From the Department of Life Science, Himeji Institute of Technology, Harima Science Garden City, Akoh-gun, Hyogo 678-1279, Japan
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ABSTRACT |
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G protein-coupled receptor kinases (GRKs) play an
important role in stimulus-dependent receptor
phosphorylation and desensitization of the receptors. Mammalian
rhodopsin kinase (RK) and -adrenergic receptor kinase (
ARK) are
the most studied members among known GRKs. In this work, we purified RK
from octopus photoreceptors for the first time from invertebrate
tissues. The molecular mass of the purified enzyme was 80 kDa as
estimated by SDS-polyacrylamide gel electrophoresis, and this was 17 kDa larger than that of the vertebrate enzymes. Unlike vertebrate RK,
octopus RK (ORK) was directly activated by
-subunits of a
photoreceptor G protein. We examined the effects of various known
activators and inhibitors of GRKs on the activity of the purified ORK
and found that their effects were different from those on either bovine
RK or
ARK. To analyze the primary structure of the enzyme, we cloned
the cDNA encoding ORK from an octopus retinal cDNA library. The
deduced amino acid sequence of the cDNA was highly homologous to
ARK over the entire molecule, including a pleckstrin homology domain located in the C-terminal region, and homology to RK was significantly lower. Furthermore, Western blot analysis of various octopus tissues with an antibody against the purified ORK showed that ORK is expressed solely in the retina, which confirmed the identity of the enzyme as
rhodopsin kinase. Thus, ORK appears to represent a unique subgroup in
the GRK family, which is distinguished from vertebrate RK.
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INTRODUCTION |
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Many G protein-coupled receptors such as rhodopsin and
-adrenergic receptors are known to be phosphorylated in a light- or agonist-dependent manner by a member of the specific
protein kinase family called G protein-coupled receptor kinases
(GRKs).1 This
stimulus-dependent phosphorylation of the receptors is
thought to be involved in the desensitization of these receptors (for reviews, see Refs. 1 and 2). The kinases responsible for phosphorylating the activated forms of rhodopsin and
-adrenergic receptors are rhodopsin kinase (RK or GRK1) (3) and
-adrenergic receptor kinases (
ARK1/2 or GRK2/3) (4), respectively. Both of these
kinases have been purified to homogeneity, and their specificities and
activities have been examined in reconstituted systems (5, 6). In
addition to RK (7) and two kinds of
ARK (8, 9), at least three other
members of the GRK family (GRK4-6) have been cloned in mammals
(10-12), and several related genes have also been cloned from other
organisms such as Drosophila (13) and Caenorhabditis
elegans (14).
It has been demonstrated that ARK is capable of phosphorylating
rhodopsin in a totally light-dependent fashion and that RK can phosphorylate the agonist-occupied
-adrenergic receptors (15).
Both kinases are insensitive to cyclic nucleotides and Ca2+
and inhibited by 0.1 M NaCl, 1 mM
ZnCl2, detergents, and polyanions (16, 17). In addition to
these similarities, some differences are present in the characteristics
of RK and
ARK. Polycations activate RK (16), but not
ARK (17).
The sequence similarity between the two kinases is not high, and the
differences are most evident in the C-terminal regions (7). RK lacks
~120 C-terminal residues present in the corresponding region of
ARK. This region in
ARK is referred to as the pleckstrin homology
(PH) domain and has been identified as the site where the enzyme
interacts with G protein
-subunits and plasma membranes upon
phosphorylation of substrate receptors (18-20). In contrast with the
case of
ARK, which interacts with and is activated by G
(21,
22), phosphorylation of receptors catalyzed by RK is not affected by
G
(23). This is reasonable since RK does not possess a PH domain.
