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 Ϸ10 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.rod ͉ photoreceptors ͉ retina ͉ phototransduction O ur visual system consists of two components: rods and cones (1, 2). These photoreceptors differ in their light sensitivity so that rods mediate twilight vision, and cones mediate daylight vision. Rods and cones are distinguished not only in their light sensitivity, but also in other response characteristics. The photoresponse time course is much briefer in cones, which improves the time resolution of our daylight vision greatly. Rods are saturated with bright background light and do not respond to more intense light (3). In contrast, cones are not saturated and respond to very bright light (4, 5). The molecular mechanisms underlying in the differences of these response characteristics are not yet known. In previous biochemical studies on the cone phototransduction mechanism, only slight quantitative differences were known in the transduction components between rods and cones (6-8).In our previous study, we obtained a large quantity of isolated cones (enough to perform biochemistry) and showed that transducin activation and cGMP phosphodiesterase activation, the reactions involved in the generation of a photoresponse, are less efficient in cones (9). These findings reasonably explained the lower light sensitivity in cones. Another remarkable difference was found in the phosphorylation of light-activated visual pigment. When light-activated, visual pigment is phosphorylated by a class of kinase known as rhodopsin kinase (rod kinase or G protein-coupled receptor kinase (GRK) 1) in...

Rotavirus (RV) and norovirus (NoV) are the 2 leading causes of acute viral gastroenteritis worldwide. We have developed a non-live NoV and RV vaccine candidate consisting of NoV virus-like particles (VLPs) and recombinant polymeric RV VP6 protein produced in baculovirus-insect cell expression system. Both components have been shown to induce strong potentially protective immune responses. As VP6 nanotubes are highly immunogenic, we investigated here a possible adjuvant effect of these structures on NoV-specific immune responses in vivo. BALB/c mice were immunized intramuscularly with a suboptimal dose (0.3 μg) of GII.4 or GI.3 VLPs either alone or in a combination with 10 μg dose of VP6 and induction of NoV-specific antibodies in sera of experimental animals were measured. Blocking assay using human saliva or synthetic histo-blood group antigens was employed to test NoV blocking antibodies. Suboptimal doses of the VLPs alone did not induce substantial anti-NoV antibodies. When co-administered with the VP6, considerable titers of not only type-specific but also cross-reactive IgG antibodies against NoV VLP genotypes not included in the vaccine composition were induced. Most importantly, NoV-specific blocking antibodies, a surrogate for neutralizing antibodies, were generated. Our results show that RV VP6 protein has an in vivo adjuvant effect on NoV-specific antibody responses and support the use of VP6 protein as a part of the NoV-RV combination vaccine, especially when addition of external adjuvants is not desirable.

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 lightactivated 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.

J. Neurochem. (2010) 115, 259–268. Abstract In the carp retina, visual pigment kinase, GRK1 (G‐protein coupled receptor kinase 1) in rods and GRK7 in cones, is inhibited by a photoreceptor neuronal Ca2+‐sensor protein, S‐modulin (or recoverin) in rods and visinin (formerly named s26) in cones. Here, we compared Ca2+‐dependent inhibition of GRK1 by S‐modulin and that of GRK7 by visinin. First, the concentrations of S‐modulin and visinin in the outer segment were estimated: the concentration of visinin (1.2 mM) was 20 times higher than that of S‐modulin (53 μM). Based on the determined concentrations of the Ca2+‐sensor proteins and the known dark Ca2+ concentrations, we estimated that in situ Ca2+‐dependent inhibition on GRK in cones would be 2.5 times higher than that in rods at the Ca2+ concentration in the dark. Because GRK activity is approximately 100 times higher in cones than in rods [Proc. Natl Acad. Sci. USA102 (2005) 21359], the range of Ca2+‐dependent inhibition on GRK activity is more than 100 times wider in cones than in rods. The inhibitory effects of S‐modulin and visinin on photoreceptor GRKs were indistinguishable, although these Ca2+‐sensor proteins are expressed in a cell‐type specific manner. The inhibition by these Ca2+‐sensor proteins was slightly higher on GRK7 than GRK1 probably because of a characteristic specific to GRK7.

GRK1 is a visual pigment kinase in rods and is essential for inactivation of light-activated rhodopsin. The GRK1 activity is inhibited by binding of the Ca(2+)-bound form of S-modulin/recoverin. We previously identified the S-modulin/recoverin site to interact with GRK1. In the present study, we identified its counterpart in GRK1. We synthesized 29 of GRK1 or GRK7 partial peptides that cover the entire sequence of GRK1/GRK7, and examined whether these peptides inhibit S-modulin/recoverin activity most probably by preoccupying the binding site for GRK1. The inhibition was the greatest with the N-terminal peptide (p1, aa 3-23 in GRK7). On mutation of each of eight amino acid residues highly conserved in the p1 region of more than 10 orthologs, the inhibition was significantly reduced in the mutation of Leu(6), Asn(12) and Tyr(15). We further examined the binding of the peptides, including mutated ones, to S-modulin/recoverin with a resonance mirror biosensor. The binding correlated well with the degree of the inhibition by a peptide. The inhibition, therefore, seemed to be due to a direct binding of the kinase peptide to the binding site of active S-modulin/recoverin. A GRK1 region close to its C-terminus also seemed to be the binding site for S-modulin/recoverin.