May 2012
Volume 53, Issue 6
Free
Biochemistry and Molecular Biology  |   May 2012
Phosphorylation of Phosducin Accelerates Rod Recovery from Transducin Translocation
Author Affiliations & Notes
  • Marycharmain Belcastro
    From the Departments of Ophthalmology,
  • Hongman Song
    From the Departments of Ophthalmology,
    and Biochemistry, West Virginia University, Morgantown, West Virginia.
  • Satyabrata Sinha
    From the Departments of Ophthalmology,
  • Chunyan Song
    From the Departments of Ophthalmology,
  • Peter H Mathers
    Otolaryngology,
    and Biochemistry, West Virginia University, Morgantown, West Virginia.
  • Maxim Sokolov
    From the Departments of Ophthalmology,
    and Biochemistry, West Virginia University, Morgantown, West Virginia.
  • Footnotes
    4  Current address: National Institute of Deafness and Other Communication Disorders, National Institutes of Health, 31 Center Drive, MSC 2320, Bethesda, MD 20892-2320.
  • Corresponding author: Maxim Sokolov, West Virginia University Eye Institute, 1 Stadium Drive, Morgantown, WV 26506; Telephone 1-304-598-6958; Fax 1-304-598-6928; sokolovm@wvuhealthcare.com
Investigative Ophthalmology & Visual Science May 2012, Vol.53, 3084-3091. doi:10.1167/iovs.11-8798
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      Marycharmain Belcastro, Hongman Song, Satyabrata Sinha, Chunyan Song, Peter H Mathers, Maxim Sokolov; Phosphorylation of Phosducin Accelerates Rod Recovery from Transducin Translocation. Invest. Ophthalmol. Vis. Sci. 2012;53(6):3084-3091. doi: 10.1167/iovs.11-8798.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: In rods saturated by light, the G protein transducin undergoes translocation from the outer segment compartment, which results in the uncoupling of transducin from its innate receptor, rhodopsin. We measured the kinetics of recovery from this adaptive cellular response, while also investigating the role of phosducin, a phosphoprotein binding transducin βγ subunits in its de-phosphorylated state, in regulating this process.

Methods.: Mice were exposed to a moderate rod-saturating light triggering transducin translocation, and then allowed to recover in the dark while free running. The kinetics of the return of the transducin subunits to the outer segments were compared in transgenic mouse models expressing full-length phosducin, and phosducin lacking phosphorylation sites serine 54 and 71, using Western blot analysis of serial tangential sections of the retina.

Results.: In mice expressing normal phosducin, transducin α and βγ subunits returned to the outer segments with a half-time (t1/2) of ∼24 and 29 minutes, respectively. In the phosducin phosphorylation mutants, the transducin α subunit moved four times slower, with t1/2 ∼95 minutes, while the movement of transducin βγ was less affected.

Conclusions.: We demonstrate that the recovery of rod photoreceptors from the ambient saturating levels of illumination, in terms of the return of the light-dispersed transducin subunits to the rod outer segments, occurs six times faster than reported previously. Our data also support the notion that the accumulation of transducin α subunit in the outer segment is driven by its re-binding to the transducin βγ dimer, because this process is accelerated significantly by phosducin phosphorylation.

