|Molecular Vision 2005;
Received 13 July 2005 | Accepted 16 December 2005 | Published 29 December 2005
Isolation and characterization of visual pigment kinase-related genes in carp retina: Polyphyly in GRK1 subtypes, GRK1A and 1B
Yoshie Shimauchi-Matsukawa,1 Yoshinobu Aman,2 Shuji
1Graduate School of Frontier Biosciences, and 2Department of Biology, Graduate School of Science, Osaka University, Yamada-oka 1-3, Suita, Osaka 565-0871, Japan
Correspondence to: Satoru Kawamura, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka 1-3, Suita, Osaka 565-0871, Japan Phone: +81-6-6879-4610; FAX: +81-6-6879-4614; email: firstname.lastname@example.org
Purpose: Visual pigment is phosphorylated and inactivated after light stimulus. The responsible enzyme is known as rhodopsin kinase or G-protein-coupled receptor kinase 1 (GRK1) in rods. We recently showed that the kinase in cones (GRK7) has much higher activity than GRK1 in rods in carp retina [1,2]. During the course of these studies, we realized that there are several subtypes of GRK1 and GRK7. In the present study, therefore, to identify the GRK1 and GRK7 subtypes expressed in carp photoreceptors, we determined their nucleotide sequences together with their expression patterns in carp retina. We also analyzed their relationships to other GRK1s and GRK7s phylogenetically.
Methods: Oligonucleotides corresponding to the amino acid sequences conserved in GRK1 or GRK7 were synthesized to screen the GRK subtypes in a carp retinal cDNA library. The isolated partial cDNAs were used to determine the full length of GRK subtypes. Genomic Southern hybridization was performed to learn whether each of the isolated GRKs is encoded by a single gene or it is an allelic variation. Tissue localization of the isolated GRKs was examined with in situ hybridization.
Results: A novel subtype of GRK1, GRK1B, was found in addition to the conventional GRK1 (called GRK1A subtype in this study) in carp retina. The GRK1A subtype, more specifically the GRK1A-1 subtype, which is related to the mammalian-type GRK1, was expressed in rods, while the GRK1B subtype, related to chicken GRK1, was expressed in cones. Since GRK7-1 was also expressed in cones, carp cones express both GRK7-1 and GRK1B. There were two paralogous genes in all of the GRK1 and GRK7 subtypes in carp retina: GRK1A-1a and 1A-1b, GRK1Ba and 1Bb, and GRK7-1a and 7-1b. Each of these genes was suggested to be encoded by a single gene in the carp genome, and each pair was found to be expressed in the same type of photoreceptors.
Conclusions: Carp rods and cones express at least two kinds of visual pigment kinases. Phylogenetic analysis suggested that GRK7-1 together with GRK1A-1 and GRK1B appeared before divergence of vertebrates and that some of these genes were lost during evolution in a species-dependent manner. This evolutional process probably explains why the expression pattern of GRK1 and GRK7 is complex among vertebrate species.
Vertebrate photoreceptor cells are morphologically classified into two types: rods and cones. Rods contribute to scotopic vision and cones supply photopic vision . In both rods and cones, light-activated visual pigment is quenched by phosphorylation of bleached pigment .
The enzyme responsible for the phosphorylation is a member of a G-protein-coupled receptor kinase (GRK) family . In all of the species examined, rods contain GRK1 [6-9]. GRK7 is expressed in cones in teleosts  and in some mammals [10,11], but in mice, GRK7 is not present and GRK1 is expressed in both rods and cones . Our recent study showed that carp GRK7 has a much higher specific activity than carp GRK1 . This higher activity of GRK7 could explain several aspects of cone photoresponses in carp. In the course of this study, we realized that carp retina expresses several kinds of GRK1 and GRK7. In the present study, we tried to identify them.
To determine the GRK1 and GRK7 subtypes expressed in carp retina, we isolated carp retinal GRK1- and GRK7-related genes. In addition to the conventional GRK1 (called GRK1A in this study) that includes mammalian GRK1, we found a novel GRK1B subtype that includes chicken GRK1 . Based on these findings, it is suggested that GRK1 and GRK7 appeared before divergence of the vertebrates, and that the GRK1 gene was further duplicated into GRK1A and GRK1B. All these genes seem to be retained until today in some animals, for example in teleosts, but lost in others. The complexity of the expression profile of GRK1 and GRK7 among animal species seems to be related to the evolution of each species.
