\set{final}

\def\Author{Zhu}
\def\author{zhu}
\def\year{2002}
\def\vol{8}
\def\anum{56}
\def\pages{462-471}
\def\txt_title{Mouse cone arrestin expression pattern: Light induced translocation in cone photoreceptors}
\def\txt_authors{Xuemei Zhu, Aimin Li, Bruce Brown, Ellen R. Weiss, Shoji Osawa, Cheryl M. Craft}

\def\rcvd{19 June 2002}
\def\accept{10 December 2002}
\def\publ{11 December 2002}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

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\| Internal defs


\article{

\title{Mouse cone arrestin expression pattern: Light induced
translocation in cone photoreceptors}

\authors{\mailto{xuemeizh@usc.edu}{Xuemei Zhu},\sup{1}
\mailto{aiminli@usc.edu}{Aimin Li},\sup{1} \mailto{bbrown@usc.edu}{Bruce
Brown},\sup{1} \mailto{erweiss@med.unc.edu}{Ellen R. Weiss},\sup{2}
\mailto{shoosawa@med.unc.edu}{Shoji Osawa},\sup{2}
\mailto{ccraft@usc.edu}{Cheryl M. Craft}\sup{1}}

\institutions{\sup{1}The 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; \sup{2}Department of Cell and Developmental Biology, The
University of North Carolina at Chapel Hill, Chapel Hill, NC}

\correspondence{Cheryl M. Craft, Ph.D., Department of Cell \and\
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

\abs_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.}

\abs_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 \i{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.}

\abs_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 \i{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.}

\abs_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

\p{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 \i{Drosophila} through the formation of stable
rhodopsin-arrestin complexes that are recruited to the cytoplasmic
compartment through clathrin-dependent endocytosis [6-9].}

\p{\beta-arrestins are ubiquitously expressed and have a similar
function to rod arrestin in the termination of GPCR signaling [10,11].
\beta-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, \beta-arrestins recruit
activated tyrosine kinase c-Src into complexes with the \beta 2
adrenergic receptor (\beta 2AR), which is involved in the activation of
MAPKs [16-18]. Recently, \beta-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].}

\p{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].}

\p{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

\subsection{Animals}

\p{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 (\i{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.}

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

\subsection{Northern blot analysis}

\p{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 [\alpha-\sup{32}P] dCTP
labeled random primed mCAR cDNA fragment. The membrane was stripped and
re-hybridized to a mouse SAG (mSAG) cDNA probe labeled with
[\alpha-\sup{32}P] dCTP and then stripped again and re-hybridized with a
\beta-actin probe.}

\subsection{In situ hybridization}

\p{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
\sup{35}S-riboprobes labeled with [\sup{35}S] UTP (uridine
5'-[\alpha-\sup{35}S] 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 \deg C, slides were hybridized with
approximately 4x10\sup{5} cpm/slide of cRNA probe at 55 \deg C
overnight. Slides were washed, dehydrated, dipped in Kodak NTB-2
photographic emulsion (diluted 1:1 with water) at 40 \deg C, and
developed at 1-, 2-, and 3-week intervals after exposure in the dark at
4 \deg 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).}

\subsection{Antisera generation}

\p{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\reg\ Kit (Pierce,
Rockford, IL) as described [29].}

\subsection{Immunoblot analysis}

\p{Soluble proteins (50 \mu g) 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].}

\p{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 \mu l 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.}

\subsection{Immunohistochemistry}

\p{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 \deg 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).}

\subsection{Phosphorylation of chicken opsins by recombinant GRK1 and GRK7}

\p{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.}

\p{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].}

\p{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
MgCl\sub{2}, 2 mM EDTA, 0.5 \mu M okadaic acid, 2 mM ATP, and 8 \mu Ci
[\gamma-\sup{32}P] ATP (6000 Ci/mmol) with no exogenous GRK (control) or
5 \mu l of exogenous recombinant GRK1, GRK7, or native bovine GRK in 5
\mu l of the extract from crude bovine ROS. The kinase reaction was
stopped with SDS sample buffer, and the \sup{32}P labeled proteins were
electrophoresed and detected in the dried gel with a phosphorimager.}