Instead of a PH domain, RK has a C terminus sequence unique within the
GRK family, the motif termed CAAX boxes (where C is
cysteine, A is an aliphatic residue, and X is any
amino acid). CAAX boxes are one of the known C-terminal
isoprenylation motifs, and bovine RK is farnesylated at its C-terminal
cysteine (24). Isoprenylation has been reported to be essential for the
expression of full enzymatic activity of RK (25). It is required for
light-induced translocation of the enzyme to the disc membranes (26)
and therefore seems to play a very important role in the physiological
action of RK. Thus,
ARK and RK, as far as the present understanding,
represent different subgroups in the GRK family in terms of both their
structure and regulatory mechanisms.
We have previously shown that octopus rhodopsin is phosphorylated in a
light-dependent manner and that light-induced
phosphorylation of octopus rhodopsin in the microvillar membranes is
markedly enhanced by GTPS (27), which suggests that octopus
rhodopsin kinase (ORK), like
ARK, could be activated by
-subunits of G protein in contrast with bovine RK. Since light
depolarizes invertebrate photoreceptor cells, whereas it hyperpolarizes
vertebrate rod and cone photoreceptor cells, the underlying
phototransduction machinery, including that for desensitization, in
invertebrate photoreceptor cells could be quite distinct from that
operating in vertebrate photoreceptors (28). In an effort to explore
the molecular and enzymatic properties of ORK, we purified the enzyme to apparent homogeneity for the first time as an invertebrate enzyme
and cloned the cDNA encoding it. Here we report the very unique
characteristics of ORK, which differ from its vertebrate counterpart
and are closer to those of
ARK.
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EXPERIMENTAL PROCEDURES |
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Materials--
Mono Q 5/5, concanavalin A-Sepharose,
CNBr-activated Sepharose 4B, the Thermo Sequenase fluorescent labeled
primer cycle sequencing kit, and the ECL chemiluminescence detection
system were purchased from Amersham Pharmacia Biotech.
Sulfate-Cellulofine was from Seikagaku Kogyo Inc. (Tokyo).
Extractigel-D was from Pierce. [-32P]ATP was from NEN
Life Science Products. Achromobacter protease I was from
Wako Pure Chemical Industries (Osaka, Japan). Taq polymerase and restriction enzymes were from TaKaRa (Otsu, Japan). Lambda ZAP II
vector was from Stratagene. pT7Blue(R) vector was from Novagen. Other
reagents used were the highest grade commercially available.
Purification of ORK from Octopus Photoreceptor Microvillar
Membranes--
Microvillar membranes of octopus photoreceptors were
prepared from eyes of Octopus dofreini as described
previously (27). Microvillar membranes were isolated by sucrose
flotation (repeated twice) from the retinal homogenate. The isolated
microvillar membranes were washed three times with 10 mM
Tris-HCl (pH 7.4), 0.4 M KCl, 10 mM
MgCl2, 1 mM dithiothreitol, and 20 µM APMSF (isotonic buffer). Rhodopsin kinase activity was
extracted by freezing and thawing the washed microvillar membranes. The
membranes were frozen in liquid nitrogen and then thawed by
homogenizing the frozen pellet in 10 mM Tris-HCl (pH 7.4),
0.4 M KCl, 1 mM dithiothreitol, and 20 µM APMSF. The extracted proteins were separated from the
membranes by centrifugation at 42,000 × g for 20 min
after each freeze-thaw cycle, and five portions of successive extract
were pooled together. The extract was diluted to 0.2 M NaCl
and applied to a sulfate-Cellulofine column (2 × 3.5 cm)
equilibrated with 20 mM Tris-HCl (pH 7.4), 1 mM
dithiothreitol, 20 µM APMSF, and 0.2 M NaCl
at a flow rate of 0.6 ml/min. After the column was thoroughly washed
with the equilibration buffer, the proteins were eluted with a linear
gradient of 0.2-1.0 M NaCl in 60 ml and collected in
fractions of 3 ml. The fractions were assayed for rhodopsin kinase
activity with rhodopsin-containing phospholipid vesicles as a
substrate. ORK was eluted at ~0.4 M NaCl as a single
peak. The fractions containing ORK were combined; dialyzed against 20 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 20 µM APMSF, and 20 mM NaCl; and applied to a
Mono Q 5/5 column equilibrated with the dialysis buffer. The proteins
were eluted with a linear gradient of 20-200 mM NaCl in 20 ml and collected in fractions of 1.5 ml. The fractions were assayed for
rhodopsin kinase activity, and the purity of the separated kinase was
estimated by SDS-PAGE on 11% gels. The purified enzyme was mixed with
an equal volume of ethylene glycol as a stabilizer and stored at
30 °C until use.