Common carp (Cyprinus carpio), 25-30 cm in length, were purchased from a local supplier. After pithing, the eye was enucleated and the retinas were dissected. We froze the isolated retinas in liquid nitrogen before RNA extraction.
Isolation of cDNA clones for carp GRK homologues
A partial carp GRK1 sequence was obtained during our search of the genes specifically expressed in carp rods. This sequence was used to screen the full length of the cDNAs of conventional GRK1s (GRK1A-1a and GRK1A-1b).
To determine the carp GRK7 sequence, we synthesized oligonucleotides that corresponded to the amino acid sequence of KGGFGEVCA (aa 199-207 in bovine GRK7) for the sense-strand, and RDMKPENVLL (aa 315-324) for the antisense. Both sequences are conserved among vertebrate GRK7s.
To search carp retinal GRK1-related genes other than the conventional GRK1s (GRK1A-1a and GRK1A-1b), we constructed oligonucleotides that correspond to VMTIMNGGD (aa 263-271 in bovine GRK1) for the sense-strand, and IPWQEEMIE (aa 513-521) for the antisense-strand. These sequences are conserved in many GRK1s and GRK7s, and yet the peptide sequence between these sequences is rather variable. Therefore, these sequences were suitable for the search of unknown GRKs such as GRK1B. Using these oligonucleotides as primers, we amplified target fragments from carp retinal cDNA by polymerase chain reaction (PCR). Using the candidate cDNA fragments, a carp retinal cDNA library was screened at low stringency. The nucleotide sequence of the cDNA clone was determined for both strands with an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA).
The 3' UTR sequence of the novel carp GRK1Ba sequence was determined by a 3' RACE reaction. Carp retinal cDNA was synthesized using an oligo dT-adaptor primer with StrataScript (Stratagene, La Jolla, CA). The 3' UTR sequence was amplified with a T7 primer and a GRK1Ba-specific primer (CTT TCA GGT GTC GTG GAG AG).
Genomic Southern hybridization
Genomic DNA was extracted from carp muscle with blood and cell culture DNA Mini Kit (Qiagen, Alameda, CA). The DNA (3 μg) was digested at 37 °C for 9 h with PstI. The digests were electrophoresed on a 0.7% agarose gel, transferred to a Hybond-N+ membrane (Amersham, Piscataway, NJ) and followed by UV-cross-linking. To avoid cross-hybridization, template DNAs were amplified around the 3' UTR region by PCR. The nucleotide sequences used were those from nt 2550-2860 of the deposited carp GRK1A-1a (GenBank accession number AB055657), nt 2264-2535 of GRK1A-1b (AB119260), nt 1275-1531 of GRK1Ba (AB119262), nt 2346-2673 of GRK7-1a (AB055658), and nt 2854-3205 of GRK7-1b (AB119261). The probes used showed no or very low cross-hybridization. The membranes were hybridized with [32P]-labeled DNA probes at 42 °C for 17 h and washed under high-stringency conditions.
Amino acid sequences of the vertebrate GRKs were aligned with Clustal W. A total of 312 confidently aligned residues were used for estimating relationships among the proteins analyzed by neighbor-joining  using the PHYLIP version 3.5c software package . Confidence in the phylogeny was assessed by bootstrap resampling of the data.
Tissue reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from carp retina with GenElute Mammalian Total RNA Miniprep Kit (Sigma, St. Louis, MO). Genomic DNA was removed by treatment with DNase I (Invitrogen, Carlsbad, CA), and cDNA was synthesized from 2.5 μg total RNA with SuperScript II (Invitrogen). cDNA concentrations were normalized with the β-actin signal intensity. Primer sequences used for reverse transcription-polymerase chain reaction (RT-PCR) were: CTG GAA CTC CAG GGT TCA (Fw) and TGT CCA CCT CTT TAG CCA (Rev) for both GRK1A-1a and GRK1A-1b, CTT TCA GGT GTC GTG GAG AG (Fw) and AGG ATC TGG GAC AAA AGG AG (Rev) for both GRK1Ba and GRK1Bb, AGC TGG CTA CAC CCC ATT (Fw) and CTC AGC AAT GTC ACC TGT ATC C (Rev) for GRK7-1a and GRK7-1b, TAT GTG GCT CTT GAC TTC (Fw) and TGG TCC AGA CTC ATC ATA (Rev) for β-actin.
