Molecular Vision 2002; 8:462-471 <http://www.molvis.org/molvis/v8/a56/> Received 19 June 2002 | Accepted 10 December 2002 | Published 11 December 2002

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Mouse cone arrestin expression pattern: Light induced translocation in cone photoreceptors

Xuemei Zhu,¹ Aimin Li,¹ Bruce Brown,¹ Ellen R. Weiss,² Shoji Osawa,² Cheryl M. Craft¹
 
 

¹The Mary D. Allen Laboratory for Vision Research, Doheny Eye Institute, Department of Cell and Neurobiology, The Keck School of Medicine of the University of Southern California, Los Angeles, CA; ²Department of Cell and Developmental Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC

Correspondence to: Cheryl M. Craft, Ph.D., Department of Cell & Neurobiology, The Keck School of Medicine of the University of Southern California, 1333 San Pablo Street, BMT 401, Los Angeles, CA, 90089-9112; Phone: (323) 442-1794; FAX: (323) 442-2709; email: ccraft@usc.edu

Abstract

Purpose: Arrestins are a superfamily of regulatory proteins that down-regulate activated and phosphorylated G-protein coupled receptors (GPCRs). Cone arrestin (CAR) is expressed in cone photoreceptors and pinealocytes and may contribute to the shutoff mechanisms associtated with high acuity color vision. To initiate a study of CAR's function in cone phototransduction, the mouse CAR (mCAR) transcript and protein expression patterns are examined and in vitro binding assays are also presented.

Methods: Tissue distribution of mCAR was determined by Northern and immunoblot analyses and its cellular localization identified by In situ hybridization and immunohistochemistry. The protein expression pattern of mCAR in the postnatal developmental and adult mouse retina was analyzed by immunoblotting in normal C57 and rd/rd mouse retinas. In vitro binding assays with in vitro translated arrestins were used to study the interaction of mCAR and mouse S-antigen (mSAG) with embryonic chicken outer segment (OS) membranes containing both rod and cone opsins.

Results: MCAR has a high level of amino acid sequence identity with orthologous sequences reported for other species except the C-terminal region, which is highly conserved between mouse and rat but divergent in other species. MCAR is expressed exclusively in the retina and the pineal gland, and unique isoforms are expressed during postnatal development of the retina and the pineal gland. The postnatal developmental expression pattern of mCAR and mSAG in the rd/rd mouse retina parallels the generation and degeneration of the cone and rod photoreceptors in these mice. In situ and immunohistochemistry both reveal cone-specific expression of mCAR in the retina. Immunofluorescent staining of retinal sections from dark-adapted or light-exposed mice suggests a light-dependent translocation of mCAR immunoreactivity from the cone inner segments (CIS) and other parts of the cell body to the cone outer segments (COS), similar to but not as dramatic as rod arrestin. In vitro binding assays show a small yet significant increase in binding of the full-length mCAR (mCARFL) to embryonic chicken OS membranes following light activation and phosphorylation of the opsins in the membranes.

Conclusions: MCAR is expressed in retinal cone photoreceptors and the pineal gland. The light-dependent translocation of mCAR immunoreactivity and the increase of mCAR binding to light-activated, phosphorylated embryonic chicken OS membranes, compared to its binding to dark, unphosphorylated membranes, suggest the possibility that mCAR is involved in shutting off the phototransduction cascade in cone photoreceptors as rod arrestin does in rod photoreceptors. However, prominent differences exist between rod arrestin and CAR, suggesting other functions for CAR.

Introduction

Members of the arrestin family are involved in G-protein coupled receptor (GPCR) desensitization, internalization, and GPCR-mediated activation of mitogen-activated protein kinase (MAPK) pathways. Rod arrestin (also called S-antigen, SAG) was the first member in the family to be molecularly characterized [1]. It is a key player in quenching the light-induced phototransduction cascade in rod photoreceptors by binding to light-activated, phosphorylated rhodopsin [2,3]. Lack of rod arrestin expression as a result of gene targeting knockout technology leads to prolonged photoresponses and increased susceptibility to light damage in rod photoreceptors [4,5]. Recent studies show that rod arrestin also participates in the molecular pathway for light-induced photoreceptor apoptosis in Drosophila through the formation of stable rhodopsin-arrestin complexes that are recruited to the cytoplasmic compartment through clathrin-dependent endocytosis [6-9].