Purification of Octopus Rhodopsin--
Octopus rhodopsin was
affinity-purified with concanavalin A-Sepharose from the detergent
extract of the microvillar membranes. The membrane extract with 1%
(w/v) sucrose monolaurate was applied to a concanavalin A column
(2 × 3.5 cm) equilibrated with 10 mM Tris-HCl (pH
7.4), 0.5 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 0.1% sucrose monolaurate, and
the column was thoroughly washed with the equilibration buffer until
the absorbance at 280 nm returned to the base-line level. Rhodopsin was
eluted from the column with 250 mM
-methyl-D-mannopyranoside in the same buffer. The amount of rhodopsin was measured by absorbance at 476 nm (molecular extinction coefficient = 30,000 (29, 30)).
Reconstitution of Octopus Rhodopsin into Phospholipid Vesicles-- Reconstitution of octopus rhodopsin into phospholipid vesicles was carried out as described by Cerione et al. (31). Briefly, the purified rhodopsin (~8 µM) in a buffer containing 2.5 mg/ml azolectin (Sigma), 1% (w/v) octyl glucoside, and 1 mg/ml bovine serum albumin was passed through a 1-ml Extractigel-D column equilibrated with 20 mM Tris-HCl (pH 7.4), 25 mM MgCl2, and 100 mM NaCl. Turbid fractions after the void volume were collected. Rhodopsin in the reconstituted vesicles retained its photoreversibility between rhodopsin and metarhodopsin.
Phosphorylation of Rhodopsin--
Phosphorylation of octopus
rhodopsin was carried out according to the method described previously
(27) with some modifications. Briefly, the rhodopsin-containing
vesicles (~3 µM rhodopsin) was incubated with the
sample containing ORK in a buffer containing 20 mM Tris-HCl
(pH 7.4), 50 mM KCl, 10 mM MgCl2,
and 0.5 mM [-32P]ATP (1 µCi/µl) at
15 °C in the dark or light. The reaction was terminated by addition
of an equal volume of electrophoresis sample buffer. Incorporation of
radioactivity into rhodopsin was visualized by autoradiography or
measured with a Fuji BioImage BAS2000 analyzer after SDS-PAGE on 11%
gels.
Amino Acid Sequencing-- The purified ORK (~1 nmol) was dialyzed against 8 M urea and digested overnight with Achromobacter protease I (EC 3.4.21.50) in 50 mM Tris-HCl (pH 9.5) and 4 M urea. The digested peptides were separated by reversed-phase high pressure liquid chromatography, and amino acid sequences of the peptides in the peak fractions were analyzed with an Applied Biosystems Model 473A Protein Sequencer.
Preparation of Octopus Photoreceptor G Protein--
One of the
octopus photoreceptor G proteins (32), Gq, was purified to
apparent homogeneity from the detergent extract of the microvillar
membranes as described previously (33). Briefly, the 1% sucrose
monolaurate extract of the microvillar membranes was applied to a
DEAE-cellulose column, and bound proteins including Gq were
eluted stepwise with a buffer containing 0.5 M NaCl and 1%
cholate. A trimeric Gq preparation was obtained after gel
filtration on Sephacryl S-300 HR (Amersham Pharmacia Biotech). To
obtain the -monomer and
-dimer, the subunits were further
separated on Mono Q PC (Amersham Pharmacia Biotech). Gq and
its subunits thus prepared were homogeneous as revealed by
SDS-PAGE.