Real-time quantitative polymerase chain reaction (PCR)
Total RNAs extracted from retinas of each individual were used. Following DNase I (Invitrogen) treatment, first-strand cDNA was synthesized with StrataScript (Stratagen) using 2.5 μM random hexamers. The primers were designed with Primer Express Software version 2.0 (Applied Biosystems). The primer sequences used were: GGA CAA GAG ACT GGG CTT CAA G (Fw) and TCC AGT TGA TTT CGC TGA AGA A (Rev) for both GRK1A-1a and GRK A-1b, CCT TTT GTC CCA GAT CCT AAG ATG (Fw) and ACC TTT GAT GGT GCT GAA TGC T (Rev) for both GRK1Ba and GRK1Bb, AAG CAT GAG TGG TTC AAG TCC AT (Fw) and CAC CCA GGG TGG ATC GAT AA (Rev) for both GRK7-1a and GRK7-1b, and TGG GAC AGA AGG ACA GCT ACG T (Fw) and CTC CAT GTC ATC CCA GTT GGT (Rv) for β-actin.
Real-time RT-PCR was performed using ABI PRISM 7300 (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. All PCR reactions were performed in triplicate. Control measurements were done in the absence of reverse transcriptase to make sure that the levels of genomic DNA contaminants and the formation of primer-dimers were negligible. Data were calibrated using a standard curve generated from known amounts of the corresponding DNA obtained by PCR amplification. In each determination, the ratio of the amount of mRNA of one type of GRK to that of an endogenous reference (β-actin) was determined. This normalized value was compared among the GRK subtypes.
In situ hybridization
Plasmids containing carp GRK cDNAs were used as templates for in vitro transcription. Sense and antisense riboprobes were synthesized by run-off transcription from the T3 or T7 promoter with DIG RNA Labeling Mix (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions.
Isolated carp retinas were fixed in 3.7% formaldehyde/5% sucrose in 0.1 M phosphate buffer (PB, pH 7.4) at room temperature (RT) for three days, and embedded in 33% OCT compound diluted with 20% sucrose in PB . The cryosections 10 μm-thick were mounted on aminopropylsilane (APS)-coated slides. In situ hybridization was done as described [15,16]. Briefly, the sections were dried at 50 °C for 15 min, soaked in chloroform for 5 min, and air-dried at RT. The sections were post-fixed with 4% paraformaldehyde in PB for 30 min and then treated with 0.2 N HCl for 10 min. Acetylation was carried out in 0.1 M triethanolamine (pH 8.0) supplemented with 0.25% acetic anhydride for 10 min. Proteinase K (10 μg/ml) treatment was carried out for 30 min at RT. The sections were hybridized with 0.1-2.0 μg/ml cRNA probes. The hybridization buffer contained 50% formamide, 6X SSC, 5X Denhardt's solution, 0.1 mg/ml yeast tRNA, and 1 μM EDTA. The hybridization signal was visualized using alkaline phosphatase.
Isolation of carp retinal cDNAs encoding GRK1 or GRK7
Using partial carp GRK1 sequences, carp retinal cDNA library was screened, and two different sequences were obtained. The full-length nucleotide sequences of the two GRK1s (GRK1A-1a, GRK1A-1b; GenBank accession numbers AB055657 and AB119260, respectively) were determined (Figure 1). They share 68.8% identity in the overall nucleotide sequence and 95.7% in the deduced amino acid sequence (Table 1). Their amino acid sequences were similar to that of medaka OlGRK-R (Table 1, 87.9% identity in GRK1A-1a and 87.0% in GRK1A-1b).