Introduction
Phosducin (Pdc) is a cytosolic phosphoprotein implicated in the regulation of heterotrimeric G proteins based on its high affinity towards Gβγ subunits. 15 Pdc is highly expressed in the rod and cone photoreceptors of the vertebrate retina. 6,7 Knockout of the Pdc gene causes a reduction in the cellular levels of and a partial mislocalization of the visual G protein, transducin, in rod photoreceptors. 8,9 An in depth analysis of phosducin knockout mice also revealed the role of phosducin in regulating synaptic transmission, 10 hypothesized previously. 11 Some evidence suggests that Pdc also may be expressed in a variety of other tissues, however, at much lower levels. 12,13 The affinity of Pdc towards Gβγ is down-regulated by phosphorylation and consequent binding of the 14-3-3 protein. 11,14 In photoreceptors, the phosphorylation status of Pdc depends on the history of illumination. Two phosphorylation sites of Pdc, serine 54 and 73 (71 in the mouse sequence), are regulated by the concerted interplay of the activities of PKA, CaMKII, and PP2A, resulting in Pdc's dynamic phosphorylation in the dark and dephosphorylation in the light. 1418 The physiological significance of light-dependent Pdc phosphorylation has remained unknown. 
We tested the hypothesis that phosphorylation of phosducin regulates the trafficking of transducin subunits to the rod outer segment. For that, we generated transgenic mice expressing normal Pdc or a Pdc phosphorylation mutant in rod photoreceptors, and analyzed the kinetics of the return of the light-dispersed transducin subunits to the rod outer segments in these models. Our study reveals the physiological rate of the trafficking of transducin subunits to the outer segment, and the role phosducin phosphorylation has in accelerating this process. 
Methods
Generation of Pdc-FLAG and PdcS54A/S71A-FLAG Transgenic Mice
All experiments involving animals were performed according to the procedures approved by the Animal Care and Use Committee of West Virginia University, and in adherence with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. To prepare the wild-type mouse phosducin (Pdc) construct with a 3′-FLAG tag, total RNA was isolated first from the retina of a 129Sv mouse using the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA), and the RNA was reverse transcribed using the mouse Pdc gene-specific RT primer 5′-CCC GAG TTT AAA TAG CC with the AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Stratagene). The polymerase chain reaction, using the Easy-A High-Fidelity PCR Master Mix (Stratagene) and the following primers, was used to amplify the coding sequence of the subsequent Pdc cDNA, and to add a Kozak sequence (GCCACCAUGG) to the 5′ end and a FLAG sequence to the 3′ end of Pdc, as well as two new stop codons: forward primer 5′-GCC ACC ATG GAA GAA GCC GCC AGC CAA AGC and reverse primer 5′-CTA CTA CTT GTC ATC GTC GTC CTT GTA ATC TTC AAT GTC CTC GTC TTC CAT GTT GG. The PCR product originally was cloned into the StrataClone PCR Cloning Vector pSC-A (Stratagene), but ultimately subcloned into the pBAM4.2 vector containing a 4.4 kb mouse rhodopsin promoter and a mouse protamine I polyadenylation sequence (MPI). 19 PCR amplification of the Kozak-WT Pdc-FLAG sequence from its pSC-A template was done using the forward primer 5′-GAG AGT CGA CGC CAC CAT GGA AGA AGC CGC C to add a SalI site to the 5′ end, and using the reverse primer 5′-GCC TGG ATC CCT ACT ACT TGT CAT CGT CGT CC to add a BamHI site to the 3′ end. The subsequent PCR product and the pBAM4.2 vector were digested with SalI and BamHI restriction endonucleases, gel purified, and ligated so the Kozak-WT Pdc-FLAG sequence would be inserted after the mouse rhodopsin promoter and before the mouse protamine I polyA sequence, creating pRhop4.4k-Pdc. An additional serine-to-alanine variant transgene of mouse Pdc at positions 54 and 71 was created from the pRhop4.4k-Pdc vector by using a PCR-based strategy and the QuikChange Site-Directed Mutagenesis Kit (Stratagene) with the following primers in subsequent order: For S54A conversion – forward primer 5′-CCT CAG ACA AAT GGC CTC TCC TCA GAG CAG and reverse primer 5′-CTG CTC TGA GGA GAG GCC ATT TGT CTG AGG. For S71A conversion – forward primer 5′-GAA AGA ATG AGC AGA AAG ATG GCC ATT CAA GAA TAT GAA CTC ATT C and reverse primer 5′-GAA TGA GTT CAT ATT CTT GAA TGG CCA TCT TTC TGC TCA TTC TTT C (with all underlined bases above indicating those changed from the wild-type sequence). The integrity of both constructs was confirmed by sequence analysis, and the final transgenes (4.4 kb rhodopsin promoter-Kozak-WT Pdc-FLAG-MPI polyA or 4.4 kb rhodopsin promoter-Kozak-PdcS54A/S71A-FLAG-MPI polyA) were removed from pRhop4.4k-Pdc and pRhop4.4k- PdcS54A/S71A, respectively, by digestion with restriction endonucleases KpnI and SpeI, gel purified, and submitted to the West Virginia University Transgenic Animal Core Facility, where they were injected into the pronuclei of zygotes from superovulated FVB females. Transgene integration was determined in the offsprings by PCR analyses of tail DNA, and then transgenic expression of Pdc-FLAG or PdcS54A/S71A -FLAG in the retinas was confirmed by Western blotting. Two independent transgenic lines expressing epitope-tagged Pdc of each kind were established by crossing selected founders with Pdc−/− mice. 8,9  
Quantification of Proteins by Western Blotting
Each retina was homogenized in 0.2 mL of urea sample buffer (USB) by short ultrasonic pulses, and the resulting extract was cleared by centrifugation. The total protein concentration was determined on a Nanodrop ND-1000 spectrophotometer, and then 5 mM N-ethylmaleimide (Sigma, St. Louis, MO ) was added. To prepare SDS-PAGE samples, the protein concentration in each of the compared samples was adjusted to 0.5 mg/mL, and bromophenol blue tracking dye and 5% β-mercaptoethanol were added. Then, 5 μL of each retinal extract from Pdc-FLAG, PdcS54A/S71A-FLAG and wild-type (129Sv) mice were separated side-by-side on a 10% Tris-HCl acrylamide gel. Quantification of the specific bands corresponding to transducin α and β subunits, phosducin and β-tubulin, was performed simultaneously in each sample using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) according to the manufacturer's protocols. The fluorescence values of the transducin α, β and phosducin bands in each sample were normalized first to that of the β-tubulin band used as the loading control. Then, normalized bands in the adjacent wild-type, Pdc-FLAG, and PdcS54A/S71A-FLAG samples were compared. Data from two independent measurements using four mice of each genotype each time were averaged.  
Pull Down of Epitope-Tagged Phosducin
Two retinas of each type were homogenized by short ultrasonic pulses delivered from a Microson ultrasonic cell disruptor equipped with a 3 mm probe (Misonix, Farmingdale, NY) in 0.2 mL of buffer containing 10 mM Tris/HCl, pH 7.0, 1.5% Igepal CA-630 (Sigma), and 5 mM N-ethylmaleimide. Cellular insoluble parts were cleared by centrifugation, and each supernatant was incubated with 10 μL of anti-FLAG M2-Agarose beads (Sigma) for 1 hour at room temperature. Beads were washed 4 times for 3 minutes with 1.0 mL of buffer, and bound proteins were eluted with 0.1 mL of USB buffer containing 125 mM Tris/HCl, pH 6.8, 4% SDS, and 6M urea. The samples then were analyzed by Western blotting using antibodies against transducin β and phosducin. 
