 (sc-134344) and anti-␤-actin (sc-47778) antibodies were from Santa Cruz Biotechnology. Rabbit anti-PSMD1 (ab140682) antibody was from Abcam. Rabbit anti-G␤ 1 (GTX114442) was from GeneTex. The specificity of the anti-G␣ t antibody in the context of retinal tissue was directly tested in G␣ t knock-out animals. The specificity of other antibodies was assumed per manufacturer descriptions. Secondary goat or donkey antibodies for Western blotting conjugated with Alexa Fluor 680 and 800 were from Invitrogen. Protein bands were visualized and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Western blotting
For quantitative Western blot analysis of G␣ t and G␤ 1 protein levels, two mouse retinas per sample were solubilized in 150 l of 1% Triton X-100 in PBS. Total protein concentration was measured using the DC Protein Assay kit (Bio-Rad), and samples were diluted with SDS-PAGE sample buffer to achieve a protein concentration of 1 g/l. Aliquots from G␥ 1 knock-out mice containing 10 g of total protein were separated by SDS-PAGE along with a serial dilution of WT retinal lysate.

Histology and microscopy
Agarose-embedded retinal cross sections were prepared as previously described (Lobanova et al., 2010), collected in 24-well plates, and incubated for 2 h with Alexa Fluor 594 conjugate of wheat germ agglutinin (In-vitrogen) in PBS containing 0.1% Triton X-100. Sections were washed three times in PBS, mounted with Fluoromount G (Electron Microscopy Sciences) under glass coverslips, and visualized using a Nikon Eclipse 90i Confocal Microscope.
Plastic-embedded retinal cross sections (1 m thick) were prepared as previously described (Sokolov et al., 2004) and stained with toluidine blue for light microscopy. Tiled images of whole retina cross sections were obtained using the Olympus IX-81 Inverted Fluorescence Microscope, and aligned and stitched using the Olympus cellSens Dimension software. The number of photoreceptor nuclei in representative segments of outer nuclear layer (ONL) was quantified as a quantitative measure of surviving photoreceptors. The number of nuclei in a 400 m segment of the ONL, located at 1 mm from each side of the optic nerve, was counted by hand.

Rod outer segment isolation
Rod outer segments were isolated from WT and G␥ 1 Ϫ/Ϫ retinas as previously described (Tsang et al., 1998), with minor modifications. Briefly, retinas from 6 -10 animals were removed from the eyecups and placed in 150 l of 8% OptiPrep Density Gradient Medium (Sigma-Aldrich) in mouse Ringer's buffer (130 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl 2 , 1.2 mM CaCl 2 , 10 mM HEPES, and 0.02 mM EDTA, pH 7.4). The tubes were vortexed at maximum speed for 60 s and centrifuged at 200 ϫ g for 60 s, and the supernatant containing rod outer segments was gently collected. Two hundred microliters of fresh 8% OptiPrep solution was added to the retinal pellet, and vortexing/ sedimentation was performed again. This sequence was repeated at least five times. The combined supernatant was loaded on a step gradient made with 10% and 18% OptiPrep in a 4 ml centrifuge tube and centrifuged in a swing-bucket rotor at 115,000 ϫ g for 30 min. Rod outer segments were collected from the interface between 10% and 18% OptiPrep, diluted with 4 ml of Ringer's solution, and centrifuged at 100,000 ϫ g for 1 h. The pellet containing rod outer segments was rinsed once with 200 l of Ringer's solution, resuspended in 200 l of PBS, snap frozen in liquid N 2 , and stored at Ϫ80°C until use.

