Molecular Vision 2003; 9:231-237 <http://www.molvis.org/molvis/v9/a34/> Received 11 April 2003 | Accepted 5 June 2003 | Published 9 June 2003

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Light-dependent redistribution of visual arrestins and transducin subunits in mice with defective phototransduction

Houbin Zhang,¹ Wei Huang,² Haikun Zhang,² Xuemei Zhu,³ Cheryl M. Craft,³ Wolfgang Baehr,^1,2,4 Ching-Kang Chen^2,5,6
 
 

Departments of ¹Neurobiology and Anatomy, ²Ophthalmology and Visual Sciences, ⁴Biology, ⁵Human Genetics, and ⁶Pharmacology and Toxicology, University of Utah, Salt Lake City, UT; ³The Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA

Correspondence to: Ching-Kang Chen, Moran Eye Center, EIHG 3110A, 15 North 2030 East, University of Utah Health Science Center, Salt Lake City, UT, 84112-5330; Phone: (801) 585-5258; FAX: (801) 585-3501; email: jason@hmbg.utah.edu

Abstract

Purpose: The light-dependent redistribution of phototransduction components in photoreceptor cells plays a role in light adaptation. Upon illumination, rod and cone arrestins (Arr and cArr) translocate from the inner to the outer segments while transducin subunits (Tα, Tβγ) translocate in the opposite direction. The underlying translocation mechanisms are unclear. This study examines these previously demonstrated translocation in mice with defective phototransduction.

Methods: The distribution of Arr, cArr, Tα, and Tβγ was examined using immunoblotting and immunocytochemistry in dark- and light-adapted single knockout mice lacking G-protein coupled receptor kinase 1 (Grk1^-/-) and double knockout mice lacking GRK1 and transducin α subunit (Grk1^-/-/Gnat1^-/-), or lacking GRK1 and arrestin (Grk1^-/-/Arr^-/-).

Results: Arr redistributed in the light to the outer segments in Grk1^-/- mice as well as in Grk1^-/-/Gnat1^-/- double knockout retinas. Immunoblotting revealed that approximately 25-50% of Arr associated with the membrane in light-adapted wild-type, Grk1^-/- and Gnat1^-/-/Grk1^-/- mouse retinas. In contrast, cArr did not stably associate with light-adapted membranes in either wild-type or Grk1^-/- retinas under our experimental conditions, but redistributed to the cone outer segments in a light-dependent manner. The redistribution of transducin subunits to the inner segments in light occurred in both wild-type and Grk1^-/-/Arr^-/- double knockout photoreceptors. However, Tβγ subunits did not redistribute in the absence of Tα, suggesting that transducin only translocates as an intact heterotrimer.

Conclusions: We conclude that in rods, Arr redistribution requires neither rhodopsin phosphorylation nor phototransduction, suggesting the presence of another light-dependent pathway to trigger translocation. In cones, the light-dependent movement of cArr appears to be independent of stable association with the cone pigments. The light-dependent translocations of Arr and transducin subunits in opposite directions appear to be based on independent mechanisms.

Introduction

Retinal photoreceptors are highly differentiated sensory neurons where conversion of light energy into neuronal signals takes place through a mechanism called phototransduction [1-3]. In rod photoreceptors, the absorption of photons elicits a response culminating in rapid hydrolysis of cGMP, the second messenger of phototransduction, and hyperpolarization of the cell. Rod photoreceptors are able to retain light responsiveness at different levels of illumination, a process known as light adaptation that requires feedback mechanisms to control the sensitivity and gain of the phototransduction cascade. Most of these mechanisms operate on a fast timescale using Ca²⁺ as an internal messenger [4,5]. Another slower mechanism of light adaptation (within minutes) involves the light dependent redistribution of distinct phototransduction proteins such as arrestin and transducin in and out of the outer segment where phototransduction occurs. This phenomenon was initially reported by several groups in the late 80's [6-11], but only recently unambiguously confirmed in both vertebrate and invertebrate photoreceptors [12,13]. It was shown that such movements enable the photoreceptor to modulate its signaling capacities by changing the effective concentrations of these proteins in the outer segment. The mechanism for this light-dependent redistribution is unclear, although the involvement of cytoskeleton [14] and lipid rafts [15] has been postulated.