Light Conditioning of Mice and Analysis of Protein Localization in Rod Photoreceptors
Mice were anesthetized with a 2% isoflurane/98% oxygen mixture, and their pupils were dilated with one drop per eye of a mixture of 1.25% phenylephrine hydrochloride and 0.5% tropicamide ophthalmic solution for 10 minutes. Anesthetized mice were exposed to a pre-set 200 cd/m2 white background light inside the light dome of a UTAS-E4000 Visual Electrodiagnostic Test System (LKC Technologies, Gaithersburg, MD), equivalent to 600 ± 50 photopic lux luminance at the cornea as measured by a traceable light meter (Fisher Scientific, Pittsburgh, PA) for 10 minutes. Following light conditioning, mice were allowed to awaken from gas anesthesia, which usually took a minute, and then they were placed in their original cages to recover in the dark. Mice were sacrificed at different time points of dark recovery, and the protein localization in rod photoreceptors was analyzed using Western blotting of serial tangential sections of the frozen retina, according to the latest modification 20 of a previously described procedure. 21 For indirect immunofluorescence detection of rod transducin, eyes were enucleated first, and fixed for 10 minutes with Oculo Fix (BBC Biochemical, Pittsburgh, PA). The anterior portions of the fixed eyes then were removed, and the eyecups were fixed further with Oculo Fix for 3 hours. Fixed eyecups then were cryoprotected with 30% sucrose in 1X PBS overnight. The eye cups then were transferred to a 1:1 tissue freezing medium (TFM; TBS Triangle Biomedical Sciences, Inc., Durham, NC) −30% sucrose solution for 2 hours, and then embedded in TFM, and frozen. The frozen blocks then were cut radially in 16 μm sections on a Leica CM1850 Cryostat, and the sections stored at −20°C. Before immunostaining, frozen sections were thawed at room temperature for 10 minutes, hydrated in 1× PBS for 5 minutes, and blocked with goat-serum containing 5% Triton-X 100 for 30 minutes. Sections were probed first with primary antibody against transducin α at a 1:1000 dilution overnight, and then probed with Alexa Fluor 488 or 568 conjugated secondary antibody (Invitrogen) for 1 hour. The tissue sections also were counterstained with either propidium iodide (EMD Biosciences/Calbiochem, San Diego, CA) or 100 ng/mL DAPI (Roche, Indianapolis, IN) for rod nuclear staining. Images were acquired on a Zeiss LSM 510 Meta confocal microscope after adjusting the detector gain for all the fluorescent channels, and the amplifier offset for background corrections, and while keeping the amplifier gain of the lasers to a minimum. Identical parameters were used when comparing samples. 
Determination of the Levels of Rhodopsin
A protocol modified from that of Song et al. 18 was used as follows. All procedures were performed under dim red light. Each retina was harvested, and homogenized in 0.2 mL of water containing 2.5% n-octyl β-D-glucopyranoside (BP585-1; Fisher Scientific) and 1.25% cetyltrimethylammonium chloride (292,737, Sigma) by short ultrasonic pulses. The extract was cleared by centrifugation on a Centrifuge 5415 D (Eppendorf, Hauppauge, NY) at 16,000 revolutions per minute for one minute. The absorption 400–700 nm spectrum of supernatant then was obtained in a UV-Mini 1240 spectrophotometer (Shimadzu, Columbia, MD), and assigned as a baseline, after which the sample was bleached for 1 minute with bright white light, and the spectrum was obtained again. Rhodopsin concentration was determined from a 500 nm negative peak. The average level of rhodopsin bleaching was calculated by comparing light-conditioned and dark-adapted mice using retinas from at least 4 mice of the same age and genetic background in each group. 
Antibodies
We used primary antibodies against transducin α (Gtα1) (sc-389; Santa Cruz Biotechnology, Santa Cruz, CA), transducin β (PA1-725; Applied BioReagents; Golden, CO), Gβ (sc-378; Santa Cruz Biotechnolgy), Gγ1 (sc-373; Santa Cruz Biotechnology), β-tubulin (T0198; Sigma Aldrich, St. Louis, MO), visual arrestin (V4389; Sigma), and cytochrome c oxidase subunit I (COX I; MS404, MitoSciences, Eugene, OR). Antibody against rod outer segment membrane protein (rom1) was provided generously by Andrew Goldberg, Oakland University. Pan-specific (Pdc-pan) and phospho-specific (Pdc54p and Pdc71p) antibodies against phosducin were generated in the lab. 18  
Results
Generation of Transgenic Mouse Models Expressing Normal Phosducin or a Phosducin Phosphorylation Mutant in Rods
DNA constructs encoding epitope-tagged full-length mouse phosducin (Pdc-FLAG) and phosducin carrying serine-to-alanine substitutions at residues 54 and 71 (Pdc S54A/S71A-FLAG, Fig. 1A) were expressed in transgenic mice under the control of a 4.4 kb rhodopsin promoter 19 targeting expression of the constructs to rod photoreceptors. Founders expressing similar protein levels of epitope-tagged and endogenous Pdc were backcrossed with Pdc-null mice for several generations. As a result, we established several transgenic lines (Tg-Pdc-FLAG+/−;Pdc−/− and Tg-PdcS54A/S71A-FLAG+/−;Pdc−/− hereafter designated as Pdc-FLAG and PdcS54A/S71A-FLAG, respectively) where the endogenous photoreceptors' Pdc was substituted with transgenic Pdc-FLAG or PdcS54A/S71A-FLAG (Fig. 1B). Pdc-FLAG mice expressed their epitope-tagged Pdc at 46 ± 2% (SEM, n = 4) of the normal Pdc level. Such levels of Pdc expression were deemed normal, since Pdc+/− mice expressing only half the normal Pdc level essentially have a wild-type phenotype (data not shown). The expression level of PdcS54A/S71A-FLAG was 141 ± 5% (SEM, n = 4) compared to that of wild-type Pdc. Western blot analysis using antibodies specific to Pdc phosphorylated at serine 54 and serine 7118 revealed robust phosphorylation of both sites in the retinas of Pdc-FLAG mice incubated with the protein phosphatase inhibitor, okadaic acid, in the dark (Fig. 1C). This phosphorylation was completely absent in the retinas of PdcS54A/S71A-FLAG mice (Fig. 1C). This result confirmed further that the mutations introduced in PdcS54A/S71A-FLAG eliminated both principal phosphorylation sites of Pdc that are regulated in a light-dependent manner in vivo. 18,22 All other tested properties of Pdc-FLAG and PdcS54A/S71A-FLAG were similar. Like the endogenous Pdc, epitope-tagged Pdc was present in all cellular compartments of the rod cell (data not shown). In light-adapted retinas, when Pdc is dephosphorylated, Pdc-FLAG and PdcS54A/S71A-FLAG co-precipitated with significant amounts of Gβ1, indicating that the C-terminal epitope-tagging does not affect the high affinity of Pdc towards G-protein β subunits (Fig. 1D). Additional evidence of the functional activity of transgenic Pdc was the rescue of the Gβ1 expression in rods, reduced as a result of the Pdc gene knockout. 8,9 As such 94 ± 2% and 97 ± 3% (SEM, n = 4) of the wild-type Gβ1 levels were found in the rods of the Pdc-FLAG and PdcS54A/S71A-FLAG transgenic lines, respectively. The level of transducin α subunit, slightly reduced in Pdc-null rods, 9 also was restored completely in Pdc-FLAG and PdcS54A/S71A-FLAG lines. Thus, both types of epitope-tagged phosducin, Pdc-FLAG and PdcS54A/S71A-FLAG, were similarly efficient in the restoration of transducin levels in vivo, indicating that phosphorylation of Pdc at serines 54 and 71 does not have a significant role in the regulation of this Pdc function. 
Figure 1. 
 
