PRIORITY PAPER
Ascidian arrestin (Ci-arr), the origin of the visual and nonvisual
arrestins of vertebrate
Masashi Nakagawa
1
, Hidefumi Orii
1
, Norihiro Yoshida
2
, Eri Jojima
3
, Takeo Horie
1
, Reiko Yoshida
1
,
Tatsuya Haga
3
and Motoyuki Tsuda
1
1
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Kamigori, Akoh-Gun, Hyogo, Japan;
2
Department of Neurochemistry, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Japan;
3
Institute for Biomolecular
Science, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo, Japan
Arrestin is one of the key proteins for the termination of G
protein signaling. Activated G protein-coupled receptors
(GPCRs) are specifically phosphorylated by G protein-
coupled receptor kinases (GRKs) and then bind to arrestins

meric G proteins by catalyzing the exchange of GDP for
GTP on the G protein a-subunit, followed by initiation of
the intracelluar signal transduction cascade. The active form
of GPCR must be inactivated within a proper time range for
cells to adapt to the changing environment. A two-step
process is involved in the inactivation mechanism. First the
activated GPCR is specifically phosphorylated by G
protein-coupled receptor kinase (GRK). Subsequently, a
soluble regulatory protein, arrestin, binds with high affinity
to the phosphorylated receptor to sterically inhibit the
interaction of the receptor with G protein, resulting in the
shutting-down of the downstream signaling [1–4].
Vertebrate arrestins can be divided into two classes; visual
arrestins and nonvisual, b-arrestins. Visual arrestins are
composed of rod- and cone-arrestins, both of which are
expressed exclusively in photoreceptor cells in the retina and
pineal organs and are thought to play a role in the
regulation of photo-signal transduction. Alternatively,
b-arrestins, which can be subdivided into b-arrestin1 and
b-arrestin2, are expressed ubiquitously and regulate various
GPCRs [4,5].
Some agonist-activated GPCRs are translocated from the
cell surface membrane to intracellular compartments. This
phenomenon is referred to as internalization or sequestra-
tion. b-Arrestin, but not the visual arrestins, plays a central
role in this internalization. b-Arrestin bound to the phos-
phorylated receptor recruits other key proteins, clathrin and
the AP2 complex, via distinct binding sites which are
conserved in the C-terminus of b-arrestin, leading to
endocytosis of the GPCRs [6–10]. Recent evidence showed

In this study, we isolated cDNA encoding arrestin (Ci-
arr) from the larvae of the ascidian Ciona intestinalis.The
deduced amino acid sequence suggests that Ci-arr is more
closely related to vertebrate arrestins than to other inver-
tebrate ones. Interestingly, this arrestin possesses character-
istics of both visual and b-arrestin; the gene is expressed only
in the photoreceptor of the ocellus, but promotes agonist-
induced internalization in the cell culture HEK293tsA201.
We will discuss the relationship between the diversity and
function of the arrestin family.
MATERIALS AND METHODS
Cloning and sequencing of Ci-arr cDNA
cDNA was synthesized from Ciona larvae mRNA with
First-strand cDNA Sythesis Kit (Amersham Pharmacia
Biotech). The cDNA was amplified by PCR using a set of
degenerate primers for the conserved region among various
arrestins: the forward primer (5¢-TG
AAGCTTYMGITAY
GGIMGIGARGA-3¢: underline is HindIII site) and reverse
primer (5¢-AGG
AGATCTTGIARCATIACISWRCAN
GG-3¢: underline is BglII site) corresponded to the amino
acid sequences FRYGRED and PCSVMLQ, respectively.
Finally, based on the nucleotide sequence of the PCR
products, specific primers were synthesized and used for
5¢-and3¢-RACE. Based on the sequence of 5¢-and3¢-
flanking region of ORF of Ci-arr (Ar-full-N (5¢-CTGT
G
CTCGAGTTTGTACTCTGTCTAAC-3¢: underline is
XhoI site) and Ar-full-C (5¢-ACA