Immunoprecipitation
Samples were prepared for immunoprecipitation as previously described (Pearring et al., 2015). Briefly, purified rod outer segments were thawed and their protein content was determined using the DC Protein Assay Kit. Samples were diluted to 0.5 g/l protein in the immunoprecipitation buffer (0.1% n-dodecyl-␤-maltoside in PBS with protease inhibitor cocktail, Sigma-Aldrich), vortexed, and centrifuged at 108,000 ϫ g for 30 min. The supernatant was removed for use in immunoprecipitation. Protein A Mag Sepharose beads (catalog #28944006, GE Healthcare Life Sciences) and Protein G Mag Sepharose Xtra beads (catalog #28967066, GE Healthcare Life Sciences) were used for immunoprecipitation with rabbit and mouse antibodies, respectively. In both cases, 5 l beads were incubated with 5 g of antibody for 2 h under rotation at room temperature. The beads were rinsed and incubated with 12.5 g of rod outer segment lysate over-night under rotation at 4°C. After the beads were rinsed, bound proteins were eluted by boiling in the SDS-PAGE loading buffer (2% SDS) at 95°C for 10 min and analyzed by Western blotting.

Mass spectrometry
Peptide mixes obtained from in-gel tryptic digests were analyzed using a nanoACQUITY UPLC System coupled to a Synapt G2 Mass Spectrometer (Waters). Peptides were separated on a 75 m ϫ 150 mm column with 1.7 m C18 BEH (Ethylene Bridged Hybrid) particles (Waters) using a 90 min gradient of 6 -32% acetonitrile with 0.1% formic acid at a flow rate of 0.3 l/min at 35°C. For each sample, we conducted data-dependent analysis (DDA) using a 0.8 s mass spectrometry (MS) scan followed by tandem MS (MS/MS) acquisition on the top three ions. MS/MS scans for each ion used an isolation window of ϳ3 Da and a dynamic exclusion window of 90 s within 1.2 Da. DDA data were converted to searchable files using ProteinLynx Global Server 2.5.1 (Waters) and searched against the Uniprot mouse database using Mascot Server version 2.5 with the following parameters: maximum one missed cleavage site, carbamidomethylation at Cys residues as fixed modification and Met oxidation, Asn, Gln deamidation, and protein N-acetylation as variable modifications. Precursor ion mass tolerance was set to 20 ppm, and fragment mass tolerance was set to 0.25 Da.

Statistical analysis
For quantification of rod outer segment content of G␣ t and G␤ 1 subunits and comparative analysis of retinal morphology, data are presented as the mean Ϯ SD. All statistical analyses were performed with GraphPad Prism 7.04. Morphologic data were analyzed using the nonparametric Mann-Whitney U test, and results were considered statistically significant at p Ͻ 0.05. For the MS identification of G-protein ␥-subunits present in rod outer segments, Mascot data were imported into Scaffold 4.8 (Proteome Software) to merge all of the data for a sample represented by multiple gel bands, to estimate a confidence score for protein identification, and to perform a relative protein quantification based on the sum of intensities of the constituent peptides.

Results
Hereafter, we will refer to the two G␥ 1 Ϫ/Ϫ mouse strains analyzed in this study as "Deltagen" and "StL" G␥ 1 Ϫ/Ϫ mice, as the mouse described in the study by Lobanova et al. (2008) was produced by and licensed from Deltagen and the mouse characterized in the study by Kolesnikov et al. (2011) was produced at Saint Louis University.