(A) Transgenic construct used for the generation of Pdc-FLAG and PdcS54A/S71A-FLAG mutant mice. Partial amino acid sequence alignment of human and mouse phosducin showing the positions of serine 54 and 71 (*), which were substituted with alanine in the PdcS54A/S71A-FLAG mutant. (B) Representative Western blot shows the levels of epitope-tagged or endogenous phosducin in whole retina extracts of Pdc-FLAG, PdcS54A/S71A-FLAG, and wild-type (129Sv) mice. (C) Freshly dissected retinas from dark-adapted mice of the indicated genotypes were incubated with 10 nM okadaic acid in the dark for 20 minutes, and the phosphorylation of phosducin at serines 54 and 71 was assayed by Western blotting, using pan-specific (Pdc-pan) and phospho-specific antibodies against Pdc phosphorylated at serine 54 (Pdc54p) and serine 71 (Pdc71p). (D) Phosducin was pulled-down by its FLAG tag from retinal extracts, and Gβ1 protein in the pull-downs was detected by Western blotting using an anti-Gβ1 antibody. Pdc-pan antibody blots show equal levels of the FLAG-tagged Pdc in the extracts.
Figure 1. 
 
(A) Transgenic construct used for the generation of Pdc-FLAG and PdcS54A/S71A-FLAG mutant mice. Partial amino acid sequence alignment of human and mouse phosducin showing the positions of serine 54 and 71 (*), which were substituted with alanine in the PdcS54A/S71A-FLAG mutant. (B) Representative Western blot shows the levels of epitope-tagged or endogenous phosducin in whole retina extracts of Pdc-FLAG, PdcS54A/S71A-FLAG, and wild-type (129Sv) mice. (C) Freshly dissected retinas from dark-adapted mice of the indicated genotypes were incubated with 10 nM okadaic acid in the dark for 20 minutes, and the phosphorylation of phosducin at serines 54 and 71 was assayed by Western blotting, using pan-specific (Pdc-pan) and phospho-specific antibodies against Pdc phosphorylated at serine 54 (Pdc54p) and serine 71 (Pdc71p). (D) Phosducin was pulled-down by its FLAG tag from retinal extracts, and Gβ1 protein in the pull-downs was detected by Western blotting using an anti-Gβ1 antibody. Pdc-pan antibody blots show equal levels of the FLAG-tagged Pdc in the extracts.
Comparative Analysis of Transducin Translocation in Pdc-FLAG and PdcS54A/S71A-FLAG Mice
We used Pdc-FLAG and PdcS54A/S71A-FLAG transgenic mice to assess the effect of Pdc phosphorylation on the light-driven translocation of rod transducin. To initiate translocation of transducin from the rod outer segment, all mice were subjected to a standard 10 minutes of light-conditioning with 600 lux of white light under gas anesthesia, after which animals were allowed to wake up and recover in the dark while free running. Mice were euthanized at 0, 60, 120, and 840 minutes of dark recovery, and their retinas were harvested and frozen. The protein distribution in rods was analyzed quantitatively by Western blotting serial sections of the retina (Fig. 2A). This technique, originally described by Sokolov et al., 21 was modified recently by us for the sectioning of mouse retina. 20 In these experiments, we monitored simultaneously localization of arrestin and transducin Gtα1 and Gβ1γ1 subunits. Two protein markers, rom1 and COX I, were used to identify the rod outer/inner segment boundaries, and β-tubulin was used as a loading control for all compartments except rod outer segment (Fig. 2B). To quantify the data, the fluorescence of a specific band in each section was plotted as a percentage of the combined fluorescence in all sections (Fig. 2C), and then the fraction present in the outer segment was calculated. 21  
Figure 2. 
 
(A) Schematic illustration of how a flat-mounted fragment of retina was sectioned. Inset: cellular compartments of an individual mouse rod expressing GFP (unpublished image obtained in this laboratory) and their distribution in serial sections of the retina. (B) Representative blots showing the distribution of protein markers of different rod cellular compartments (β-tubulin, COX I, and rom1), and arrestin, and transducin α and β subunits throughout a set of serial tangential sections (1–14) of the retinas of a mouse dark-adapted for 840 minutes (DARK) following 10 minutes of light conditioning with 600 lux (LIGHT). (C) Quantification of arrestin (triangles), and transducin α (circles) and β (squares) subunits distribution in blots shown in (B). The fluorescence value of a specific band in each section on the corresponding blot was plotted as a percentage of the total fluorescence value of all bands in all sections on the blots, dark-adapted mouse (closed symbols), light-conditioned mouse (open symbols).
Figure 2. 
 
(A) Schematic illustration of how a flat-mounted fragment of retina was sectioned. Inset: cellular compartments of an individual mouse rod expressing GFP (unpublished image obtained in this laboratory) and their distribution in serial sections of the retina. (B) Representative blots showing the distribution of protein markers of different rod cellular compartments (β-tubulin, COX I, and rom1), and arrestin, and transducin α and β subunits throughout a set of serial tangential sections (1–14) of the retinas of a mouse dark-adapted for 840 minutes (DARK) following 10 minutes of light conditioning with 600 lux (LIGHT). (C) Quantification of arrestin (triangles), and transducin α (circles) and β (squares) subunits distribution in blots shown in (B). The fluorescence value of a specific band in each section on the corresponding blot was plotted as a percentage of the total fluorescence value of all bands in all sections on the blots, dark-adapted mouse (closed symbols), light-conditioned mouse (open symbols).
We found that when Pdc-FLAG mice were dark-adapted overnight, on average 74% of Gtα1, and 67% of Gβ1γ1 localized to the rod outer segments (Table 1, dark recovery 840 minutes), and a nearly identical distribution of these proteins was observed in PdcS54A/S71A-FLAG mice, indicating that Pdc phosphorylation has no significant effect on the equilibrium distribution of transducin during prolonged dark-adaptation. 
Table 1.  
 
Fractions of Protein Found in the Rod Outer Segment at Different Times of Dark Recovery after Light Conditioning
Table 1.  
 