,100m
M
NaCl].
Sequence data source
Sequence data used in the present analyses were taken from
GenBank, EMBL, SWISS-PROT or NCBI databases, with
following accession numbers: human rod (P10523), cone
(P36575), b-arrestin1 (P49407), and b-arrestin2 (P32121);
bovine rod (P08168), b-arrestin1 (P17870), and b-arrestin2
(P32120); mouse rod (P20443), rat rod (P15887), b-arrestin1
(P29066), and b-arrestin2 (P29067); bullfrog rod arrestin
(X92399); leopard frog rod (X92398) and cone (X92400)
arrestins; clawed frog rod (U41623) and cone (L40463)
arrestins; rainbow trout red blood cell arrestin (P51466);
killifish rod1 (AB002554), rod2 (AB029392), and cone
(AB002555) arrestins; fruit fly arrestin1 (P15372) and
arrestin2 (P19107); bluebottle fly arrestin1 (X79072) and
arrestin2 (X79073); migratory locust arrestin (S57174);
horseshoe crab arrestin (U08883); fruit fly kurtz
(AF221066); planarian arrestin (Orii et al. unpublished);
nematode arrestin (P51485).
Southern blot hybridization
Three types of the probes, a full-length Ci-arr cDNA (F),
5¢-region probe (N, XhoI–PstI fragment) and 3¢region (C,
PstI–BamHI fragment) were labeled with [
32
P]dCTP using a
BcaBest labeling kit (Takara) and used as probes for
hybridization.
Total DNA was isolated from the ovary of a single adult

-AR, generously provided by
R. J. Lefkowitz (Duke University Medical Center, Durham,
USA). HEK293tsA201 cells, generously provided by C. J.
van Koppen (Institute fur Pharmakologie Universitatskli-
nikum Essen, Germany) were grown on 60-mm plates as
described elsewhere [19] and transfected with 0.5 lgof
pEF-b
2
-AR, in addition to either 2 lg of pEF-Ci-arr, pEF-
Ci-arr(V55D), or pEF-BOS(control vector), by LipofectA-
MINE2000 (Gibco) according to the manufacturer’s
instructions. After 24 h transfection, cells were replated on
poly
L
-lysine-coated 24-well plates and allowed to reattach
and grow for further 24 h.
Then cells were incubated with 10 l
M
isoproterenol for
the indicated times (time course experiment) or with the
indicated concentrations of isoproterenol for 30 min (dose-
dependence experiment). After washing with ice-cold phos-
phate-buffered saline, cells were incubated with 12 n
M
[
3
H]CGP-12177 (NEN Life Science Products) in 25 m
M
Ó FEBS 2002 Origin of vertebrate visual and nonvisual arrestins (Eur. J. Biochem. 269) 5113
Hepes-buffered Dulbecco’s modified Eagle’s medium/F-12

of Ci-arr. Among them, nine introns are same in their
positions as those of vertebrate visual and b-arrestin genes
(Fig. 1), supporting close relationship between Ci-Arr and
vertebrate arrestins.
Fig. 1. Comparison of the amino acid sequences of Ci-Arr with some types of arrestins. The alignment of the deduced amino acid sequence of Ci-arr
and the typical animal arrestins was performed using a
CLUSTALW
program [20]. Since the insertion sequence of C-terminus apparently disturbed the
sequence alignment in the C-terminal portion, we manually optimized it from 350 to the end of the sequence. Dashes indicate gaps introduced to
optimize the alignment. Asterisks and dots under the sequences represent the identical and similar amino acids in eight sequences. Open and solid
wedges indicate the position of introns in Ci-arr and human visual arrestin genes, respectively. The box indicates the clathrin binding domain [21,22]
which may be responsible for the receptor internalization. White letters indicate AP2 binding site in b-arrestins [23]. White and black arrows indicate
the splicing sites in Ci-arr and bovine rod arrestin, respectively [24,25].
5114 M. Nakagawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Comparison of two types of cDNA and genomic DNA
revealed that splicing of intron 11 resulted in the shift of the
reading frame in the truncated form, in which Asn387 in
Ci-Arr was replaced with Lys followed by stop codon (Fig. 1
white arrowhead). Similar truncated form of arrestin, termed
p44, in which the last 35 amino acids are replaced by a single
Ala, was detected in bovine retinal rod cells [24,25] (Fig. 1
black arrowhead). Since p44 was present abundantly in the
outer segment of a rod photoreceptor cell and inhibited
the activity of phosphodiesterase much more effectively than
the full-length form, p44 is thought to be a key component in
phototransduction [24,26]. There is no report on such
alternative splicing in the C-terminus of b-arrestin.
In the C-terminus, b-arrestins contain a characteristic
domain (371–379 for bovine b-arrestin2; arrestin3), which
visual arrestins lack [27]. This domain is known as the