In this report we use mice lacking proteins involved in phototransduction or inactivation of the visual pigments [16-19] to study the light-dependent redistribution of rod transducin subunits and visual arrestins. The mutant mice used are either single knockouts lacking expression of GRK1 [19], or double knockouts lacking GRK1/Arr [18] or GRK1/ Tα expression [17]. We show that the redistribution of rod arrestin and transducin does not depend on one another and that phosphorylation of the receptor is not necessary for either rod or cone arrestin translocation. Furthermore, rod arrestin translocates in the absence of a functional phototransduction cascade. We also show that the Tβγ subunits of rod transducin do not translocate in the absence of Tα, suggesting that Tα is necessary for the transport of Tβγ to photoreceptor outer segments. Our data predict the existence of unidentified light-dependent pathways in retinal photoreceptors that couple the absorption of photons to the redistribution of visual arrestins and rod transducin.

Methods

Animals

All experimental procedures involving the use of laboratory mice complied with NIH guidelines as approved by the Institutional Animal Care and Use Committee of the University of Utah. All knockout mice used were in a mixed background of C57BL/6 and 129SvEv, except for Grk1^-/-/Gnat1^-/- mice which were in a mixed C57BL/6, 129SvEv, and BALB/c background. These mice were raised in the dark to preserve their retinal photoreceptors. The wild type mice, in a mixed background of C57BL/6 and 129SvEv, were raised in 12/12 light-dark cycles. Mice in the age of 1-3 months were used. To dark adapt, the control mice were placed in the dark for more than 12 h. Approximately 20 min prior to light exposure, the pupils were dilated by topical treatment of 1% tropicamide (Bausch & Lomb, Tampa, FL) and 2.5% phenylephrine (Alcon, Ft. Worth, TX). The mice were then exposed to about 2500 lux fluorescent white light for 30 min at room temperature with unrestricted access to food and water. The mice were euthanized by CO₂ inhalation. All dark procedures were performed under infrared illumination.

Protein quantification and immunoblot analysis

Retinas derived from dark- and light-adapted mice were homogenized in eppendorf tubes using disposable pestles (Kontes, Vineland, NJ) followed by sonication in RIPA buffer (150 mM NaCl, 1% Nonidet P-40 (NP40), 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 7.2). The protein levels in the resulting retinal extracts were determined by BioRad Protein Assay dye using bovine serum albumin (BSA) as standards. Precisely 70 μg of protein was subject to 10% SDS-PAGE followed by immunoblot analysis. To measure the relative amount of Arr and cArr bound to the membrane, retinas derived from dark- and light-adapted mice were homogenized in 100 μl/retina Buffer A (phosphate buffered saline (PBS) containing 5 mM ATP, 1 mM DTT and protease inhibitors (Roche Applied Science, Mannheim, Germany)) either as described above or by 30 s homogenization in eppendorf tubes with disposable pestles on ice. The resulting protein extracts were subject to ultracentrifugation either at 250,000x g at 25 °C for 10 min or at 20,000x g at 4 °C for 10 min. The pellets were resuspended in 100 μl/retina Buffer A. Approximately one twentieth of the supernatant and resuspended pellet derived from two retinas were applied onto 10% SDS-PAGE gels [20], and the relative amounts of Arr and cArr in soluble and membrane fractions were determined by immunoblotting using C10C10 (anti-Arr [21], used at 1:5,000 dilution) and LUMIJ (anti-cArr [22], used at 1:5,000 dilution) antibodies, followed by horseradish peroxidase (HRP) conjugated donkey anti-rabbit (for LUMIJ, used at 1:2000; Amersham, Piscataway, NJ) and HRP conjugated goat anti-mouse (for C10C10, used at 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA) secondary antibodies. The Western Lightning Chemiluminescence Reagent Plus Kit (PerkinElmer Life Sciences, Boston, MA) was used to detect the signals which were then digitized and quantified using Kodak 1D image analysis program on a Kodak IS440 imaging station (PerkinElmer Life Sciences).

Immunocytochemistry

Mouse eyeballs were enucleated and fixed for 2 h in 4% paraformaldehyde in PBS at room temperature. After fixation, the cornea and the lens were removed and the eyecups were immersed in 10% sucrose prepared in PBS for 1 h, 20% sucrose in PBS for 1 h, and 30% sucrose in PBS for overnight at 4 °C. The eyecups were embedded in TBS (Triangle Biomedical Sciences, Durham, NC) and 14 μm frozen sections were cut and mounted onto Superfrost/Plus slides (Fisher Scientific, Pittsburgh, PA). The sections were blocked with PBS containing 1% BSA, 0.1% Triton X-100 and 10% goat serum, and probed with various primary antibodies, including A9C6 (anti-Arr [21], 1:400 dilution), LUMIJ (anti-cArr [22], 1:250), Tα1A (anti-Tα [16], 1:500), BN1 (anti-Tβ [23], 1:800) and GN2 (anti-Tγ [24], 1:1,200), respectively. Rhodamine-conjugated goat-anti-rabbit (for LUMIJ, Tα1A, BN1 and GN2) and goat-anti-mouse (for A9C6) secondary antibodies (Southern Biotechnology Associates, Birmingham, AL) were used (1:2,000 dilution) to visualize the signals under a Nikon Eclipse E600 fluorescent microscope.