\subsection{In vitro binding assay}

\p{The mCAR isoforms [27] and mSAG in the pBluescript vector were used
to make the \sup{35}S-labeled proteins by in vitro
transcription/translation with the T\sub{N}T T3 Coupled Reticulocyte
Lysate System (Promega) and [\sup{35}S]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.}

\p{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.
\sup{35}S-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 \deg C. After
centrifugation at 27,000x g for 10 min at 4 \deg 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 \sup{35}S-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 \pom\ SE. Statistical analysis was performed using
the unpaired t test.}

\resultsdisc

\subsection{Analysis of mCAR transcripts}

\p{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 (\figref{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
(\figref{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 \beta-actin
cDNA radioactive labeled probe (\figref{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.}

\p{No detectable mCAR mRNA expression was seen in the pineal gland or
other tissues tested (\figref{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
(\figref{2}{A,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 (\figref{2}{C,D}).}

\subsection{mCAR protein analysis}

\p{The open reading frame of the mCAR full-length cDNA, mCARFL (GenBank
accession number \genbankdna{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 \genbankdna{U03626},
\genbankdna{D85340}, \genbankdna{AF203328}, \genbankdna{X92401},
\genbankdna{X92400}, \genbankdna{L40463}, and \genbankdna{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].}

\p{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 \genbankdna{U03628}; data not
shown) but highly divergent from that of the other species, suggesting
that rodent's CAR may have additional functions.}

\p{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 \i{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 (\figref{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 (\figref{4}{C}). Protein
bands at different molecular weights were detected in other tissues
(\figref{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.}

\subsection{Analysis of mCAR protein expression in the developing mouse
retina and pineal gland}

\p{Retinal extracts from both normal C57 mice and retinal degeneration
(\i{rd/rd}) mice, which have defects in the \beta-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 \figref{4}{A}, 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 \i{rd/rd}
mouse retina (\figref{4}{B}), 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 \i{rd/rd} mouse retina. MSAG is not
detected until P5 but its expression levels increased more rapidly with
age in both normal and \i{rd/rd} mouse retinas (\figref{4}{A,B}). It
reached its peak level at P9 and remained constant until adult in normal
C57 mice. In the \i{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 (\figref{4}{A,B}).}

\p{The expression pattern of mCAR in adult and P5 mouse retina was
different from that in the pineal gland (\figref{4}{C}). 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 (\figref{4}{A}). This 43 kDa protein may be one of
the alternatively spliced forms of mCAR, possibly mCAR\Delta E14 [27],
estimated from the molecular weight.}

\subsection{Light-dependent translocation of mCAR in cone photoreceptors
of the mouse retina}

\p{Examination of adult mouse retina by immunohistochemistry with LUMIJ
demonstrated that mCAR localized specifically in cone photoreceptors
(\figref{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
(\figref{5} and \figref{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 (\figref{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 (\figref{5}). The different
distribution of mCAR in the COS and CIS between light and dark was more
clearly demonstrated in \figref{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.}

\p{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 \i{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
\beta-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).}

\p{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.}

\subsection{Binding of mCAR to light-activated, phosphorylated chicken
cone opsins}

\p{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.}

\p{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.}

\p{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 (\figref{7}).}

\p{We could easily express all the mCAR isoforms by in vitro
transcription/translation except mCAR\Delta E4 and mCAR\Delta E13\and
14. The failure to express mCAR\Delta E4 and mCAR\Delta E13\and 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 (\figref{8}{A})
selectively bound to light-activated, phosphorylated chicken opsins (L+)
compared to the dark, unphosphorylated membranes (D-, P\lt 0.01). This
binding selectivity of mCARFL was similar to that of mSAG
(\figref{8}{D}), which also showed light- and phosphorylation-dependent
binding (L+ versus D-, P\lt 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.}