β-arrestins are ubiquitously expressed and have a similar function to rod arrestin in the termination of GPCR signaling [10,11]. β-arrestins also participate in signaling to downstream effectors [12,13] because of their ability to act as adaptors to facilitate clathrin-mediated endocytosis [14,15]. Moreover, β-arrestins recruit activated tyrosine kinase c-Src into complexes with the β2 adrenergic receptor (β2AR), which is involved in the activation of MAPKs [16-18]. Recently, β-arrestin 2 has been identified as a binding partner of c-Jun amino terminal kinase 3 (JNK3) and was suggested to act as a receptor-regulated MAPK scaffold for the activation of JNK3 [19].

Cone arrestin (CAR, also known as X-arrestin or arrestin 4) is the newest member of the arrestin superfamily, and the human CAR (hCAR) gene was mapped to the X chromosome [20,21]. Numerous CAR orthologs have been cloned from other vertebrates, including killifish [22], bullfrog [23], leopard frog [23], clawed frog [24], bovine [25] and salamander [26]. Its high sequence homology to other arrestins and its cone photoreceptor localization suggest that CAR may play as important a role in the modulation of phototransduction in cones as rod arrestin does in rods; however, its actual function is still unknown. Bovine CAR failed to bind rhodopsin or heparin, predicted to be due to the difference in the structure of the C-terminal domain between cone and rod arrestins [25]. Likewise, salamander CAR, SalArr2, has a 50 fold lower affinity for rhodopsin than its rod counterpart, SalArr1 [26].

Recently, we characterized the gene structure, alternatively spliced cDNA isoforms, and the 5' regulatory region of the mouse CAR (mCAR) [27]. Although the organization and the exon/intron boundary sequences of the mCAR gene and its proximal promoter region are in good agreement with those of the hCAR gene, two transcription start sites and 5 alternatively spliced cDNA isoforms were identified in mouse [27], which is different from the hCAR gene [28]. In this study, we further characterize the tissue and developmental expression pattern of mCAR and initiate functional analysis of the mCAR isoforms using in vitro binding assays.

Methods

Animals

All animals were treated according to the guidelines established by the Institute for Laboratory Animal Research (Guide for the Care and Use of Laboratory Animals). Normal C57Bl/6J mice and mice with inherited retinal degeneration (rd/rd) were exposed to controlled illumination during postnatal development. Animals were reared on a 12 h:12 h Light/Dark (L/D) cycle (6:00 AM, lights on and 6:00 PM, lights off) and were killed midday. For the light-dependent translocation experiments, mice were dark-adapted overnight; lights were turned on, and the mice were killed at selected times.

For RNA isolation and immunoblot analysis, tissues were dissected and flash frozen on dry ice and stored at -80 °C until use. For immunohistochemistry, eyecups were immersion-fixed in 4% paraformaldehyde at 4 °C.

Northern blot analysis

Total RNA was extracted from various mouse tissues using the RNA STAT60 total RNA isolation reagent (Tel-Test Inc., Friendswood, TX) following the manufacturer's instructions. A pool of 8-10 retinas or 30 pineal glands was used for each RNA preparation. Northern blot analysis was performed as described [21], using an [α-³²P] dCTP labeled random primed mCAR cDNA fragment. The membrane was stripped and re-hybridized to a mouse SAG (mSAG) cDNA probe labeled with [α-³²P] dCTP and then stripped again and re-hybridized with a β-actin probe.

In situ hybridization

In situ hybridization was performed as described previously [21]. Briefly, either mCAR cDNA clone 12 [27] or mSAG full-length coding region in the PCR 2.1 vector was linearized. Sense and antisense ³⁵S-riboprobes labeled with [³⁵S] UTP (uridine 5'-[α-³⁵S] triphosphate, 250Ci/mmol, Amersham Corp. Piscataway, NJ) were generated with T7 and T3 RNA polymerase (Promega), respectively. Tissues were deproteinized and acetylated. After prehybridization for 2 h at 55 °C, slides were hybridized with approximately 4x10⁵ cpm/slide of cRNA probe at 55 °C overnight. Slides were washed, dehydrated, dipped in Kodak NTB-2 photographic emulsion (diluted 1:1 with water) at 40 °C, and developed at 1-, 2-, and 3-week intervals after exposure in the dark at 4 °C. The tissue sections were viewed and photographed using a Nikon light microscope (Microphot-fxa, Nikon Inc., Melville, NY). Photomicrographs were taken using the Spot Camera (Diagnostic Instruments Inc, Sterling Heights, MI). The digitized images were prepared as graphics by using Adobe Photoshop (version 5.0).