Identification of transducin ␣and ␤-subunit levels in G␥ 1
؊/؊ retinal lysates We used quantitative Western blotting to directly compare the levels of G␣ t and G␤ 1 in the two G␥ 1 knock-out models. Retinas were harvested at 1 month of age and lysed, and proteins from the lysates were separated by SDS-PAGE alongside serial dilutions of retinal lysates from WT mice (Fig. 1A). G␣ t and G␤ 1 were then detected by immunoblotting with specific antibodies against each protein. The standard curves relating band intensity to total protein amount were obtained from WT lysates and used to calculate relative contents of G␣ t and G␤ 1 in each model (Fig. 1B).
This analysis revealed an identical reduction in the contents of transducin subunits in the retinas of both mouse strains (Fig. 1C). G␣ t was present at 3.3% and 3.0% of the WT amount in Deltagen and StL retinas, respectively, whereas G␤ 1 was present at 6.9% and 7.1% of the WT amount. a These numbers are very close to those previously reported for the Deltagen mouse (3.9% remaining G␣ t and 10.6% G␤ 1 ; Lobanova et al., 2008), a conclusion reinforced by the fact that the standard curves in the study by Lobanova et al. (2008) were generated based on an alternative methodology using purified protein standards rather than serial dilutions of WT retinal extracts. It is worth noting that the difference between the conclusions of the two studies could not be attributed to different states of light-dependent transducin translocation, a phenomenon taking place in WT rods (Sokolov et al., 2002), because transducin does not translocate in G␥ 1 Ϫ/Ϫ rods (Lobanova et al., 2008). These data clearly demonstrate that the amount of transducin remaining in retinas of the StL mouse was overestimated and that this aspect of the G␥ 1 Ϫ/Ϫ phenotype is strain independent. We next calculated how much G␣ t and G␤ 1 is present in rod outer segments of G␥ 1 Ϫ/Ϫ mice, taking into account that approximately one-half of each subunit is mislocalized from G␥ 1 Ϫ/Ϫ rod outer segments and a large (ϳ82%) fraction of G␤ 1 in this mouse is expressed in the inner retina (Lobanova et al., 2008; Table 1). Therefore, rod outer segments of Deltagen G␥ 1 Ϫ/Ϫ mice contain between ϳ1.5% (based on the lower value of 3.0% measured in Statistical Table   Line Figure/table  Data  The range for the G␤ 1 amount in G␥ 1 Ϫ/Ϫ outer segments is between 0.6% and 1% of its WT content (based on the total retinal amounts of 7% and 10.6%, respectively, obtained in the current and previous study). The corresponding amounts for the StL mice derived from the values reported in Fig. 1 are 1.5% and 0.7% of WT, respectively, for G␣ t and G␤ 1 ( Table 1).

Consideration of transducin activation mechanism in G␥ 1
؊/؊ rods Precise determination of the degree of transducin subunit loss in the outer segments of G␥ 1 Ϫ/Ϫ rods allows critical evaluation of the two hypotheses explaining light signaling in these cells: G␣ t activated alone versus G␣ t activation assisted by an alternative G␤␥. As mentioned above, the reduction in photoresponse sensitivity documented for these cells was consistent between both studies (ϳ67-fold in Lobanova et al., 2008; ϳ90-fold in Kolesnikov et al., 2011). This represents 1.1-1.5% of WT Ϫ/Ϫ mice. A, Retinal lysates from Deltagen and StL mouse strains containing 10 g of total protein were separated by SDS-PAGE alongside serial dilutions of WT retinal lysates and were immunoblotted with antibodies against each transducin subunit. Data are taken from one of three similar experiments. B, The relative contents of G␣ t and G␤ 1 in retinal lysates analyzed in A were determined from the calibration curves obtained from the WT lysate dilutions (G␣ t regression equation: y ϭ 18,600 ϫ x Ϫ 623; G␤ 1 regression equation: y ϭ 8130 ϫ x ϩ 665).
• ϭ WT, OE ϭ Deltagen G␥ 1 Ϫ/Ϫ , and छ ϭ StL G␥ 1 Ϫ/Ϫ . C, The amounts of transducin subunits in G␥ 1 Ϫ/Ϫ retinas averaged across three independent experiments are expressed as percentages of their amounts in WT retinas (mean Ϯ SD). The amount of G␤ 1 was corrected to reflect that only 18% of G␤ 1 in G␥ 1 Ϫ/Ϫ retinas is expressed in rods, whereas the rest is expressed in the inner retina (Lobanova et al., 2008). The mean values for protein content in retinas were taken from Fig. 1 and the study by Lobanova et al. (2008) and were corrected for the outer segment fraction of each protein, as described in the text. Relative light sensitivities were derived from the single-cell recordings in the studies by Lobanova et al. (2008) and Kolesnikov et al. (2011) by dividing the value of half-saturating light intensity of WT rods by the corresponding value for G␥ 1 Ϫ/Ϫ rods. sensitivity and is very close to the degree of G␣ t and G␤ 1 reduction determined in the previous section (Table 1). Because photoreceptor light sensitivity is directly proportional to the concentration of transducin heterotrimer on the membranes of outer segment discs (Pugh and Lamb, 1993;Heck and Hofmann, 2001;Sokolov et al., 2002;Arshavsky and Burns, 2014), the comparable reduction in G␣ t and photoresponse sensitivity indicates that the G␣ t remaining in G␥ 1 Ϫ/Ϫ rod outer segments is activated by rhodopsin at nearly the same efficiency as in WT outer segments. On the other hand, the efficiency of G␣ t activation by rhodopsin without G␤␥ is at least an order of magnitude lower than with G␤␥ (Fung, 1983;Kisselev et al., 1999;Herrmann et al., 2006). This argues that G␣ t activation in G␥ 1 Ϫ/Ϫ rods is supported by a complex between G␤ 1 and a G-protein ␥-subunit replacing G␥ 1 in these rods. We therefore set up a search for this alternative G␤ 1 ␥ complex in G␥ 1 Ϫ/Ϫ rods.