Fractions of Protein Found in the Rod Outer Segment at Different Times of Dark Recovery after Light Conditioning
Mouse Model Protein Time of Dark Recovery (Percent of Total Present in the Outer Segment)
0 Mins. 60 Mins. 120 Mins. 840 Mins.
Pdc-FLAG Transducin α 12 ± 3% 64 ± 3% 79 ± 4% 74 ± 8%
Transducin β 33 ± 2% 59 ± 1% 71 ± 6% 67 ± 4%
Arrestin 64 ± 7% 28 ± 3% 16 ± 4% 6 ± 1%
Pdc S54A/S71A-FLAG Transducin α 23 ± 6% 42 ± 5% 53 ± 4% 75 ± 2%
Transducin β 39 ± 5% 62 ± 10% 49 ± 5% 62 ± 1%
Arrestin 59 ± 1% 27 ± 3% 11 ± 2% 9 ± 1%
Light triggered translocation of Gtα1 and Gβ1γ1 subunits from the rod outer segments in both types of mice (Table 1, dark recovery 0 minutes). Immediately after the light conditioning, the fraction of Gtα1 in the outer segment of Pdc-FLAG mice was reduced to 12% (6-fold reduction), whereas 23% of Gtα1 (3-fold reduction) was retained in the outer segment of PdcS54A/S71A-FLAG mice. The corresponding fractions of the Gβ1γ1 subunits complex were 33% (2-fold reduction) and 39% (1.6-fold reduction) in Pdc-FLAG and PdcS54A/S71A-FLAG mice, respectively. We applied the same light conditioning protocol to Gtα1 −/− mice. In these mutants, characterized by the complete absence of Gtα1 expression, 23 the free Gβ1γ1 subunits become unable to accumulate in the outer segment under any condition of illumination. 24,25 We found that 30 ± 4% (SEM, n = 4) of Gβ1γ1 dimer was present in the outer segments of Gtα1 −/− mice under this condition. Therefore, these data suggest the light conditioning used here evoked the maximum possible translocation of transducin in mouse rods, apparently approaching the equilibrium distributions of fully dissociated Gtα1 and Gβ1γ1 subunits in the context of these cells. 
When this process was reversed by allowing mice to recover in the dark, Pdc-FLAG and PdcS54A/S71A-FLAG mice showed profound differences in the rates of Gtα1 subunit return to the outer segment (Table 1, dark recovery 60 and 120 minutes). When fitted with an exponential rise to maximum, this process had a half-time (t1/2) of 24 minutes in Pdc-FLAG and 95 minutes in PdcS54A/S71A-FLAG mice (Fig. 3A), thus demonstrating that Gtα1 returned to the outer segment four times faster if Pdc could undergo phosphorylation. In all these experiments, the accumulation of Gβ1γ1 dimer in the rod outer segment has similar kinetics in both types of mice during the initial 60 minutes of dark recovery (Fig. 3B). At 120 minutes of dark recovery, the movement of Gβ1γ1 in PdcS54A/S71A-FLAG mice became noticeably irregular and deviated significantly from the exponential kinetics observed in Pdc-FLAG mice (Fig. 3B). These data demonstrate that phosphorylation of Pdc primarily controls the rate of Gtα1 return to the outer segment, while also affecting some later stages of the Gβ1γ1 accumulation. Importantly, subcellular movement of rod arrestin, which also is controlled by illumination, though in the opposite direction of transducin, 26 appeared to have identical kinetics in Pdc-FLAG and PdcS54A/S71A-FLAG mice (Table 1 and Fig. 3C), indicating that this effect of Pdc phosphorylation was specific for the trafficking of transducin. 
Figure 3. 
 
(A) Data for the transducin α subunits from Table 1 for Pdc-FLAG mice (white circles) and PdcS54A/S71A-FLAG mice (black circles) were fitted with exponential rise to maximum function y = yo + Image not available . The half-time ( Image not available ) of transducin α return to the rod outer segment was 24 and 95 minutes in Pdc-FLAG mice and in PdcS54A/S71A-FLAG mice, respectively. (B) Similar analysis as in (A) revealed t1/2 = 29 minutes for the transducin β movement in Pdc-FLAG mice. Movement of this subunit in the PdcS54A/S71A-FLAG strain did not obey exponential kinetics (dashed line) and its t1/2 could not be determined. (C) Fitting of the arrestin data from Table 1 was performed using an exponential decay function Image not available . Arrestin moves from the outer segment with t1/2 = 44 and 38 minutes in Pdc-FLAG and PdcS54A/S71A-FLAG mice, respectively. For all data a t-test was applied: no asterisk indicates a P value > 0.1; (**) indicates a P value < 0.05.
Figure 3. 
 
(A) Data for the transducin α subunits from Table 1 for Pdc-FLAG mice (white circles) and PdcS54A/S71A-FLAG mice (black circles) were fitted with exponential rise to maximum function y = yo + Image not available . The half-time ( Image not available ) of transducin α return to the rod outer segment was 24 and 95 minutes in Pdc-FLAG mice and in PdcS54A/S71A-FLAG mice, respectively. (B) Similar analysis as in (A) revealed t1/2 = 29 minutes for the transducin β movement in Pdc-FLAG mice. Movement of this subunit in the PdcS54A/S71A-FLAG strain did not obey exponential kinetics (dashed line) and its t1/2 could not be determined. (C) Fitting of the arrestin data from Table 1 was performed using an exponential decay function Image not available . Arrestin moves from the outer segment with t1/2 = 44 and 38 minutes in Pdc-FLAG and PdcS54A/S71A-FLAG mice, respectively. For all data a t-test was applied: no asterisk indicates a P value > 0.1; (**) indicates a P value < 0.05.
To rule out the possibility that the slower rate of transducin movement observed in PdcS54A/S71A-FLAG mice was caused by the approximately 3-fold transgene overexpression in this versus Pdc-FLAG animals (46% and 142% of the endogenous level, respectively), we analyzed mice overexpressing wild-type phosducin. These mice were generated by back-crossing Pdc-FLAG mice into a 129Sv background (Tg-Pdc-FLAG+/−;Pdc+/+ designated as Pdc-FLAG x WT). Although the combined level of phosducin in their photoreceptors exceeded that in PdcS54A/S71A-FLAG mice by 2.5-fold (Fig. 4A), following standard light conditioning and 60 minutes dark recovery, their transducin α subunits still returned to the rod outer segments appreciably faster than in PdcS54A/S71A-FLAG mice (Fig. 4B). We concluded, therefore, that the ability of Pdc to undergo phosphorylation, rather than its intracellular level, is responsible primarily for the acceleration of the trafficking of transducin to the rod outer segments. 
Figure 4. 
 
