Different phosphorylation rates among vertebrate cone visual pigments with different spectral sensitivities.

Cone photoreceptor subtypes having different spectral sensitivities exhibit different recovery kinetics in their photoresponses in some vertebrates. Phosphorylation by G protein-coupled receptor kinase (GRK) is essential for the rapid inactivation of light-activated visual pigment, which is the rate-limiting step of the cone photoresponse recovery in salamander. In this study we compared the rate of light-dependent phosphorylation by GRK7 of carp green- and blue-sensitive cone visual pigments. Blue pigment was phosphorylated significantly less effectively than green pigment, suggesting that the difference in the pigment phosphorylation rate is responsible for the difference in photoresponse kinetics among cone photoreceptor subtypes.

Highly efficient retinal metabolism in cones.

After bleaching of visual pigment in vertebrate photoreceptors, all-trans retinal is reduced to all-trans retinol by retinol dehydrogenases (RDHs). We investigated this reaction in purified carp rods and cones, and we found that the reducing activity toward all-trans retinal in the outer segment (OS) of cones is >30 times higher than that of rods. The high activity of RDHs was attributed to high content of RDH8 in cones. In the inner segment (IS) in both rods and cones, RDH8L2 and RDH13 were found to be the major enzymes among RDH family proteins. We further found a previously undescribed and effective pathway to convert 11-cis retinol to 11-cis retinal in cones: this oxidative conversion did not require NADP(+) and instead was coupled with reduction of all-trans retinal to all-trans retinol. The activity was >50 times effective than the oxidizing activity of RDHs that require NADP(+). These highly effective reactions of removal of all-trans retinal by RDH8 and production of 11-cis retinal by the coupling reaction are probably the underlying mechanisms that ensure effective visual pigment regeneration in cones that function under much brighter light conditions than rods.

Identification of differentially expressed genes in carp rods and cones.

PURPOSE: Rods and cones differ in their photoresponse characteristics, morphology, and susceptibilities to certain diseases. To contribute to the studies at the molecular level of these differences, we tried to identify genes expressed preferentially in rods or cones. METHODS: From purified carp rods and cones, we extracted their RNA and obtained corresponding cDNA pools (rod cDNA and cone cDNA). We employed the suppression subtractive hybridization method to identify the genes expressed preferentially in rods or cones. Cone cDNA was subtracted from rod cDNA to obtain cDNA, which ideally contained cDNA expressed preferentially in rods (R/c cDNA). Similarly, rod cDNA was subtracted from cone cDNA to obtain C/r cDNA. With differential array screening, we screened candidate genes that were expressed mainly or exclusively in rods or cones. The nucleotide sequences of the positive genes were determined. In some of them, their mRNA localizations were confirmed by in situ hybridization. RESULTS: R/c cDNA contained genes already known to code rod specific proteins, such as cGMP gated channel, transducin beta1, and rhodopsin. In sharp contrast, C/r cDNA contained genes that code proteins of which functions are mostly unknown. Among them, N-myc downregulated gene 1-like (NDRG1L) and aryl hydrocarbon receptor 2 (AhR2) were most abundant, and by in situ hybridization, they were proven to be expressed specifically in cones. CONCLUSIONS: Using purified rods and cones, we identified mRNAs expressed preferentially in rods or cones. Of particular interest is the specific expression of NDRG1L and AhR2 in cones.

Molecular mechanisms characterizing cone photoresponses.

In the vertebrate retina, rods mediate twilight vision and cones mediate daylight vision. Their photoresponse characteristics are different. The light-sensitivity of a cone is 10(2)-10(3) times lower than that of a rod. In addition, the photoresponse time course is much faster in cones. The mechanism characterizing cone photoresponses has not been known mainly because of the difficulty in isolating cones in large quantities to perform biochemistry. Recently, we developed a method to purify cones from carp retina using a density gradient, which made it possible to analyze the differences in the molecular mechanism of phototransduction between rods and cones. The results showed that signal amplification in cones is less effective, which explains the lower light-sensitivity of cones. The results also showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. The rapid phosphorylation in cones is attributed to a very high total kinase activity in cones. Because of this high activity, cone pigment is readily phosphorylated even at very high bleaching levels, which probably explains why cone photoresponses recover quickly. Based on these findings, the molecular mechanisms of the differences in the photoresponse characteristics between rods and cones are outlined.

Highly effective phosphorylation by G protein-coupled receptor kinase 7 of light-activated visual pigment in cones.

Cone photoreceptors show briefer photoresponses than rod photoreceptors. Our previous study showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. Here, we measured the early time course of this rapid phosphorylation with good time resolution and directly compared it with the photoresponse time course in cones. At the time of photoresponse recovery, almost two phosphates were incorporated into a bleached cone pigment molecule, which indicated that the visual pigment phosphorylation coincides with the photoresponse recovery. The rapid phosphorylation in cones is attributed to very high activity of visual pigment kinase [G protein-coupled receptor kinase (GRK) 7] in cones. Because of this high activity, cone pigment is readily phosphorylated at very high bleach levels, which probably explains why cone photoresponses recover quickly even after a very bright light and do not saturate under intense background light. The high GRK7 activity is brought about by high content of a highly potent enzyme. The expression level of GRK7 was 10 times higher than that of rod kinase (GRK1), and the specific activity of a single GRK7 molecule was approximately 10 times higher than that of GRK1. The specific activity of GRK7 is the highest among the GRKs so far known. Our result seems to explain the response characteristics of cone photoreceptors in many aspects, including the nonsaturation of the cone responses during daylight vision.

Isolation and characterization of visual pigment kinase-related genes in carp retina: polyphyly in GRK1 subtypes, GRK1A and 1B.

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. 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.