and WT rod outer segments
To elucidate the molecular composition of the putative G␤ 1 ␥ complexes supporting visual function in the absence of G␥ 1 , we first conducted MS identification of all G-protein ␥-subunits present in rod outer segments of WT and G␥ 1 Ϫ/Ϫ mice. Outer segments were purified from each mouse type, and their proteins were separated by SDS-PAGE. Gel fragments containing G-protein ␥-subunits were excised, and proteins were subjected to in-gel tryptic digestion followed by liquid chromatography-MS/MS analysis of the resulting peptides. Of the 14 G-protein ␥-subunits encoded in the mouse genome (Milligan and Kostenis, 2006), only 3 were found in WT and 2 were found in G␥ 1 Ϫ/Ϫ outer segments ( Table 2). As expected, WT but not G␥ 1 Ϫ/Ϫ outer segments contained G␥ 1 . The other two subunits, G␥ 2 and G␥ 3 , were found in both preparations, although the confidence score for G␥ 3 in the WT preparation was low. b A rough estimate, based on comparing the total ion intensity produced by all peptides representing each G␥ subunit type, suggested that for every 82 molecules of G␥ 1 present in WT outer segments there are 3 molecules of G␥ 2 and 1 molecule of G␥ 3 . A similar estimate performed for G␥ 1 Ϫ/Ϫ rod outer segments suggested a molar ratio of ϳ1.7 between G␥ 2 and G␥ 3 . These results narrowed the list of potential G␤ 1 binding partners in G␥ 1 Ϫ/Ϫ outer segments to these two ␥-subunits.
Therefore, we sought to directly demonstrate that G␤ 1 forms complexes with G␥ 2 and/or G␥ 3 in G␥ 1 Ϫ/Ϫ rod outer segments by coimmunoprecipitating these putative complexes using antibodies against G␥ 2 and G␥ 3 . We have found that a significant fraction of G␤ 1 in G␥ 1 Ϫ/Ϫ rod outer segments was precipitated with antibodies specifically recognizing G␥ 2 or G␥ 3 ( Fig. 2A,B). These results confirm that G␤ 1 remaining in the rod outer segments of G␥ 1 Ϫ/Ϫ mice indeed forms complexes with both of these ␥-subunits. We also attempted to conduct a reciprocal coprecipitation experiment, but unfortunately were not able to identify a precipitating antibody against G␤ 1 .
We next sought to determine whether these alternative G␤␥ complexes only form in the absence of G␥ 1 or whether they are also present in WT rods. To test this, we repeated coimmunoprecipitation experiments using rod outer segments from WT mice (Fig. 2C,D). This analysis  revealed that, even in the presence of abundant amounts of its cognate partner G␥ 1 , a small portion of G␤ 1 binds G␥ 2 or G␥ 3 . This result suggests that G␤ 1 normally forms complexes with these two alternative ␥-subunits in addition to its canonical G␥ 1 binding partner, although these complexes are not likely to be functionally significant due to their low abundance.
Together, our experiments demonstrate that G␥ 2 and G␥ 3 are naturally present in complexes with G␤ 1 in the WT rod outer segment. When G␥ 1 is knocked out, these alternative G␤ 1 ␥ 2 and G␤ 1 ␥ 3 complexes can support light signaling in mutant photoreceptors.