Results

Light-dependent membrane association of rod arrestin

In mouse retinas, G protein coupled receptor kinase 1 (GRK1) is required for the deactivation of light activated visual pigments in rods and cones [19,25,26]. In rods, the phosphorylation of activated rhodopsin (R*) by GRK1 facilitates its interaction with rod Arr, which prevents further interaction of R* with transducin, a key event in turning off the cascade [27,28]. Stable and light-dependent binding of Arr to phosphorylated pigment and hence the reduction of soluble Arr in the outer segment may create a gradient that triggers redistribution of Arr from the inner segment to the outer segment. We tested the light-dependent binding of Arr in Grk1^-/- photoreceptors in which rhodopsin cannot be phosphorylated, and in Grk1^-/-/Gnat1^-/- photoreceptors lacking both phosphorylation and a functional phototransduction cascade. The expression level of Arr in wild-type and mutant retinas was not affected by either the inactivation of the Grk1 gene alone, or by the inactivation of both the Grk1 and the Gnat1 genes (Figure 1A). In the dark-adapted state, regardless of the genetic background, Arr was nearly completely soluble (>95%, Figure 1B, upper panel). In light-adapted retinas, in contrast, the amount of soluble Arr appeared to depend on the genetic background (Figure 1B, lower panel). Approximately 53% of Arr in wild-type, 74% in Grk1^-/-, and 57% in Grk1^-/-/Gnat1^-/- mouse photoreceptors remained soluble (Figure 1B, bargraph). These results indicate that the deletion of the Grk1 gene reduces the binding of Arr to rhodopsin in light-adapted retina, but does not completely eliminate the binding. The more efficient binding of Arr to the Grk1^-/-/Gnat1^-/- membrane, as compared with Grk1^-/- retina, may result from the lack of competition from Tα to bind to unphosphorylated R* in the Grk1^-/- /Gnat1^-/- double knockout retina.

Light-dependent redistribution of Arr in mouse Grk1^-/- and Grk1^-/-/Gnat1^-/- photoreceptors

It was shown that Arr and transducin translocated within photoreceptors upon illumination in opposite directions [7,14,29]. To examine whether the reciprocal redistribution of Arr and transducin is coupled and the relationship between rhodopsin phosphorylation and Arr redistribution, we compared the redistribution of Arr in wild-type mice with that in Grk1^-/- and Grk1^-/-/Gnat1^-/- mice (Figure 2). We found that in dark-adapted wild type, Grk1^-/- and Grk1^-/-/Gnat1^-/- retinas, the majority of Arr was localized to rod inner segments (RIS) where biosynthesis takes place (a small fraction of Arr was localized to synaptic terminals). In the light-adapted retinas, the majority of Arr redistributed to rod outer segments (ROS). These results show that the light-dependent redistribution of Arr does not require the phosphorylation of light-activated rhodopsin. The weaker association of Arr with membranes in Grk1^-/- retina appeared to have little effect on the light-dependent redistribution of arrestin. Since Tα is an essential component of the phototransduction cascade, the light-dependent redistribution of arrestin in Grk1^-/-/Gnat1^-/- retina indicates that downstream signaling events in phototransduction are not required for Arr redistribution.

Light-dependent movement of cone arrestin in Grk1^-/- mice

It was previously reported that bovine [30] or salamander cArr [31] have low binding affinities compared to rod Arr for light-activated phosphorylated and unphosphorylated rhodopsin. It was also shown that cArr did not bind to heparin [30] as rod Arr does, although rod and cone arrestin amino acid sequences are highly conserved suggesting a closely related structure. The light-dependent redistribution of cArr in mouse and bovine cones has been observed [22,32] suggesting that cArr may quench the cone phototransduction cascade similarly as Arr does in rods. Because GRK1 is expressed in both rod and cone photoreceptors in mouse [25], and it has recently been shown that GRK1 is required for light-dependent phosphorylation of mouse cone visual pigments [33], we examined the light-dependent membrane association and photoreceptor redistribution of cArr in Grk1^-/- mice. We found that the majority of cArr remained in the soluble fraction in both wild-type and Grk1^-/- retinas independent of light history under our experimental conditions (Figure 1C), but the light-dependent redistribution of cArr was evident and indistinguishable between wild type and Grk1^-/- retinas (Figure 3). These results suggest that, similar to what we observed for Arr, neither stable membrane association of cArr nor phosphorylation of light-activated cone opsin by GRK1 is required for light-dependent cArr redistribution.