Antisera generation

Rabbit antisera against the peptide of mCAR (369-381; CEEFMQHNSQTQS; Luminaire Junior, LUMIJ) at the C-terminus of the mCAR protein was made by Zymed Laboratories Inc. (South San Francisco, CA) and affinity purified against the peptide with the SulfoLink® Kit (Pierce, Rockford, IL) as described [29].

Immunoblot analysis

Soluble proteins (50 μg) from selected mouse tissues were electrophoresed on 11.5% SDS-PAGE, transferred to Immobilon-P (Millipore Corp., Bedford, MA), incubated with LUMIJ (1:1000) and horseradish peroxidase (HRP) conjugated anti-rabbit secondary antibody and visualized by an Enhanced Chemiluminescence (ECL) Kit (Amersham, Arlington Heights, IL) [30].

For the retinal developmental study, 6 to 18 mouse retinas from each odd postnatal day (P) from P1 to P17 and adult (at least three months old) were homogenized in 600 μl of 50 mM potassium phosphate buffer, pH 6.8 with protease inhibitors. The retinal homogenates were centrifuged at 13,000x g for 10 min. The supernatants were each assayed for total protein (BioRad) and an equal amount of protein for each postnatal age was applied to SDS-PAGE gels and transferred to membranes prior to analysis of mCAR and mSAG by immunoblot analysis.

Immunohistochemistry

We followed the protocol for immunohistochemistry published elsewhere [29] with minor modifications. Briefly, frozen sections were heated in 0.01% sodium citrate buffer (pH 6.0) in a boiling water bath for 20 min in an antigen retrieval step, then incubated in blocking buffer (3% bovine serum albumin, 5% normal goat serum, and 0.2% Triton X-100 in PBS) for 30 min, and then with LUMIJ (1:1,000) and C10C10 (anti-rod arrestin monoclonal antibody, kindly provided by Dr. Larry A. Donoso (Wills Eye Hospital, Philadelphia, PA); 1:10,000) overnight at 4 °C. Following the washing step, sections were reacted with Texas Red-conjugated anti-rabbit IgG (1:100; Vector Laboratories, Inc., Burlingame, CA) and Fluorecin-conjugated anti-mouse IgG (1:100) for 1 h at room temperature. After washing, the slides were mounted and photographed with a laser scanning confocal microscope equipped with multiphoton technology (Zeiss LSM-510, Carl Zeiss, Inc., Thornwood, NY).

Phosphorylation of chicken opsins by recombinant GRK1 and GRK7

Chicken outer segments (OS), including both rod and cone outer segments, were isolated from embryonic day 19 (E19) chicken retinas as described [31] with a few modifications. Briefly, E19 chicken eyes were dark-adapted for 2 h, and the retinas were dissected under infrared light and shaken in 40% sucrose buffer to float the OS, which were then diluted and pelleted.

GRK1 and GRK7 were expressed in COS-7 cells by transient transfection, and cell lysates, which were used as the source of kinase, were prepared as described [32,33]. Native bovine GRK (bGRK) was extracted from crude bovine rod outer segments (ROS) as previously described [34].

The OS membranes were phosphorylated in the dark or light for 10 min at room temperature in the presence of 20 mM Tris HCl, pH 7.5, 6 mM MgCl₂, 2 mM EDTA, 0.5 μM okadaic acid, 2 mM ATP, and 8 μCi [γ-³²P] ATP (6000 Ci/mmol) with no exogenous GRK (control) or 5 μl of exogenous recombinant GRK1, GRK7, or native bovine GRK in 5 μl of the extract from crude bovine ROS. The kinase reaction was stopped with SDS sample buffer, and the ³²P labeled proteins were electrophoresed and detected in the dried gel with a phosphorimager.