interact with b
2
-ARs [27]. We constructed the correspond-
ing mutant for Ci-Arr (V55D). This mutant, however,
showed that the extent of the internalization was almost the
same as that of the control (Fig. 2B) in spite of the same
levels of the expression (Fig. 2A). This result will be
discussed later. The isoproterenol concentration dependence
also showed that Ci-Arr induced about twofold sequestra-
tion of the receptors compared with control and V55D
(Fig. 2C). These results indicated that Ci-Arr was able to
promote the agonist-induced receptor internalization. It
should be noted that Ci-Arr promoted the internalization of
not only b
2
-AR, but also m2- and m4- muscarinic receptors
(data not shown).
The internalization of b
2
-ARs has been reported to be
unaffected by coexpression of b-arrestin1 but significantly
attenuated by coexpression of b-arrestin1 (V53D) [7,28]. We
confirmed these observations by experiments carried out in
parallel with the experiments on Ci-Arr (data not shown).
These results indicate that the Ci-Arr (V55D) does not
function in a dominant negative manner for endogenous
b-arrestin or related proteins, and that Ci-Arr enhances the
internalization of b
2
-ARs by the mechanism independent of

indicated period (B) or with the indicated concentrations of isopro-
terenol for 30 min (C). Data are the mean ± SE from triplicate
experiments.
Ó FEBS 2002 Origin of vertebrate visual and nonvisual arrestins (Eur. J. Biochem. 269) 5115
arrestin family, we constructed a molecular phylogenetic
tree. This tree demonstrates that triploblastica arrestins can
be classified into two major classes: vertebrate and protos-
tome arrestins (Fig. 4). Vertebrate arrestins are subdivided
into two groups, visual arrestins and b-arrestins. The tree
showed that Ci-Arr was more closely related to the
vertebrate arrestins than to protostome arrestins and that
it was branched from a common root of the vertebrate
visual and b-arrestins. This result allows us to propose a
hypothesis that Ci-Arr is the prototype of vertebrate arrestin
and that in the evolutionary process to vertebrates the
duplication of the prototype resulted in two different types
of arrestins, visual and b-arrestins. If so, Ciona must have
only one gene for arrestin.
Fig. 5. Genomic Southern blot analysis. (A) The probes used for
hybridization were schematically drawn under Ci-arr cDNA. It should
be noted that there is no SpeIsiteinCi-arr cDNA. (B) Genomic DNA
extracted from an individual adult of Ciona was digested with SpeIor
PstI. Each lane was loaded with a digest of 10 lg DNA. The same blot
was hybridized repeatedly with a full length probe (F), 5¢-region probe
(N) or 3¢-region probe (C) of Ci-arr cDNA in the low stringency
condition (6 · NaCl/Cit, 60 °C).
Fig. 4. Phylogenetic tree of arrestin family. The molecular phylo-
genetic tree was constructed by the neighbor-joining method [30] based
on the amino acid sequences of various arrestins using
CLUSTALW

search of Ci-arr against JGI Ciona
genome project database indicated that all of 18 hits of
genomic DNA fragments were almost identical to Ci-arr
and that there are no hits with some similarity except for
them. This result also supports the suggestion that Ciona
does not contain any arrestin genes other than Ci-arr.
DISCUSSION
Gene duplication is thought to have occurred two times in
the early process of vertebrate evolution [31]. This hypothesis
is in agreement with our present results; the first gene
duplication of the prototypical arrestin gene like Ci-arr might
result in the ancestral visual and b-arrestins, the second
duplication might result in cone and rod arrestins from the
former, and b-arrestin1 and b-arresin2 from the latter.
Recently, it was reported that Drosophila visual arrestin
(Arr2) was involved in the apoptosis of the photoreceptor
cells [32,33]. In the photoreceptors of mutant flies whose
retinae degrade in light dependent manner, Arr2 was tightly
bound to photoactivated and phosphorylated rhodopsin
and the complex was dynamin-dependently internalized to
induce the apoptotic cell death of the photoreceptors,
although Arr2 dose not contain the consensus sequence for
clathrin binding (Fig. 1). In contrast, vertebrate visual
arrestin does not have the ability to bind clathrin and cannot
induce receptor internalization [6,8]. Ci-Arr retaining this
function is apparently close from the viewpoint of the amino
acid sequence similarity and the position of the intron to the
vertebrate type of arrestin (Fig. 1). Therefore, it may be
presumed that the function was lost in vertebrate visual
arrestin during the evolution.

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