Evaluation of photoreceptor degeneration in two strains of G␥ 1
؊/؊ mice Another discrepancy reported between the Deltagen and StL G␥ 1 Ϫ/Ϫ mouse strains was that photoreceptor degeneration in the former progressed significantly faster than in the latter. We therefore performed a quantitative side-by-side comparison of the rate of retinal degeneration in these animals. This was accomplished by counting the number of photoreceptor nuclei in representative segments of the ONL at 1, 3, and 6 months of age. Although this method is more labor intensive than commonly used alternatives (e.g., measuring the ONL thickness or count-ing the number of nuclei per ONL stack), it provides the most reliable information on the actual number of photoreceptors remaining "alive" in a degenerating retina. This is because nuclear stacks in degenerating retinas could become distorted and hard to quantify, whereas the ONL thickness may change nonproportionally to the actual cell loss.
Retinas from both G␥ 1 Ϫ/Ϫ mouse lines were embedded in plastic, cross-sectioned, and stained with toluidine blue (Fig. 3A). Photoreceptor nuclei were then counted in two 400 m segments per eye, one on each side of the optic nerve, and the values were averaged across at least three mice of each age (Fig. 3B). These data indicated that the rates of photoreceptor degeneration in both knock-out strains were nearly identical at all tested ages. Formal analysis suggested that a statistically significant difference in nuclear counts existed in 3-month-old mice (p ϭ 0.065 c at 1 month; p ϭ 0.041 d at 3 months; p ϭ 0.24 e at 6 months). However, the absolute difference of Ͻ10% is hard to consider physiologically significant. This analysis showed that approximately half of the photoreceptor cells were lost by the age of 6 months, a rate of degeneration that falls somewhere in the middle between the two previous reports. One potential source of this discrepancy is that the first study (Lobanova et al., 2008) did not assess Ϫ/Ϫ mice. A, Representative images of toluidine blue-stained, plastic-embedded 1-m-thick retinal cross sections from each mouse strain at the indicated ages. Scale bar, 25 m. B, The number of photoreceptor nuclei counted in 400 m outer nuclear layer (ONL) segments located 1 mm from either side of the optic nerve. The number of nuclei in each segment was counted by hand. Data were averaged across nuclear counts obtained from each side of the optic nerve from three or four animals of each age and genotype, and are shown as the mean Ϯ SD. Each dot represents a single data point. C, Accumulation of the Ub G76V -GFP reporter of proteasomal activity (green) in rods of 3-month-old Deltagen and StL G␥ 1 Ϫ/Ϫ mice. A Ub G76V -GFP-expressing WT retina is shown as a control. Wheat germ agglutinin staining is shown in red. Scale bar, 10 m.
New Research this parameter quantitatively and apparently provided more dramatic examples of cellular loss than average. On the other hand, the rate of photoreceptor degeneration in the study by Kolesnikov et al. (2011) may have been underestimated due to the use of the nuclear stack counting methodology, which may be more arbitrary than the total nuclear count.