Cloning and Sequencing of the ork Gene-- Poly(A)+ RNA was prepared from an octopus retina with Oligotex-dT30 Super (TaKaRa) and used to construct a cDNA library in Lambda ZAP II vector. A pair of PCR primers were synthesized on the basis of the partial amino acid sequences determined from peptide fragments generated by Achromobacter protease I digestion of the purified ORK: ork-f1, 5'-CCIGA(AG)(CA)GICA(AG)CA(TC)AA(AG)-3'; and ork-r1, 5'-CA(AG)AA(AG)TT(TC)TC(AG)TAIA(AG)(TC)TG-3' (where I is deoxyinosine). DNA fragments were amplified by PCR using this pair of primers and the octopus retinal cDNA library as a template. The PCR products that matched the predicted size between the primer regions (~360 base pairs) were cloned into the plasmid vector pT7Blue(R), and the inserts were analyzed for their DNA sequences. DNA sequence was determined according to the chain termination procedure using the Thermo Sequenase fluorescent labeled primer cycle sequencing kit and an SQ-3000 DNA sequencer (Hitachi, Tokyo). All nucleotide sequences were determined for both strands from several independent clones. All PCR clones for which the insert DNA sequence was determined had an identical insert, and they contained the sequence corresponding to the peptide fragment obtained from the purified protein (clone P-1). To determine the amino- and carboxyl-terminal sequences, 5'- and 3'-PCRs were carried out with the same cDNA library as a template. For the amino-terminal sequence, the first 5'-PCR was carried out with a gene-specific primer, ork 5'-1 (5'-ATCTCCACTGACATGGAATC-3'), and a vector-specific primer, P8 (TOYOBO, Tokyo), with the retinal cDNA library as a template. The amplified DNA was then used as a template DNA for the second 5'-PCR with a gene-specific primer, ork 5'-2 (5'-GACATTCATTGTCATAGTCA-3'), and a vector-specific primer, SK (TOYOBO). The PCR fragments obtained after the second amplification were cloned and sequenced. For the carboxyl terminus, the 3'-PCR was likewise carried out using ork 3'-1 (5'-GCAGATGCTTTTGATATTGG-3') and P7 (TOYOBO) and then ork 3'-2 (5'-GATGATACAAAAGGAATCAG-3') and a vector-specific primer, T7, for the promoter sequence of T7 polymerase (TOYOBO). The PCR clone that contained the entire coding region, named ork, was finally obtained by cloning the PCR products amplified with two sets of primers corresponding to 5'- and 3'-noncoding regions, ork-N1 (5'-ACGAGGGTAAAGCTCTCAAG-3') and ork-C1 (5'-GTAGGAAATGGTTGGCAACA-3') and ork-N2 (5'-ATACCAGAAGTACGAAATCC-3') and ork-C2 (5'-CCAATATCTAGAAGTCTCT A-3'), and then its nucleotide sequences were determined for both strands from several independent clones.
Production and Purification of Antibody-- The purified ORK was mixed with Freund's complete adjuvant and used to subcutaneously inoculate rabbits (~0.1 mg each). The rabbits were boosted 3 weeks after the first inoculation (incomplete adjuvant was used for the booster) and bled 1-2 weeks after the booster. The IgG fraction was collected from the antisera by ammonium sulfate precipitation. Anti-ORK antibody was then affinity-purified on a Sepharose 4B column coupled with the purified ORK.
Electrophoresis and Immunoblot Analysis-- SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (34). Protein blotting onto polyvinylidene difluoride membrane was performed following the method of Towbin et al. (35) using a transfer buffer containing 0.1% (w/v) SDS and 15% (v/v) methanol. For immunological detection, horseradish peroxidase-conjugated anti-IgG antibodies and the ECL chemiluminescence detection system were used according to the manufacturer's instructions.