\p{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,
mCAR\Delta E14 (\figref{8}{B}) and mCAR clone12 (\figref{8}{C}), do not
show either light- or phosphorylation-dependent binding, suggesting
different functional roles for these other isoforms.}

\p{Our in vitro binding data are consistent with the light-dependent
translocation data shown by immunohistochemistry (\figref{5} and
\figref{6}). With immunohistochemistry (\figref{5} and \figref{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 (\figref{8}{A}). 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 (\figref{5}{B}), 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.}

\p{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 \i{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

\p{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-J\uuml rgen F\uuml lle), 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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}

\beginfigures

\figfile{1}{
\figtitle{1}{Northern blot analysis of mCAR mRNA in mouse tissues}

\p{Total RNA (20 \mu g) from various mouse tissues was resolved on a 1%
denaturing agarose gel, transferred to a nylon membrane and hybridized
to a mCAR cDNA radioactive labeled probe. The membrane was striped and
re-hybridized to a mouse rod arrestin (mSAG) cDNA radioactive probe, and
striped again and rehybridized to a mouse \beta-actin probe, as
described in the procedures. Sizes of the hybridization bands are
indicated. Light retina, retinas from mouse killed at midday under room
light; Dark retina, retinas from mouse killed at midnight under infrared
light.}

\ctr{\jpgimage{1}{372}{518}{21}}

}

\figfile{2}{
\figtitle{2}{In situ hybridization of mouse retina}

\p{In situ hybridization of mouse retina using either the mCAR or mSAG
cRNA probes. (\panel{A}) mCAR antisense cRNA probe. The probe labels a
population of cells consistent with the location, appearance, and
distribution of cone photoreceptors (arrows). (\panel{B}) mCAR sense
cRNA probe. No specific hybridization signals. (\panel{C}) mSAG
antisense cRNA probe. The probe labels rod inner segment and outer
nuclear layers. (\panel{D}) mSAG sense cRNA probe. Note the lack of
hybridization signal over the photoreceptors. OS, outer segments; IS,
inner segments; ONL, outer nuclear layer; INL, inner nuclear layer. Bar
represents 10 \mu m.}

\ctr{\jpgimage{2}{834}{896}{161}}

}

\figfile{3}{
\figtitle{3}{Tissue distribution of the mCAR protein}

\p{Soluble proteins (50 \mu g) from various mouse tissues were subjected
to immunoblot analysis with LUMIJ and anti-rabbit secondary antibody
using an ECL kit. The molecular markers are indicated on the left.}

\ctr{\jpgimage{3}{767}{534}{36}}

}

\figfile{4}{
\figtitle{4}{Developmental Study of mCAR expression}

\p{C57 (\panel{A}) and \i{rd/rd} mice (\panel{B}) were killed at P1, 3,
5, etc. and adult (Ad), and retinas were dissected. Fifty micrograms of
soluble retinal proteins were applied to an 11.5% SDS-PAGE and processed
for immunoblot analysis with primary antibodies LUMIJ for mCAR, C10C10
for rod arrestin (mSAG), and polyclonal antibody 1948 for creatine
kinase (mCK) sequentially. Molecular weights of proteins are indicated
on the right. (\panel{C}) The expression pattern of mCAR in normal adult
and P5 mouse retina and the pineal gland. Soluble proteins (50 \mu g)
from the retina and the pineal gland of either adult (Ad) or P5 C57 mice
were subjected to immunoblot analysis with LUMIJ as described in
(\panel{A}).}

\ctr{\jpgimage{4}{606}{470}{44}}

}

\figfile{5}{
\figtitle{5}{Immunofluorescent staining of adult mouse retinal sections
with LUMIJ and C10C10}