In vitro binding assay

The mCAR isoforms [27] and mSAG in the pBluescript vector were used to make the ³⁵S-labeled proteins by in vitro transcription/translation with the T_NT T3 Coupled Reticulocyte Lysate System (Promega) and [³⁵S]methionine following the manufacturer's instructions. A centrifuge binding assay described by McDowell et al. [35] was used to test the interaction of mCAR and mSAG with chicken opsins in the OS preparation.

E19 chicken OS were phosphorylated for 30 min in the light with recombinant GRK1 using the conditions described above but with only cold ATP (phosphorylated), along with a control containing no ATP or GRK (unphosphorylated). Both samples were returned to the dark and the opsins were regenerated at room temperature with 11-cis-retinal for 1 h. ³⁵S-labeled mCAR isoforms or mSAG were added to the phosphorylated and unphosphorylated OS in the dark. The arrestins were allowed to bind to the membranes in the light or dark for 10 min at 37 °C. After centrifugation at 27,000x g for 10 min at 4 °C, the membranes were rinsed once with ice cold buffer, solubilized in SDS sample buffer, and electrophoresed next to a lane containing the total amount of arrestin added to each binding tube. The gels were dried and subjected to phosphorimager detection of ³⁵S-labeled products. The intensity of the bands was quantitated with the ImageQuant software (Molecular Dynamics), and the percent bound (bound/total) arrestin calculated. The experiment was repeated 6-10 times for each arrestin, and the data are presented as means ± SE. Statistical analysis was performed using the unpaired t test.

Results & Discussion

Analysis of mCAR transcripts

We previously characterized the gene structure, alternative splicing, and the 5'-flanking region of mCAR [27]. To further confirm its tissue distribution and mRNA expression pattern, we performed Northern blot and RT-PCR analyses. Two major retina-specific mCAR transcripts with molecular weights of about 1.5 and 2.3 kb and two minor ones of about 4.5 and 1.0 kb were identified in mouse retinas by Northern blot analysis (Figure 1, upper panel). No significant difference was seen in the intensity of the 1.0, 1.5, and 2.3 kb bands between light and dark retinas, consistent with the primer extension results described by us [27]. However, the 4.5 kb band was stronger in the light than in the dark. mSAG also showed a higher expression in light than in dark (Figure 1, middle panel), which is consistent with the results reported by us and others previously [36-38]. Equal loading of RNA between light and dark retinal samples and RNA integrity in all the samples were confirmed by hybridizing the same membrane to a β-actin cDNA radioactive labeled probe (Figure 1, lower panel). Since the full-length mCAR cDNA is 1,311 bp [27], its transcript is predicted to be the 1.5 kb band. The other alternatively spliced mRNA isoforms are all similar in size to the full-length one [27], so it was not possible to resolve them by Northern blot analysis. The two larger hybridization bands were either intermediate RNA forms with unspliced introns or different gene products with high homology to CAR. Alternatively, these bands could be the same mCAR gene products by using alternate promoters and/or polyadenylation signals.

No detectable mCAR mRNA expression was seen in the pineal gland or other tissues tested (Figure 1); however, on a separate Northern blot and with RT-PCR, the mCAR mRNA was observed in the pineal gland (data not shown). The original CAR was cloned from rat pineal gland [21], and mCAR is detectable in the mouse pineal gland at the protein level (see Figures 3 and 4, discussed below). The reason we did not detect mCAR mRNA in the pineal gland on this Northern blot is probably because the mouse pineal gland is 4 fold smaller than rat, and the pineal glands used for RNA isolation contained surrounding tissues, so that the mCAR mRNA was diluted and was not at a level high enough to be detected by Northern blot analysis. In situ hybridization revealed that mCAR mRNA was localized in the inner segment of retinal cone photoreceptors (Figure 2A,B), in a pattern similar to that observed in the rat retina [21]. The transcripts of mSAG, however, were diffused throughout the whole IS layer of the retina (Figure 2C,D).

mCAR protein analysis

The open reading frame of the mCAR full-length cDNA, mCARFL (GenBank accession number AF156979) [27], encodes a predicted protein of 381 amino acid residues with an estimated molecular weight of 41.921 kDa and an isoelectric point of 5.99 compared to its human orthologue, which has an estimated molecular weight of 42.519 kDa and an isoelectric point of 5.71. Amino acid alignment indicates the mCAR protein shares 82%, 78%, 65%, 60%, 61%, 59% and 55% homology with human, bovine, salamander, bull frog, leopard frog, clawed frog and killifish CAR sequences (Genbank accession numbers U03626, D85340, AF203328, X92401, X92400, L40463, and AB002555), respectively (data not shown). The reported structural domains for arrestins and CARs [24] were found in mCAR. The shared epitope for the arrestin antibody, mAb 5C6.47 [21] was also identified, in agreement with previous reports regarding hCAR [20,21].