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RESULTS |
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Purification of ORK-- About half of the rhodopsin kinase activity in the microvillar membranes was extracted by freeze-thaw methods. The extracted proteins were loaded onto a heparin-like polyanion affinity matrix sulfate-Cellulofine column. As shown in Fig. 1, all of the rhodopsin kinase activity was bound to the column (no rhodopsin kinase activity was detected in the flow-through fractions) and eluted as a single activity peak on a linear gradient of 0.2-1.0 M NaCl. ORK was eluted at ~0.4 M NaCl, and fractions 11-17 contained an 80-kDa protein as a major component (data not shown). These fractions were pooled, dialyzed to reduce salt concentration, and further purified by Mono Q chromatography. Fig. 2 shows the elution profile of ORK on a Mono Q 5/5 column. The proteins were eluted by a linear gradient of 20-200 mM NaCl. ORK was eluted at ~100 mM NaCl as a single peak. The peak fractions eluting from the Mono Q column consisted of only one detectable protein with an apparent molecular mass of 80 kDa as determined by SDS-PAGE followed by Coomassie Blue staining (Fig. 3). Purification to apparent homogeneity was thus achieved at this step. The summary of the purification is documented in Table I and Fig. 3.
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Activation of ORK by G Protein Subunits--
Since phosphorylation
of octopus rhodopsin in the membrane preparation markedly increased
with addition of GTPS (27), a photoreceptor G protein could be
involved in regulation of rhodopsin phosphorylation. Thus, we
investigated the effects of octopus photoreceptor Gq on
rhodopsin phosphorylation in a well defined reconstituted system using
highly purified proteins. Phosphorylation of the purified octopus
rhodopsin in phospholipid vesicles by the purified ORK was examined in
the presence or absence of the subunits of Gq isolated from
the microvillar membranes. As demonstrated in Fig.
5, addition of the purified
-subunits of Gq increased rhodopsin phosphorylation
2.5-fold. On the other hand, the purified
-subunit of Gq
showed no effect regardless of the presence of GDP or GTP
S. The
heat-denatured
-subunits were completely inactive, showing that
the functionally intact subunits are required for activation. These
results agree with those obtained with
ARK, which possesses a PH
domain, but are quite different from the results obtained with RK.
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Comparison of Effects of Various Activators and Inhibitors of
Mammalian GRKs on ORK Activity--
To characterize ORK biochemically,
we studied the effects of various compounds known to affect mammalian
GRKs on ORK activity. Polycations such as polyamines and polylysine act
as activators of bovine RK (16), but they were potent inhibitors of ORK
(Fig. 6A). Spermine weakly (up
to 20% activation) activated ORK at low concentrations, but showed
strong inhibition at higher concentrations. Its IC50 was
~5 µM. Spermidine (Fig. 6A) and polylysine
(data not shown) did not show any activation, and their
IC50 values were 10 µM and 50 µg/ml,
respectively. Polyanions such as heparin, dextran sulfate, and
polyglutamic acid inhibit both bovine RK (16) and ARK (17), and they
also exhibited similar inhibition of ORK; heparin and dextran sulfate
were strong inhibitors, and polyglutamic acid was a weak inhibitor
(Fig. 6B). The IC50 values for heparin and
dextran sulfate were ~1 and 0.3 µg/ml, respectively. The
receptor-mimetic peptide mastoparan, which acts as an activator of both
RK (36) and
ARK (37), was, on the contrary, a potent inhibitor of
ORK (Fig. 6C), and its IC50 was ~0.1
mM.
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Cloning and Sequencing of the cDNA Encoding ORK--
To
isolate the octopus cDNA clone encoding ORK for determination of
its primary structure, PCR was conducted with an octopus retinal
cDNA library as a template. Degenerative primers for screening of
the library were synthesized on the basis of the partial amino acid
sequences determined from peptide fragments generated by Achromobacter protease I treatment of the purified ORK.