Carp retinal GRK7-related sequences were searched in the carp retinal cDNA library under low stringency conditions, and two different sequences (GRK7-1a and GRK7-1b; GenBank accession numbers AB055658 and AB119261, respectively) were found (Figure 2). They showed high identity to each other: 88.3% in the nucleotide sequence, and 94.2% in the amino acid sequence (Table 2). Both amino acid sequences share high identity with medaka OlGRK-C (Table 2, 81.5% in GRK7-1a, and 81.0% in GRK7-1b).
To investigate whether GRK1A-1a and GRK1A-1b, or GRK7-1a and GRK7-1b, are paralogous genes that are separately encoded on a genome or they are allelic variations from the same locus, genomic Southern hybridization was performed (Figure 3). The genomic DNA was isolated from five individuals (Figure 3, lanes 1-5), and the probe was synthesized at the sequence specific to each GRK examined. Each probe showed signals in all of the animals tested.
If the paired genes, for example GRK1A-1a and GRK1A-1b, are allelic variations, either of the paired genes is not present in some of the individual fish. However, in Figure 3, we observed positive signals in all five individuals. We, therefore, concluded that GRK1A-1a and GRK1A-1b, and also GRK7-1a, and GRK7-1b, are distinct genes and that each of the subtypes is encoded separately in the carp genome.
Novel GRK1-related genes in carp retina
In medaka, two types of GRK1, OlGRK-R1 (originally OlGRK-R ) and OlGRK-R2 , were found. The GRK1 subtypes we identified previously, GRK1A-1a and GRK1A-1b, showed higher identity to medaka OlGRK-R1 than OlGRK-R2 (87-88% and 78%, respectively; Table 1), which suggested that there might be OlGRK-R2-related genes in carp. Using the oligonucleotides suitable for detection of unknown GRKs, we obtained 108 PCR fragments of the same size (about 800 bp). Among them, 106 clones contained GRK sequences (80 GRK1, 15 GRK4, 5 GRK6, and 6 GRK7).
The 80 GRK1-related sequences were classified into four groups: two of them were those of GRK1A-1a (35 clones) and GRK1A-1b (28 clones), and the other two were classified into a novel group (17 clones, GRK1Ba and GRK1Bb). Novel GRK1Ba and GRK1Bb showed relatively low amino acid sequence identity to medaka OlGRK-R2 (Table 1; 60.4% in GRK1Ba and 63.6% in GRK1Bb). As previously stated, the PCR fragments contained GRK4- and GRK6-related genes that show a much lower amino acid identity (46%-52%) to medaka OlGRK-R2. Even under this low stringency, we could not find the carp homologues of medaka OlGRK-R2 subtype (GRK1A-2 subtype; see "Complexity of the expression pattern of GRK1 and GRK7" in Discussion). The GRK1Ba nucleotide sequence (GenBank accession number AB119262) was obtained by screening the carp retinal cDNA library, but unfortunately, for GRK1Bb, only a partial sequence was obtained (Figure 1).
In a genomic Southern blot analysis, the GRK1Ba probe having 53-54% nucleotide identity with GRK1A-1a and GRK1A-1b showed a distinct hybridization pattern from that of GRK1A-1a and GRK1A-1b in all of the individuals tested (Figure 3). This result indicated that the GRK1Ba gene is different from the GRK1A-1a and GRK1A-1b genes. The GRK1Ba probe showed many bands in individual animals. Because the probe used had a high degree of nucleotide sequence identity (91%) between GRK1Ba and GRK1Bb, cross-hybridization might have occurred.
Phylogenic relationships among GRK1- or GRK7-related genes
To analyze phylogenic relations among GRK1 and GRK7 subtypes, we constructed a molecular phylogenetic tree including GRK1-related and GRK7-related sequences in fugu and zebrafish (Figure 4). As seen in Figure 4, in both GRK1 and GRK7 subtypes, there are warm-blooded animal and fish clusters (groups I and IV, and groups II and V, respectively, in Figure 4). Although the clustering probability is not 100% (i.e., 90%), in GRK1, there is another cluster which includes chicken GRK1 and carp GRK1Ba (group III in Figure 4). The result suggests that the vertebrate GRK1 gene duplicated prior to the diversion of vertebrates and each vertebrate species lost some of them during the evolutionary process.