Light-dependent movement of transducin in the absence of arrestin and GRK1

In addition to arrestin, light also triggers the translocation of transducin subunits from the outer segment in the dark, to the inner segment in the light (Figure 4, top panels). Transducin, the heterotrimeric G protein of the rod phototransduction cascade, is activated by interacting with R*. The phosphorylation of R* weakens the interaction between transducin and rhodopsin, and the binding of Arr to P-R* prevents further interaction between rhodopsin and transducin. To investigate how phosphorylation of R* and arrestin binding to P-R* affect the movement of Tα, we examined light-dependent redistribution of Tα in Grk1^-/-/Arr^-/- retinas. As shown in Figure 4, the majority of Tα resides in ROS in dark-adapted retina, whereas in the light-adapted state Tα is found throughout the photoreceptor layer, including the RIS and the synapse, similar to the distribution of Tα in wild type mouse retinas. These results suggest that Tα redistribution is independent of R* phosphorylation and Arr binding to phosphorylated rhodopsin.

The distribution of Tβγ subunits in Gnat1^-/- mice

It was previously shown that all three subunits of transducin (Tα, Tβγ), undergo light-dependent redistribution in wild type retina [29]. To test the hypothesis that these three subunits translocate as a heterotrimeric complex Tαβγ, we examined the light-dependent distribution of Tβγ subunits in Grk1^-/-/Gnat1^-/- and Grk1^-/-/Arr^-/- double knockout mice (Figure 5 and Figure 6). We stained the dark- and light-adapted double knockout retinas with anti-Tβ and Tγ antibodies, respectively (Figure 5 and Figure 6). The results demonstrate that Tβγ subunits reside throughout the rod photoreceptors of Grk1^-/-/Gnat1^-/- mice, independent of light history, while the distribution Tβγ subunits in Grk1^-/-/Arr^-/- resembles closely that of wild-type retina. These results show that the translocation of Tβγ from inner segments to outer segments requires Tα, and suggest that transducin translocates as an intact heterotrimer.

Discussion

The purpose of this study was to provide further insight into the mechanisms of translocation of components of the phototransduction cascade using an arsenal of single and double knockout mice. The light-dependent movement of Arr from inner segment to outer segment was observed nearly two decades ago [8], and that of Tα and Tβ from outer segments to inner segments was reported shortly after [6,7]. However, the movement particularly of transducin was challenged as an artifact of epitope masking [9], a problem that hampered the progress toward understanding the movement mechanism for more than a decade. Immunoblotting of tangentially sectioned photoreceptor layers, however, proved that the light-dependent translocation of transducin indeed occurs on a time scale of minutes [12].

In the dark-adapted wild-type mouse, the majority of Arr is distributed in RIS and synaptic terminals, only small amounts of Arr are present in the ROS. Under this condition we found that >95% of Arr stays in the soluble fraction (Figure 1). Upon illumination, the majority of Arr redistributed to ROS and about 47% of Arr associated with membranes. Arr binds tightly with activated and phosphorylated rhodopsin, a process necessary for the shutoff of rod phototransduction and timely recovery of the dark-adapted state [28]. Mechanisms involved in triggering the translocation and the transport itself are unclear. It is conceivable that Arr binding to phosphorylated rhodopsin draws the soluble Arr from the inner segment to the outer segments. Therefore, the observed translocation may simply be the result of mass action and diffusion of Arr, an event secondary to the high affinity binding of Arr to rhodopsin after photobleaching. However, in Grk1^-/- mice where light-dependent rhodopsin phosphorylation is abolished [19], we found that the membrane affinity of Arr was significantly reduced (from about 47% to 25%), but the light-dependent translocation of Arr still occurred. Because the translocation of Arr can be uncoupled from its membrane association, another light dependent mechanism that triggers massive redistribution must be present in photoreceptors. This uncoupling of translocation and membrane association is even more pronounced for cArr. GRK1 was shown to be the only GRK in mouse cones that participates in the deactivation of cone phototransduction [25,26]. Under our experimental conditions in which light-dependent membrane association of Arr can be readily observed (Figure 1B), the light-dependent membrane association of cArr was not evident (Figure 1C). It is worth noting, however, under different experimental conditions using hypotonic buffer (50 mM sodium phosphate buffer, pH 6.8) and milder homogenization techniques [33] the light-dependent membrane association of cArr can be detected in wild-type mice. Under those conditions, however, the light-dependent association of cArr was not detectable in Grk1^-/- mice. Because the light-induced redistribution of cArr to cone outer segments occurs (Figure 3) in the absence of detectable light-dependent membrane association of cArr, these two light-dependent events in cone photoreceptors clearly occur independent of each other.