Sequences from several peptide fragments showed homology to ARK, and
two regions were selected to synthesize the degenerative primers (data not shown).
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Tissue Distribution of ORK-- To examine the tissue distribution of this enzyme, Western blot analysis was conducted with an antibody raised against the purified kinase. As shown in Fig. 8, ORK was abundantly expressed in the retina and was not detected in all the other tissues tested (brain, optic lobe, testis, liver, muscle, salivary gland, and skin). Thus, ORK was specifically, or at least predominantly, expressed in the retina, which is consistent with its identity as rhodopsin kinase.
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DISCUSSION |
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In this paper, we report the first rhodopsin kinase that has been
purified from invertebrate photoreceptors to apparent homogeneity using
octopus photoreceptors. Rhodopsin kinase activity is present in both
the soluble and membrane fractions of octopus retinal homogenate. As we
have previously reported, rhodopsin kinase activity is present even in
thoroughly washed microvillar membranes (27). This indicates that a
considerable amount of ORK remains as a membrane-bound form in the
washed microvillar membrane preparation; thus, we intended to isolate
the enzyme from the membrane extract. We found that roughly more than
half of the rhodopsin kinase activity is detached from the washed
microvillar membranes by freeze-thawing the membranes in isotonic
buffer. Since this freeze-thaw extract contains relatively small number
of proteins and is free of detergents that interfere with
phosphorylation of rhodopsin, it is suitable as a starting material for
purification. We used sulfate-Cellulofine, a sulfated cellulose resin,
for the affinity chromatography since it gave better resolution of
proteins than the widely used immobilized heparin under our
experimental conditions. By two steps of successive chromatography on
sulfate-Cellulofine and Mono Q columns, ORK was purified to apparent
homogeneity. The apparent molecular mass of the purified ORK was
estimated as 80 kDa by SDS-PAGE, which differs from that of RK (67 kDa)
(5), but is similar to that of ARK (80 kDa) (6). It is also
consistent with the molecular mass predicted from the sequence of the
ork gene (80 kDa).
ORK also resembles mammalian ARK in terms of regulation of activity.
ORK is activated by
-subunits of a photoreceptor G protein,
Gq. This is consistent with our previous observation that
phosphorylation of rhodopsin in the microvillar membrane preparation is
enhanced in the presence of GTP (27), which suggests regulatory action
of a G protein. The result that ORK is activated by G
implies
that the C-terminal region of ORK can serve as an interface domain
between G
, as expected from its sequence similarity to the PH
domain present in
ARK. RK does not possess a PH domain (7), and it
is not activated by G
(23) either. Isoprenylation, which occurs
at the C terminus of RK but not of the octopus enzyme, plays an
important role in regulation of the enzymatic activity (25, 26). Thus,
the mode of regulation of rhodopsin kinase seems to be quite different
between vertebrate and invertebrate photoreceptors.
Mammalian rhodopsin couples with the photoreceptor-specific transducin,
whereas octopus rhodopsin couples with multiple photoreceptor G
proteins (38), including the more widely expressed Gq (33). ORK also has many properties common to ARK, which is expressed in a
wide range of tissues. Together with the fact that the depolarizing photoresponses of invertebrate photoreceptors are the same as those of
the typical mammalian neurons, these findings show that invertebrate
photoreceptors have adopted signaling machinery common to typical
mammalian neurons in their evolutionary process.