Localization of GRK1A-1, GRK1B, and GRK7-1 in carp retina
Tissue localization of GRK1 transcripts in carp adult organs was revealed by RT-PCR (Figure 5). The primers were designed to detect both paralogues: GRK1A-1 primers detect both GRK1A-1a and GRK1A-1b, GRK1B primers detect GRK1Ba and GRK1Bb, and GRK7-1 primers detect GRK7-1a and GRK7-1b. It was found that all of the GRK subtypes examined are expressed specifically in retina.
Localization of the subtypes in carp retina was investigated by in situ hybridization in retinal cryosections (Figure 6). Control sections hybridized with sense probes did not show signals above the background level (Figure 6D). GRK1A-1a (Figure 6A,E) and GRK1A-1b (data not shown) signals were detected throughout the outer nuclear layer where the signals of rhodopsin mRNA were observed (Figure 6H). The result, therefore, implied that GRK1A-1a and GRK1A-1b are expressed in rods. GRK7-1a (Figure 6C,G) and GRK7-1b (data not shown) signals were observed in a narrow restricted layer of the myoid region of cones (Figure 6G, arrow). This hybridization pattern was the same when antisense probes of red sensitive opsin were used (Figure 6I, arrow). The result, therefore, suggested that GRK7-1a and GRK7-1b are expressed in cones. The signals of the above mentioned subtypes of GRK1A-1 and GRK7-1 were almost exclusively observed in photoreceptors. However, GRK1Ba signal was detected rather dispersedly (Figure 6B). In photoreceptors, the signal was observed in the cone myoid region (Figure 6F, arrow), although the signal was very weak when compared with that of GRK1A-1a or GRK7-1a. Because we performed the hybridization under the same conditions in all of the studies in Figure 6A-G, it is suggested that the transcription level of GRK1B in photoreceptor cells is lower than those of GRK1A-1 and GRK7-1. In agreement with this, our semiquantitative RT-PCR analysis showed that the relative abundance of mRNAs of GRK1A-1 (GRK1A-1a plus GRK1A-1b), GRK1B (GRK1Ba plus GRK1Bb) and GRK7-1 (GRK7-1a plus GRK7-1b) was 12.4:1:2.4 in carp retina (Figure 7).
In the present study, we showed that carp retina expresses two subtypes of GRK1, GRK1A-1 and GRK1B, and one type of GRK7 (Figure 1, Figure 2, Table 1, Table 2). Each subtype consists of a pair of paralogous genes, each of which is encoded separately in the carp genome (Figure 3). All of the subtypes are expressed specifically in carp retina (Figure 5) and GRK1A-1 is expressed in rods and GRK1B together with GRK7 are expressed in cones (Figure 6). Phylogenetic analysis showed that the GRK1B subtype is a member of a family that includes chicken GRK1 but not other mammalian species (Figure 4).
Expression of paralogous genes of GRK1 and GRK7 in carp photoreceptors
As shown in the present study, carp photoreceptors express a pair of paralogous genes: GRK1A-1a and GRK1A-1b, GRK1Ba and GRK1Bb, and GRK7-1a and GRK7-1b. The amino acid sequence identity between each member of the paralogous genes is very high (>94%; Table 1, Table 2). In the regions of the catalytic domains of GRK1 and GRK7 (blue lines in Figure 1 and Figure 2), the amino acid identities are even higher than the overall identities (97.2% between GRK1A-1a and GRK1A-1b, and 96.4% between GRK7-1a and GRK7-1b). All these similarities suggest that the catalytic activities of the paralogues of GRK1A-1 or GRK7-1 are similar. In other words, they are probably expressed redundantly.
Characteristics of carp GRK1B
In the present study, we found a novel subtype of GRK1, GRK1B. It is expressed in cones, and therefore, carp cones express both GRK7-1 and GRK1B. However, as shown in Figure 7, the relative expression level of GRK1B (GRK1Ba plus GRK1Bb) is lower than that of GRK7-1 (GRK7-1a plus GRK7-1b). The catalytic activity of a single molecule of GRK7 is almost ten times higher than that of GRK1A-1a . This higher specific activity together with much higher expression level of GRK7-1a over GRK1B, the contribution of GRK1B in visual pigment phosphorylation in cones could be residual. It is, however, possible that GRK1B could have a different role other than the phosphorylation of visual pigment. Further study is required to determine the actual role of GRK1B.