The homology of CAR among different species exists throughout the mCAR sequence except for the C-terminus, which is the region noted with the highest divergence among arrestins [20,21,24]. It is interesting that the C-terminus of the mCAR protein from the conserved arrestin signature domain to the end of the C-terminus is 91.3% identical to that of rat CAR (GenBank accession number U03628; data not shown) but highly divergent from that of the other species, suggesting that rodent's CAR may have additional functions.

A polyclonal antibody (LUMIJ) against the C-terminal mCAR specific sequence in all the isoforms except clone 12 [27] was prepared, and its specificity was confirmed by immunoblot analysis using a 6xHis-tagged mCARFL recombinant protein in crude E. coli extract (data not shown). Immunoblot analysis of mCAR protein expression in selected mouse tissues revealed a single 44 kDa protein band in adult mouse retina and pineal, which was not detected in any other tissues examined, confirming the specificity of the antibody (Figure 3). On separate immunoblots, a 43 kDa band that is recognized by LUMIJ is also detected at much lower levels in both retina and pineal in adult mouse (Figure 4C). Protein bands at different molecular weights were detected in other tissues (Figure 3). These were non-specific cross-reaction of the antibody because Northern blot and RT-PCR analysis did not detect mCAR expression in these tissues.

Analysis of mCAR protein expression in the developing mouse retina and pineal gland

Retinal extracts from both normal C57 mice and retinal degeneration (rd/rd) mice, which have defects in the β-subunit of the rod cGMP-phosphodiesterase gene [39,40], at age P1 to P17 and adult (Ad) were analyzed by immunoblot analysis with LUMIJ, the rod arrestin monoclonal antibody C10C10 and a polyclonal antibody against creatine kinase (mCK), Pab1948, sequentially. As shown in Figure 4A, a 43 kDa band that is recognized by LUMIJ was present from P1 at low levels and remained at a constant level throughout the various ages we examined, while the 44 kDa mCAR band did not appear until P9 but increased with age from then until adult in the C57 mouse retina. In the rd/rd mouse retina (Figure 4B), both the 43 and 44 kDa bands showed the same expression patterns as in the C57 mouse from P1 to P17; however, both bands disappeared in the adult rd/rd mouse retina. MSAG is not detected until P5 but its expression levels increased more rapidly with age in both normal and rd/rd mouse retinas (Figure 4A,B). It reached its peak level at P9 and remained constant until adult in normal C57 mice. In the rd/rd mouse retina, mSAG reached its peak level also at P9 but decreased from P11 and disappeared in adult. These results are consistent with the published observations that rod degeneration begins on about P10, with most of the cells lost by P21 and almost none surviving by P36 [41], and that the majority of cones are still present at P21, but most of these are lost by 2-4 months of age, with a few surviving for the lifetime of the animal [41]. Creatine kinase (mCK), which is expressed throughout the mouse retina from early embryonic developmental stages until adult at constant levels, was detected to show the loading variation among samples (Figure 4A,B).

The expression pattern of mCAR in adult and P5 mouse retina was different from that in the pineal gland (Figure 4C). In retina, the 44 kDa band, supposedly the mCARFL, was not detectable at P5 but was the major form in adult. In contrast, in the pineal gland, the 44 kDa protein is much higher at P5 than in adult. The 43 kDa protein recognized by LUMIJ is present at both P5 and adult at low levels in both tissues, consistent with the observation from our retinal developmental study (Figure 4A). This 43 kDa protein may be one of the alternatively spliced forms of mCAR, possibly mCARΔE14 [27], estimated from the molecular weight.