Photoreceptors from both strains of G␥ 1
؊/؊ mice experience proteostatic stress Using the Deltagen strain, Lobanova et al. (2013) showed that photoreceptor cell death in G␥ 1 Ϫ/Ϫ mice is associated with proteostatic stress arising from the necessity to degrade large amounts of G␤ 1 that is unable to fold without its constitutive G␥ 1 partner. We now demonstrate that the same is true for the StL strain. StL mice were crossed with the mouse ubiquitously expressing the Ub G76V -GFP proteasome activity reporter, which consists of GFP fused to an uncleavable ubiquitin (Lindsten et al., 2003). This reporter undergoes efficient polyubiquitination and proteasomal degradation in healthy cells, including WT photoreceptors, but accumulates in cells suffering from impairment or insufficiency of the ubiquitin-proteasome system. Consistent with the phenotype of the Deltagen strain, photoreceptors of StL mice also displayed robust intracellular accumulation of Ub G76V -GFP, as documented by detecting GFP fluorescence in retinal cross sections (Fig. 3C). This result indicates that both mouse strains suffer from abnormal proteostasis, contributing to their photoreceptor degeneration.

Discussion
The data presented in this study demonstrate that the two lines of G␥ 1 knock-out mice currently available to the scientific community display essentially identical phenotypes. Not only do they produce similar responses to light, as reported earlier, but they also contain identical amounts of G␣ t and G␤ 1 subunits in their rods, undergo retinal degeneration at a similar rate, and share the same underlying pathobiological mechanism. Therefore, these strains could be used interchangeably in future studies of this mouse model.
Most importantly, resolving the discrepancy between previous estimates of the contents of transducin subunits in these strains allowed us to explain the mechanism by which photoreceptors respond to light in the absence of the transducin canonical ␤␥-subunit G␤ 1 ␥ 1 . Here we demonstrate that relatively small amounts of G␥ 2 and G␥ 3 are endogenously expressed in the outer segments of these cells where they produce complexes with G␤ 1 . These complexes are able to support vision in the absence of the G␤ 1 cognate ␥-subunit G␥ 1 .
Our findings highlight the versatility of G-protein signaling by showing the exchangeability of individual G-protein subunits in performing an important physiologic function. Mouse and human genomes contain genes encoding 5 G-protein ␤-subunits and 14 ␥-subunits (Milligan and Kostenis, 2006), and their expression patterns in different tissues vary widely (Largent et al., 1988;Gautam et al., 1990;Cali et al., 1992;Liang et al., 1998;Morishita et al., 1998). Given that many possible G␤␥ combinations exist, a key question in G-protein signaling is whether heterotrimers composed of distinct subunits can fulfill the same physiologic role. Many examples documented in cell culture and in vitro show that G␤␥ complexes using different ␥-subunits are able to activate the same signaling cascades (Wickman et al., 1994;Ikeda, 1996;Ruiz-Velaso and Ikeda, 2000). This includes at least three cell culture studies specifically demonstrating the functional interchangeability of G␤ 1 ␥ 1 , G␤ 1 ␥ 2 , and G␤ 1 ␥ 3 (Hawes et al., 1995;Dingus et al., 2005;Poon et al., 2009).
Examination of ␥-subunit exchangeability in the context of the whole animal has thus far been limited, but results obtained in several mouse knock-out studies have suggested that there may be a more stringent requirement for ␥-subunit specificity in vivo than in cell culture. For example, G␥ 7 is indispensable for adenylyl cyclase signaling through the A 2A receptor in the striatum (Schwindinger et al., 2003(Schwindinger et al., , 2010, whereas G␥ 13 is required for olfactory signal transduction (Li et al., 2013).
We now provide a compelling example of the functional interchangeability of G-protein ␥-subunits in vivo. To our knowledge, G␥ 1 Ϫ/Ϫ represents the first case to directly demonstrate that multiple G␤␥ complexes can perform the same function in a living animal. The only caveat is that the expression levels of alternative ␥-subunits in photoreceptors are lower than those of the conventional G␥ 1 and, therefore, the G␤ 1 ␥ 1 complex drives the majority of phototransduction unless G␥ 1 is absent.