It has been shown that there is a consensus sequence of a lipid modification, known as a CaaX motif, at the C-termini of GRK1 and GRK7 . In most of GRK1 including carp GRK1A-1 (groups I and II in Figure 4), X in the CaaX motif is serine (Figure 1, Figure 2), and in this case, the cysteine residue (C) in the CaaX motif is modified by farnesylation. In GRK7 (groups IV and V), X is leucine, and in this case, the cysteine residue is geranylgeranylated. In the member of group III including carp GRK1B and chicken GRK1, X is leucine, and therefore geranylgeranylation is expected at the C-terminus of this family. The members in group III are classified into the GRK1 group from overall similarities of their amino acid sequences, but their lipid modification at the C-terminus is a GRK7-type. Although the functional significance of the difference between these lipid modifications on GRKs is not known yet, the geranylgeranylation found in both GRK1B and GRK7 may have important roles in cones as revealed in rod transducin .
Complexity of the expression pattern of GRK1 and GRK7
There are two GRK1 subtypes (GRK1A and GRK1B) in carp retina. Carp GRK1A is a homolog of mammalian GRK1, and GRK1B is a homolog of chicken GRK1 (Figure 4). In chicken, GRK1A subtype has not been reported. As summarized in Figure 8, these results could be explained by assuming that both GRK1A and GRK1B subtype were present before these species emerged and that GRK1A subtype was lost in chicken. In mammals, GRK1B subtype has not been reported, which could be explained similarly with the loss of GRK1B subtype during evolution of mammals. In contrast to these species, both GRK1A and GRK1B are conserved in carp. Loss of genes has also been suggested in the evolution of opsin genes [19,20].
GRK1A subtype seems to have been duplicated before the appearance of Teleostei to result in the expression of GRK1A-1 and GRK1A-2 subtypes (Figure 4, both in group II). Both subtypes are present in medaka and fugu genome (Figure 4, arrowheads and open arrows, respectively). Similar gene duplication is seen in fish GC3 and GC5 genes . Such gene duplication could be the result of putative whole-genome duplication events suggested to have occurred in a ray-finned ancestor .
In carp retina, however, GRK1A-2 subtype was not found. It is possible that, in this animal, this subtype was lost from the genome after duplication of GRK1A, or that its transcriptional elements necessary for its expression in retina were lost after duplication. Instead, in carp, GRK1A-1 subtype was duplicated into GRK1A-1a and GRK1A-1b (Figure 4, arrows). Similar gene duplication is seen in carp GRK1Ba and GRK1Bb, and GRK7-1a and GRK7-1b in addition to the genes of rhodopsin , c-myc , and complement components . Because carp is allotetraploid  and none of the subtypes of GRK1A-2 were found in carp retina, it is probable that these paralogous genes derived from a whole-genome duplication after the loss of GRK1A-2 subtype in carp retina.
In rods, GRK1 is expressed in general in vertebrates. However, the expression profile of visual pigment kinase in cones is complex. In carp, GRK7 is expressed in cones , but in addition to this, GRK1B is also expressed (this study). In mouse cones, GRK1, not GRK7, is expressed . In primates, both GRK1 and GRK7 are expressed in cones [10,11]. The reason for this complexity is not known, but in addition to the loss of genes (Figure 8), some other genetic events must have occurred in a species-dependent manner during evolution of these animals. Obviously, the expression pattern of GRK1 and/or GRK7 would be reflected in the photoresponse characteristics in cones in these animals.
This research was supported by grants from the Japan Society for the Promotion of Science (JSPS) to SK. (number 15370068) and ST (number 06770114), Human Frontier Science Program to SK, and Senri Life Science Foundation to ST. YS-M is supported by JSPS Research Fellowships for Young Scientists. We thank Dr. S. Semple-Rowland for providing a chicken retinal cDNA library.
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