Light-dependent translocation of mCAR in cone photoreceptors of the mouse retina

Examination of adult mouse retina by immunohistochemistry with LUMIJ demonstrated that mCAR localized specifically in cone photoreceptors (Figure 5), consistent with the localization of hCAR [42-44]. In dark-adapted mice, mCAR immunostaining was defuse throughout the cone photoreceptors from the synaptic terminals to the cone OS (COS), with the most intense staining in the synapses in the outer plexiform layer (OPL) and similar staining intensity in the cone IS (CIS) and COS (Figure 5 and Figure 6). Rod arrestin localized mainly to the IS and the outer nuclear layer in the dark, with very weak staining in the OS and almost no staining in the synaptic terminals (Figure 5). After light exposure for 30 s, both rod arrestin and CAR have started moving to the OS because the botton part of the OS has stronger staining than the IS. After 5 and 15 min light exposure, the immunostaining of both rod arrestin and CAR moved further toward the tip of the OS. After light adaptation for 4 h, the staining concentrated in the OS, with weak staining in the IS and the cell body (perinuclear region and axon). Interestingly, there was still strong mCAR staining in the synaptic terminals in the light-adpted retina (Figure 5). The different distribution of mCAR in the COS and CIS between light and dark was more clearly demonstrated in Figure 6, when the images are enlarged. These results suggest a light-dependent translocation of mCAR immunoreactivity to the OS of the cone photoreceptors, similar to but not as dramatic as rod arrestin.

The dramatic light-dependent movement of immunoreactivity to the OS has been observed for rod arrestin in normal mouse and rat retinas [37,38,45-48], and recently in Xenopus laevis retinas [48]. Our data suggest that CAR may have a similar function to rod arrestin in the interaction with light-activated, phosphorylated opsins and may be involved in the termination of the phototransduction cascade in cone photoreceptors. However, intriguing differences exist between cone and rod arrestins, including a higher sequence homology of CAR to β-arrestins, which clearly have other regulatory functions [12-19], in addition to down regulating activated, phosphorylated GPCRs [10,11], and the fact that mCAR has multiple splice variants. Moreover, the light-dependent translocation of mCAR is not as dramatic as rod arrestin, as shown above. The intense staining of mCAR in the synapses of the cone photoreceptors in the dark-adapted retinas may imply functional involvement of mCAR in synaptic transmission. We have identified a potential mCAR functional partner, which is expressed in the retinal photoreceptors only in the dark, from a mouse dark retinal library through a yeast two-hybrid screen with mCAR as bait (unpublished data).

One explanation for the incomplete traslocation of mCAR to the COS following light adaptation is that, under the room lighting conditions, rods are saturated, and most of rod arrestin is needed in the OS to shut off the rod transduction cascade, which may account for the near compete translocation of rod arrestin. However, under the same lighting conditions, cones are not saturated, so only part of the CAR protein is needed in the COS to shut off the cone phototransduction cascade.

Binding of mCAR to light-activated, phosphorylated chicken cone opsins

Arrestins quench GPCR-mediated signal transduction by binding to and blocking the catalytic activity of photo (or ligand)-activated and then GRK-phosphorylated membrane receptors. To explore whether the mCAR isoforms may have analogous functions in cone photoreceptors, we compared mCAR isoforms and mSAG in a direct binding assay using in vitro translated arrestin proteins.

It is very difficult to get large quantities of functional cone opsins to do similar binding assays as rhodopsin. We chose embryonic chicken OS as the source of opsins for the binding assay because the chicken retina is cone-rich, and it is relatively easy to obtain hundreds of embryonic chicken retinas. In addition, the temporal expression pattern of each chicken opsin mRNA during embryonic development has been reported [49]. The long wavelength cone opsins, red and green, were first detected at embryonic day 14 (E14), and rhodopsin was first seen at E15, while the short wavelength cone opsins, blue and violet, were not detected until E16. By E19, all opsins are present in abundance in the embryonic chicken retina.

Cone opsin phosphorylation has been demonstrated in the all-cone retina of lizard [50], by in vitro phosphorylation of chicken iodopsin (red opsin) by bovine rhodopsin kinase [51] and recently by in vitro phosphorylation of membranes from purified cone cells of the carp retina by its endogenous GRK [52]. In our preliminary study with the rodless neural retina leucine zipper (Nrl) knockout mice, we have demonstrated light-dependent phosphorylation of both S and M opsins (unpublished data). We used recombinant GRK1 and GRK7 and purified bGRK as the source of kinase to phosphorylate chicken OS membranes and observed light-dependent phosphorylation of chicken opsins by both recombinant GRK1 and GRK7 as well as bGRK, as reflected by light-dependent phosphorylation bands of 35-40 kDa (Figure 7).