(A) Representative Western blot showing the levels of epitope-tagged (*) and endogenous (**) phosducin in the whole retina extracts of Pdc-FLAG mice back-crossed on the wild-type background (Pdc-FLAG × WT) and PdcS54A/S71A-FLAG mice. The combined level of phosducin (* and **) in Pdc-FLAG × WT retinas was 248 ± 17% (SEM, n = 4) of that of the mutant phosducin (*) expressed in PdcS54A/S71A-FLAG retinas. (B) Subcellular localization of transducin α subunit (green), as determined by immunofluorescence microscopy, in Pdc-FLAG × WT and PdcS54A/S71A-FLAG mice dark-adapted for 60 minutes following 10 minutes of light conditioning with 600 lux light. Retinal layers are abbreviated as OS (outer segment), IS (inner segment), and ONL (outer nuclear layer).
Figure 4. 
 
(A) Representative Western blot showing the levels of epitope-tagged (*) and endogenous (**) phosducin in the whole retina extracts of Pdc-FLAG mice back-crossed on the wild-type background (Pdc-FLAG × WT) and PdcS54A/S71A-FLAG mice. The combined level of phosducin (* and **) in Pdc-FLAG × WT retinas was 248 ± 17% (SEM, n = 4) of that of the mutant phosducin (*) expressed in PdcS54A/S71A-FLAG retinas. (B) Subcellular localization of transducin α subunit (green), as determined by immunofluorescence microscopy, in Pdc-FLAG × WT and PdcS54A/S71A-FLAG mice dark-adapted for 60 minutes following 10 minutes of light conditioning with 600 lux light. Retinal layers are abbreviated as OS (outer segment), IS (inner segment), and ONL (outer nuclear layer).
Accessing the Effect of Rhodopsin Bleaching on the Rate of Transducin Return to the Outer Segment
We compared the rates of recovery from transducin translocation in rods of wild-type mice conditioned with dim and bright light. For that, we used our standard experimental setup to initiate transducin translocation by exposing one group of mice for 10 minutes to 140 lux light, whereas the second group of mice was conditioned with significantly brighter 3300 lux light. We determined that these light intensities caused bleaching of 37 ± 4% and 98 ± 1% of total rhodopsin in the retina, respectively (SEM, n = 4). The subcellular localization of transducin in both groups then was compared at certain time points of dark recovery (Fig. 5). Immediately after light conditioning, we observed the very characteristic pattern of transducin distribution in both groups that was reported previously by us and others. 21,2730 This subcellular distribution is consistent with transducin undergoing translocation from the rod outer segments, its predominant location in dark adapted rods (Fig. 5, 12 hours of dark recovery), all the way toward the synapse, which then makes transducin unambiguously detectable in the outer nuclear and outer plexiform layers of the retina (Fig. 5, 0 minutes of dark recovery). Transducin immunostaining in these layers became reduced appreciably after 30 minutes of dark recovery; however, only in mice conditioned with 140 but not 3300 lux light (Fig. 5, compare 30 minutes of dark recovery, upper and lower panel). Therefore, to our knowledge these results provide the first evidence that rods conditioned with moderate saturating light, resulting in the bleaching of a smaller fraction of the rod's pigment, can recover from transducin translocation faster than rods exposed to excessive illumination. 
Figure 5. 
 
Subcellular localization of transducin α subunit (red) in cross-sections of retina from wild-type mice dark-adapted for 0 minutes, 30 minutes, and 12 hours, following 10 minutes of light conditioning with 140 lux and 3300 lux light. Photoreceptor nuclei are shown in blue. OPL, outer plexiform layer.
Figure 5. 
 
Subcellular localization of transducin α subunit (red) in cross-sections of retina from wild-type mice dark-adapted for 0 minutes, 30 minutes, and 12 hours, following 10 minutes of light conditioning with 140 lux and 3300 lux light. Photoreceptor nuclei are shown in blue. OPL, outer plexiform layer.
Discussion
As a potential mechanism of long-term light adaptation, bidirectional movement of the visual G protein, transducin, from the light-sensing outer segment compartment of rod photoreceptors has been the focus of numerous studies in the field. 31,32 Our current understanding of the mechanism whereby transducin departs from the outer segment is based on the premise that the sustained dissociation of heterotrimeric transducin reduces its membrane-attachment significantly, and then separated Gtα1 and Gβ1γ1 subunits re-distribute through the rod cytoplasm by diffusion. 2729 However, the molecular mechanism of the reverse event, resulting in the accumulation of transducin in the outer segment, remains controversial. Two distinct models based either on diffusion or active transport often are debated (for review see the study of Artemeyev33), and the following observations made in our studies are pertinent to this discussion. 
First, the rate of transducin accumulation in the rod outer segments reported in our study is six times faster (t1/2 ∼30minutes) than that measured previously in rats and mice (t1/2 ∼200 minutes). 21,30 A possible explanation for this discrepancy is that in our experiments we used a much milder light to evoke transducin translocation. Because rhodopsin decay intermediates produce “equivalent light” that activates transducin, 34 transducin translocation from the outer segment could be sustained during dark recovery if these products are built up in significant amounts. Therefore, it is plausible, under our standard light conditioning (10 minutes of 600 lux light), on average resulting in only 80 ± 2% total rhodopsin bleaching (SEM, n = 4), that rods accumulate less rhodopsin decay intermediates than in the aforementioned studies. Indeed, this hypothesis is supported by our data demonstrating that movement of transducin towards the rod outer segments is delayed when a bigger fraction of rod pigment has been bleached during light conditioning (Fig. 5). Importantly, our measurements demonstrate that the transducin subunits dispersed from the outer segment by light could move back to this compartment significantly faster than known previously. 
Our central observation, that phosphorylation of Pdc accelerates the trafficking of Gtα1 to the outer segment, perhaps could be explained best in terms of a mechanism of phosphorylation-dependent regulation of heterotrimeric G proteins by Pdc, something originally proposed by Gaudet et al. 35 Based on structural 2 and biochemical 36 evidence, it was proposed that serine residues 54 and 71 of Pdc are located within a loosely assembled N-terminal domain, which makes these sites accessible to a protein kinase even in the Pdc-Gβ1γ1 complex. When such a phosphorylation event occurs, it triggers dissociation of the Pdc-Gβ1γ1 complex, which makes Gβ1γ1 competent to re-associate with Gtα1. 35 Thus, phosphorylation of Pdc within the Pdc-Gβ1γ1 complex could stimulate the re-association of Gtα1 and Gβ1γ1 subunits, which is a prerequisite for outer segment targeting. 24,25 Consistently with this mechanism, phosphorylation of both sites of Pdc in the dark occurs in the rod outer segments. 18  
It also is important to note that in our phosducin phosphorylation mutant mice, PdcS54A/S71A-FLAG, transducin Gtα1 and Gβ1γ1 subunits return to the outer segment at different rates, something not seen in control mice, where the return of both subunits occurs at similar rates. These differences noted in the rates of transducin subunit return would not be possible if transducin returns to the outer segment as a heterotrimer, but rather suggest that Gtα1 and Gβ1γ1 move to this compartment as individual entities. Intriguingly, our data also suggest that the accumulation of Gtα1 subunits in the outer segment is based predominantly on their re-binding to Gβ1γ1 subunits, either already present or moved to this compartment by another mechanism. Indeed, a nearly 3-fold molar excess of Gβ1γ1 compared to Gtα1 is found in the outer segment after light conditioning (Table 1), because more Gtα1 than Gβ1γ1 undergoes translocation from this compartment. Such a mechanism also would explain why the kinetics of Gtα1 return to the outer segment during dark conditioning shows notably more dependency on Pdc phosphorylation than that of Gβ1γ1 (Figs. 3A and 3B). Two additional lines of evidence further support this hypothesis. First, Gtα1 was found to be competent fully to undergo normal reversible translocation even when Gβ1γ1 doesn't move from the outer segments. 37 Second, rod visual signaling surprisingly appeared to be unperturbed by genetic elimination of the Gtα1 lipid modification, 38 indicating that Gβ1γ1 is fully capable to recruit Gtα1 and form a functional heterotrimer coupled to rhodopsin, even if Gtα1 lacks its membrane anchor. Combined, these data thus appear to corroborate the notion that the light-displaced Gtα1 and Gβ1γ1 subunits return to the rod outer segment as individual entities, rather than as a heterotrimer. 
Most recently, two groups reported new and exciting data on the mechanism of transducin translocation. 39,40 It was demonstrated that a photoreceptor protein, UNC119, interacts specifically with the Gtα1 lipid moiety, leading to Gtα1 subunit dissociation from Gβ1γ1 and partitioning from the membrane. This mode of action, which resembles closely that of Pdc, makes it tempting to speculate that UNC119 and Pdc act in concert to keep transducin subunits soluble during their return to the rod outer segment. However, it remains to be elucidated how Gtα1 and Gβ1γ1 subunits are released from their trafficking chaperones, which would be required for their assembly on the outer segment membranes. 
Acknowledgments
Janis Lem made a 4.4 kb mouse rhodopsin promoter and transducin α knockout mice available for our study, and Andrew Goldberg generously provided the antibody against rom1. 
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Footnotes
 Supported by National Eye Institute Grant EY019665 and an unrestricted Research to Prevent Blindness Grant awarded to West Virginia University Eye Institute. The Transgenic Animal Core Facility at WVU was supported by CoBRE Grants, RR031155 and RR016440.
Footnotes
2  These authors contributed equally to this work.
Footnotes
 Disclosure: M. Belcastro, None; H. Song, None; S. Sinha, None; C. Song, None; P.H. Mathers, None; M. Sokolov, None
Figure 1. 
 