In terms of sensitivity to activators and inhibitors, ORK differs from
both ARK and RK. Mastoparan, a wasp venom that activates
ARK (37)
and bovine RK (36), does not activate, but instead inhibits ORK. Since
mastoparan is thought to mimic the third intracellular loop of the
receptor that acts as an interface on activation of G protein by the
receptor (39), one may assume that its effects on
ARK are also alike
(37). Mastoparan does not affect GTP binding to octopus photoreceptor
Gq 2; thus, it
probably does not mimic octopus rhodopsin against both Gq
and ORK. As one may presume from their sequence differences, ORK is not
activated by activators of bovine RK such as polyamines (16). On the
contrary, polyamines are very potent inhibitors of ORK. Polyamines also
inhibit
ARK (17), although less potently than they inhibit the
octopus enzyme. Polyanions such as heparin, dextran sulfate, and
polyglutamic acid inhibit all three enzymes (16, 17), but their
potencies vary with regard to each kinase. Finally, ORK is affected by
all the drugs tested in a different way compared with both mammalian
ARK and RK.
Sequence analysis of the ork gene shows striking structural
similarity of ORK to ARK, but only moderate similarity to RK. The
amino acid sequence identity of ORK is much higher to
ARK (64%
identity) than to RK (34% identity). In addition, ORK seems to possess
a PH domain in the C-terminal region, which is thought as an
interaction domain with G protein
-subunits present in
ARK,
but absent in RK (1, 2, 40). On the other hand, the CAAX
motif for C-terminal isoprenylation, which is present in RK (7), is not
found in ORK. In addition, a phylogenetic analysis of the GRK family
reveals that ORK pairs with the
ARK group, not with the RK group
(data not shown). From these results, we conclude that ORK is
evolutionarily closely related to an ancestor of mammalian
ARK and
belongs to a family distinct from RK. Drosophila GPRK-1 (13)
has also been reported to be highly homologous to
ARK, although no
adrenergic signaling system has been identified in
Drosophila. Taken together, it is interesting to hypothesize that a
ARK-like enzyme may represent a prototype of all GRKs including RK and that ORK (possibly as well as other invertebrate rhodopsin kinases) may remain in a less differentiated structure than
highly differentiated RK.
Mammalian ARK is expressed in a wide range of tissues, and
Drosophila GPRK-1 also does not show retina-specific
expression (13). Since ORK is structurally similar to these enzymes,
its expression also may not be limited to the retina, but may range over a variety of tissues. When several octopus tissues were subjected to Western blot analysis with an antibody generated against the purified ORK, immunoreactivity to the antibody was detected only in the
retina among the tissues tested, and the expression level of ORK in the
retina was very high. This abundant and specific expression pattern of
ORK in the retina, which is quite similar to that of RK, affirms that
this enzyme is indeed "rhodopsin kinase" and is not merely one
representative of invertebrate GRKs targeting multiple receptors.
In conclusion, ORK is structurally closely related to ARK, but has
enzymatic properties that are unique among the known GRKs. Thus, we
propose that it represents a novel subgroup, possibly that of
invertebrate rhodopsin kinases, in the GRK family. Validity of this
hypothesis will be examined through characterization of the
corresponding enzymes in photoreceptors of other invertebrates.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Miyata for reconstructing a phylogenetic tree of the GRK family and helpful discussion and Drs. T. Haga and K. Palczewski for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by Research Grants 0740819 and 08257219 (to M. T.) and 9780611 (to S. K.) from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ, GenBankTM/EBI Data Bank with accession number(s) AB009875.
To whom correspondence should be addressed. Tel.: 81-7915-8-0196;
Fax: 81-7915-8-0197; E-mail: mtsuda{at}sci.himeji-tech.ac.jp.
1
The abbreviations used are: GRKs, G
protein-coupled receptor kinases; RK, rhodopsin kinase; ARK,
-adrenergic receptor kinase; ORK, octopus rhodopsin kinase; PH,
pleckstrin homology; GTP
S, guanosine
5'-(3-O-thio)triphosphate; APMSF,
4-(amidinophenyl)methanesulfonyl fluoride; PAGE, polyacrylamide gel
electrophoresis; PCR, polymerase chain reaction.
2 S. Kikkawa, unpublished data.
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REFERENCES |
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