We could easily express all the mCAR isoforms by in vitro transcription/translation except mCARΔE4 and mCARΔE13&14. The failure to express mCARΔE4 and mCARΔE13&14 in vitro was probably due to the improper folding and thus degradation of the polypeptides in the in vitro transcription/translation system, as suggested for salamander CAR (SalArr2) [26]. The mCARFL (Figure 8A) selectively bound to light-activated, phosphorylated chicken opsins (L+) compared to the dark, unphosphorylated membranes (D-, P<0.01). This binding selectivity of mCARFL was similar to that of mSAG (Figure 8D), which also showed light- and phosphorylation-dependent binding (L+ versus D-, P<0.05). Previous studies with salamander [26] and bovine CAR [25] showed either low affinity or no binding of CAR to rhodopsin, so the binding we observe is not likely between mCAR and chicken rhodopsin.

The strict binding selectivity of SAG toward phosphorylated light activated rhodopsin has been described, and a multistep model of arrestin interaction with rhodopsin has been proposed [53,54]. Recent evidence suggests that the basal conformation of arrestin and the activating mechanisms triggering arrestin transition into its high affinity receptor binding state are conserved between visual and non-visual arrestins [55]. The data presented here provide the first evidence that CAR may have a similar function to rod arrestin in the interaction with GPCRs and may be involved in the down regulation of high acuity color vision mediated by the cone photoreceptor phototransduction cascade. In contrast, the two mCAR isoforms, mCARΔE14 (Figure 8B) and mCAR clone12 (Figure 8C), do not show either light- or phosphorylation-dependent binding, suggesting different functional roles for these other isoforms.

Our in vitro binding data are consistent with the light-dependent translocation data shown by immunohistochemistry (Figure 5 and Figure 6). With immunohistochemistry (Figure 5 and Figure 6), we see obvious staining in the COS in the dark-adapted retina, and with the binding experiment, we see a high percentage (21%) binding of mCARFL to the dark, unphosphorylated membranes (Figure 8A). Also, the incomplete translocation of mCAR agrees with the small increase of mCAR binding to light-activated, phosphorylated OS membranes, compared to its binding to the dark, unphosphorylated membranes. These differences between rod and cone arrestins may reflect the intrinsic mechanistic differences between rod and cone phototransduction. Alternatively, they may suggest that CAR has other functions. The striking amount of mCAR staining in the synaptic terminals (Figure 5B), and the lack of detectable cone opsins in the adult pineal gland also suggest additional functions for CAR other than down regulating the cone opsins in the phototransduction cascade. This implies that CAR may have multiple functions in regulating other GPCR signaling pathways in cone photoreceptors.

Little is known about other G-protein signaling pathways and their functions in cone photoreceptors, but it has been suggested that many GPCRs function in photoreceptors, including receptors for dopamine, serotonin, adenosine and glutamate [56-59]. Based on the gene structure [27], we have created an mCAR gene knockout construct and are developing mCAR knockout mice. We have also characterized the mCAR 5'-flanking region that is sufficient to drive cone-specific expression in transgenic Xenopus laevis [27]. Knockout of the CAR gene and subsequent knock-in of the various mCAR isoforms will address their functional significance in the retina and the pineal gland.

Acknowledgements

This work is dedicated to Mary D. Allen for her continued generous financial support of vision research, and to the memory of our lifetime collaborator, Dr. Richard N. Lolley. The authors thank Dr. Larry A. Donoso for providing the arrestin monoclonal antibody C10C10. These studies were supported, in part, from grants EY00395 (CMC and RN Lolley), EY12224 (SO), GM43582 (ERW), EY03042 Core Vision Research Center grant (Doheny Eye Institute) and L. K. Whittier Foundation (CMC). We would also like to thank both the Tony Gray Foundation and Fred Dorie Miller for generous support of postdoctoral fellows and the Neurogenetic Analysis Core (Hans-Jürgen Fülle), created with the Howard Hughes Medical Institute Resources Grant (CMC). CMC is the Mary D. Allen Professor for Vision Research, Doheny Eye Institute.

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