(A) Transgenic construct used for the generation of Pdc-FLAG and PdcS54A/S71A-FLAG mutant mice. Partial amino acid sequence alignment of human and mouse phosducin showing the positions of serine 54 and 71 (*), which were substituted with alanine in the PdcS54A/S71A-FLAG mutant. (B) Representative Western blot shows the levels of epitope-tagged or endogenous phosducin in whole retina extracts of Pdc-FLAG, PdcS54A/S71A-FLAG, and wild-type (129Sv) mice. (C) Freshly dissected retinas from dark-adapted mice of the indicated genotypes were incubated with 10 nM okadaic acid in the dark for 20 minutes, and the phosphorylation of phosducin at serines 54 and 71 was assayed by Western blotting, using pan-specific (Pdc-pan) and phospho-specific antibodies against Pdc phosphorylated at serine 54 (Pdc54p) and serine 71 (Pdc71p). (D) Phosducin was pulled-down by its FLAG tag from retinal extracts, and Gβ1 protein in the pull-downs was detected by Western blotting using an anti-Gβ1 antibody. Pdc-pan antibody blots show equal levels of the FLAG-tagged Pdc in the extracts.
Figure 1. 
 
(A) Transgenic construct used for the generation of Pdc-FLAG and PdcS54A/S71A-FLAG mutant mice. Partial amino acid sequence alignment of human and mouse phosducin showing the positions of serine 54 and 71 (*), which were substituted with alanine in the PdcS54A/S71A-FLAG mutant. (B) Representative Western blot shows the levels of epitope-tagged or endogenous phosducin in whole retina extracts of Pdc-FLAG, PdcS54A/S71A-FLAG, and wild-type (129Sv) mice. (C) Freshly dissected retinas from dark-adapted mice of the indicated genotypes were incubated with 10 nM okadaic acid in the dark for 20 minutes, and the phosphorylation of phosducin at serines 54 and 71 was assayed by Western blotting, using pan-specific (Pdc-pan) and phospho-specific antibodies against Pdc phosphorylated at serine 54 (Pdc54p) and serine 71 (Pdc71p). (D) Phosducin was pulled-down by its FLAG tag from retinal extracts, and Gβ1 protein in the pull-downs was detected by Western blotting using an anti-Gβ1 antibody. Pdc-pan antibody blots show equal levels of the FLAG-tagged Pdc in the extracts.
Figure 2. 
 
(A) Schematic illustration of how a flat-mounted fragment of retina was sectioned. Inset: cellular compartments of an individual mouse rod expressing GFP (unpublished image obtained in this laboratory) and their distribution in serial sections of the retina. (B) Representative blots showing the distribution of protein markers of different rod cellular compartments (β-tubulin, COX I, and rom1), and arrestin, and transducin α and β subunits throughout a set of serial tangential sections (1–14) of the retinas of a mouse dark-adapted for 840 minutes (DARK) following 10 minutes of light conditioning with 600 lux (LIGHT). (C) Quantification of arrestin (triangles), and transducin α (circles) and β (squares) subunits distribution in blots shown in (B). The fluorescence value of a specific band in each section on the corresponding blot was plotted as a percentage of the total fluorescence value of all bands in all sections on the blots, dark-adapted mouse (closed symbols), light-conditioned mouse (open symbols).
Figure 2. 
 