The light-dependent redistribution of transducin in a direction opposite to arrestins appeared normal in Grk1^-/-/Arr^-/- double knockout mice, suggesting that neither receptor phosphorylation nor arrestin binding is necessary for transducin translocation, and that the redistribution of arrestin and transducin is not coupled. This hypothesis is further supported by the observation that the light-dependent redistribution of Arr is normal in Gnat1^-/-/Grk1^-/- mice. Because transducin is required for phototransduction (there is no detectable rod-derived electroretinographic response in Gnat1^-/- mice [34]), the redistribution of Arr in Gnat1^-/-/Grk1^-/- mice hence indicates that neither receptor phosphorylation nor phototransduction is required for Arr translocation.

The lack of Tβγ subunits accumulation in the dark-adapted GRK1^-/-/Gnat1^-/- ROS contrasts sharply to the normal light-dependent redistribution of arrestins. This not only supports the model that the movements of arrestin and transducin are not coupled, it also suggests that the redistribution may involve different mechanisms. This conclusion is consistent with an accumulation of arrestin and opsin, but not transducin, in the inner segments of photoreceptor specific KIF3A^-/- mice [35] and a recent finding that Tβγ, but not arrestin, selectively interacts with a Ca²⁺-binding protein called centrin which is localized in the lumen of the connecting cilium [36]. The interaction between centrin and Tβγ requires Ca²⁺-binding to centrin, an event that has been postulated to play a gate-keeping role in retaining Tαβγ in the ROS in dark adapted state when intracellular Ca²⁺ concentration in the ROS is high. It is conceivable that in the absence of Tα, the lack of phototransduction and absence of regulation of cGMP levels by light leaves a larger portion of cGMP-gated channels open and the intracellular Ca²⁺ remains high to sustain the interaction between centrin and Tβγ and prevents the trafficking of Tβγ through the lumen of the connecting cilium.

It is worth noting that we can not rule out the existence of extracellular mechanisms that regulate the movement of photoreceptor proteins. Such mechanisms play an important role in retinomotor movements in some vertebrates such as fish and amphibians [37]. Experiments using dimmer illumination are currently in progress to evaluate (1) the contribution of cone photoreceptors in the movement of Arr and transducin in rod and (2) the contribution of rod photoreceptors in the movement of cArr in cones (as the data reported here were obtained under a relatively intense illumination condition where both rod and cone photoreceptors were activated). Nonetheless, our data demonstrate qualitatively that in rod photoreceptors the light-dependent redistribution of arrestin is independent of rhodopsin phosphorylation and phototransduction, and that the redistributions of transducin and Arr are not intracellularly coupled. We also show that the translocation of Tβγ requires the presence of Tα. In addition, we show that in cone photoreceptors cArr translocates in the absence of stable membrane association in both wild type and GRK1^-/- mice. Although the exact mechanisms are unclear, these results predict the existence of novel and unidentified light-dependent signaling pathways in the retina or within the photoreceptors that govern the observed light-dependent movement of these photoreceptor proteins.

Note

While this paper was under review, Mendez et al. [38] published data similar to some of the data presented here indicating that the movement of rod arrestin is independent of rhodopsin phosphorylation and phototransduction. The Mendez data set was collected by independent investigators concurrently working on the same problem.

Acknowledgements

We thank Dr. Mel Simon for transducin antibodies and the Grk1^-/-/Gnat1^-/- double knockout mice, Dr. Janis Lem for Gnat1^-/- knockout mice, Dr. Larry Donoso for arrestin antibodies, and Bruce Brown and Dr. Aimin Li for technical assistance. CKC is a recipient of the Research to Prevent Blindness (RPB) Career Development Award. WB is a recipient of the RBP Senior Investigator Award and receives a Ralph and Mary Tuck endowment from the Department of Ophthalmology at the University of Utah. CMC is the Mary D. Allen Chair for Vision Research at the Doheny Eye Institute. This work was supported in part by grants EY013811 (CKC), EY00395 (CMC), and EY08123 (WB) from the National Eye Institute. We further acknowledge grants from the Knights Templar Eye Foundation (CKC), Research to Prevent Blindness, Inc. to the Department of Ophthalmology at the University of Utah, a Center Grant from Foundation Fighting Blindness, Inc. to the University of Utah, and a grant from the Macular Vision Research Foundation (WB).

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Typographical corrections