\p{Adult C57Bl/6J mice were dark-adapted overnight (dark) and exposed to
light for selected times before being killed, and the eyecups were
immediately dissected and immersion-fixed in 4% paraformaldehyde. Frozen
eyes were sectioned at 7 \mu m through the optic nerve. The sections
were heated for 20 min in pre-heated 0.01% sodium citrate buffer (pH
6.0). After blocking, the sections were incubated with the anti-mouse
cone arrestin polyclonal antibody LUMIJ (1:1,000) and the anti-rod
arrestin monoclonal antibody C10C10 (1:10,000) and then with Texas
Red-conjugated anti-rabbit IgG (1:100) and Fluorescin-conjugated
anti-mouse IgG. After washing, the sections were examined and
photographed as described in methods. OS, outer segments; IS, inner
segments; ONL, outer nuclear layer; INL, inner nuclear layer. Bar
represents 20 \mu m.}

\ctr{\multijpgimage{5}{346}{662}{81}}

}

\figfile{6}{
\figtitle{6}{Immunofluorescent staining of adult mouse retinal sections
with LUMIJ}

\p{Adult C57Bl/6J mice were dark-adapted overnight (dark, \panel{A} and
\panel{B}) and exposed to light for 4 h (light, \panel{C} and \panel{D})
before being killed, and the eyecups were immediately dissected and
fixed in 4% paraformaldehyde. Frozen sections were treated as in
\figref{5} and stained with the anti-mouse cone arrestin polyclonal
antibody LUMIJ (1:1,000) and with Texas Red-conjugated anti-rabbit IgG
(1:100). COS, cone outer segments; CIS, cone inner segments. Bar
represents 20 \mu m. Note that mCAR immunoreactivity is evenly
distributed throughout the whole cell body in the dark-adapted mouse
retina (\panel{A} and \panel{B}) but is more intense in the COS in the
light-adapted mouse retina (\panel{C} and \panel{D}).}

\ctr{\multijpgimage{6}{787}{607}{75}}

}

\figfile{7}{
\figtitle{7}{Phosphorylation of chicken OS membranes by GRKs}

\p{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. The OS were phosphorylated
in the dark (D) or light (L) for 10 min at room temperature in the
presence of 8 \mu Ci [\gamma-\sup{32}P] ATP (6000 Ci/mmol) with no
exogenous GRK (control) or 5 \mu l of exogenous recombinant GRK1, GRK7,
or native bovine GRK (bGRK). The kinase reaction was stopped with SDS
sample buffer, and the \sup{32}P labeled proteins were electrophoresed
and detected in the dried gel with a phosphoimager. Molecular weight
markers are shown on the left.}

\ctr{\jpgimage{7}{363}{279}{27}}

}

\figfile{8}{
\figtitle{8}{Phosphorylation- and light-dependent binding of mCARFL to
chicken OS membranes}

\p{E19 chicken OS were phosphorylated for 30 min in the light with
recombinant GRK1 and cold ATP (phosphorylated), along with a control
containing no ATP or GRK (unphosphorylated). After regeneration of the
opsins, \sup{35}S-labeled mCARFL (\panel{A}), mCAR\Delta E14
(\panel{B}), mCAR clone 12 (\panel{C}) and mSAG (\panel{D}), were added
to the phosphorylated and unphosphorylated OS in the dark and allowed to
bind to the membranes in the light or dark for 10 min at 37 \deg C.
After washing, the membranes were solubilized in SDS sample buffer, and
electrophoresed next to a lane containing the total amount of arrestin
added to each binding reaction. The gels were dried and subjected to
phosphorimager detection of \sup{35}S products. D-, dark,
unphosphorylated; D+, dark, phosphorylated; L-, light, unphosphorylated;
L+, light, phosphorylated. p\lt 0.05 (L+ versus D-); p\lt 0.01 (L+
versus D-).}

\ctr{\gifimage{8}{428}{506}{27}}

}