(A) Schematic illustration of how a flat-mounted fragment of retina was sectioned. Inset: cellular compartments of an individual mouse rod expressing GFP (unpublished image obtained in this laboratory) and their distribution in serial sections of the retina. (B) Representative blots showing the distribution of protein markers of different rod cellular compartments (β-tubulin, COX I, and rom1), and arrestin, and transducin α and β subunits throughout a set of serial tangential sections (1–14) of the retinas of a mouse dark-adapted for 840 minutes (DARK) following 10 minutes of light conditioning with 600 lux (LIGHT). (C) Quantification of arrestin (triangles), and transducin α (circles) and β (squares) subunits distribution in blots shown in (B). The fluorescence value of a specific band in each section on the corresponding blot was plotted as a percentage of the total fluorescence value of all bands in all sections on the blots, dark-adapted mouse (closed symbols), light-conditioned mouse (open symbols).
Figure 3. 
 
(A) Data for the transducin α subunits from Table 1 for Pdc-FLAG mice (white circles) and PdcS54A/S71A-FLAG mice (black circles) were fitted with exponential rise to maximum function y = yo + Image not available . The half-time ( Image not available ) of transducin α return to the rod outer segment was 24 and 95 minutes in Pdc-FLAG mice and in PdcS54A/S71A-FLAG mice, respectively. (B) Similar analysis as in (A) revealed t1/2 = 29 minutes for the transducin β movement in Pdc-FLAG mice. Movement of this subunit in the PdcS54A/S71A-FLAG strain did not obey exponential kinetics (dashed line) and its t1/2 could not be determined. (C) Fitting of the arrestin data from Table 1 was performed using an exponential decay function Image not available . Arrestin moves from the outer segment with t1/2 = 44 and 38 minutes in Pdc-FLAG and PdcS54A/S71A-FLAG mice, respectively. For all data a t-test was applied: no asterisk indicates a P value > 0.1; (**) indicates a P value < 0.05.
Figure 3. 
 
(A) Data for the transducin α subunits from Table 1 for Pdc-FLAG mice (white circles) and PdcS54A/S71A-FLAG mice (black circles) were fitted with exponential rise to maximum function y = yo + Image not available . The half-time ( Image not available ) of transducin α return to the rod outer segment was 24 and 95 minutes in Pdc-FLAG mice and in PdcS54A/S71A-FLAG mice, respectively. (B) Similar analysis as in (A) revealed t1/2 = 29 minutes for the transducin β movement in Pdc-FLAG mice. Movement of this subunit in the PdcS54A/S71A-FLAG strain did not obey exponential kinetics (dashed line) and its t1/2 could not be determined. (C) Fitting of the arrestin data from Table 1 was performed using an exponential decay function Image not available . Arrestin moves from the outer segment with t1/2 = 44 and 38 minutes in Pdc-FLAG and PdcS54A/S71A-FLAG mice, respectively. For all data a t-test was applied: no asterisk indicates a P value > 0.1; (**) indicates a P value < 0.05.
Figure 4. 
 
(A) Representative Western blot showing the levels of epitope-tagged (*) and endogenous (**) phosducin in the whole retina extracts of Pdc-FLAG mice back-crossed on the wild-type background (Pdc-FLAG × WT) and PdcS54A/S71A-FLAG mice. The combined level of phosducin (* and **) in Pdc-FLAG × WT retinas was 248 ± 17% (SEM, n = 4) of that of the mutant phosducin (*) expressed in PdcS54A/S71A-FLAG retinas. (B) Subcellular localization of transducin α subunit (green), as determined by immunofluorescence microscopy, in Pdc-FLAG × WT and PdcS54A/S71A-FLAG mice dark-adapted for 60 minutes following 10 minutes of light conditioning with 600 lux light. Retinal layers are abbreviated as OS (outer segment), IS (inner segment), and ONL (outer nuclear layer).
Figure 4. 
 
(A) Representative Western blot showing the levels of epitope-tagged (*) and endogenous (**) phosducin in the whole retina extracts of Pdc-FLAG mice back-crossed on the wild-type background (Pdc-FLAG × WT) and PdcS54A/S71A-FLAG mice. The combined level of phosducin (* and **) in Pdc-FLAG × WT retinas was 248 ± 17% (SEM, n = 4) of that of the mutant phosducin (*) expressed in PdcS54A/S71A-FLAG retinas. (B) Subcellular localization of transducin α subunit (green), as determined by immunofluorescence microscopy, in Pdc-FLAG × WT and PdcS54A/S71A-FLAG mice dark-adapted for 60 minutes following 10 minutes of light conditioning with 600 lux light. Retinal layers are abbreviated as OS (outer segment), IS (inner segment), and ONL (outer nuclear layer).
Figure 5. 
 
Subcellular localization of transducin α subunit (red) in cross-sections of retina from wild-type mice dark-adapted for 0 minutes, 30 minutes, and 12 hours, following 10 minutes of light conditioning with 140 lux and 3300 lux light. Photoreceptor nuclei are shown in blue. OPL, outer plexiform layer.
Figure 5. 
 
Subcellular localization of transducin α subunit (red) in cross-sections of retina from wild-type mice dark-adapted for 0 minutes, 30 minutes, and 12 hours, following 10 minutes of light conditioning with 140 lux and 3300 lux light. Photoreceptor nuclei are shown in blue. OPL, outer plexiform layer.
Table 1.  
 
Fractions of Protein Found in the Rod Outer Segment at Different Times of Dark Recovery after Light Conditioning
Table 1.  
 
Fractions of Protein Found in the Rod Outer Segment at Different Times of Dark Recovery after Light Conditioning
Mouse Model Protein Time of Dark Recovery (Percent of Total Present in the Outer Segment)
0 Mins. 60 Mins. 120 Mins. 840 Mins.
Pdc-FLAG Transducin α 12 ± 3% 64 ± 3% 79 ± 4% 74 ± 8%
Transducin β 33 ± 2% 59 ± 1% 71 ± 6% 67 ± 4%
Arrestin 64 ± 7% 28 ± 3% 16 ± 4% 6 ± 1%
Pdc S54A/S71A-FLAG Transducin α 23 ± 6% 42 ± 5% 53 ± 4% 75 ± 2%
Transducin β 39 ± 5% 62 ± 10% 49 ± 5% 62 ± 1%
Arrestin 59 ± 1% 27 ± 3% 11 ± 2% 9 ± 1%
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