Hiroshi Ohguro

Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein

Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.

Separation of phospho- and non-phosphopeptides using reverse phase column chromatography

Peptides containing phosphoserine, phosphothreonine or phosphotyrosine and their parent non‐phosphorylated forms were chromatographed using standard C18 reverse phase chromatography in the presence of a water/acetonitrile gradient supplemented with different counter ions. We obtained the best separation of phosphorylated from non‐phosphorylated peptides in the presence of heptafluorobutyric acid, with differences in retention times as large as ∼ 20 min. The chromatographic method was reliable in separation of the same peptides phosphorylated at different positions, acidic or basic phospho‐Ser/Thr‐peptides or phospho‐Tyr‐containing peptides. The described separation conditions are useful in studying the kinetics of phosphorylation/ dephosphorylation and in analysis of phosphorylation sites in vivo.

Mechanism of rhodopsin phosphorylation

A key reaction in the inactivation of rhodopsin is its phosphorylation by rhodopsin kinase. In recent years, extensive studies related to rhodopsin kinase function and enzymatic properties were carried out. Rhodopsin kinase is a Ser/Thr protein kinase and a member of the G protein-coupled receptor kinases sub-family (GRKs) which consists of six recently identified members. Photolyzed rhodopsin is phosphorylated by rhodopsin kinase sequentially, with the first phosphate transferred preferentially to Ser-338, and subsequent phosphates transferred to Ser-343 and Thr-336. The binding of arrestin to the receptor, and reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol limits physiologically significant phosphorylation at no more than three sites (H. Ohguro, R.S. Johnson, L.H. Ericsson, K.A. Walsh and K. Palczewski, Biochemistry, 33 (1994) 1023). A similar phosphorylation reaction is implicated in most, if not all, G protein-coupled receptors during their desensitization.

Identification of a single phosphorylation site within octopus rhodopsin.

Abstract— Light‐dependent phosphorylation of rhodopsin (Rho) is a first step in the desensitization of the signaling state of the receptor during vertebrate and invertebrate visual transduction. We found that only 358Ser of the photoac‐tivated octopus Rho (oRho*) was phosphorylated by octopus rhodopsin kinase (oRK). Tryptic truncation of the C‐terminal PPQGY repeats of oRho that follow the phosphorylation region did not influence spectral or G‐protein activation properties of oRho but abolished phos phorylation. Despite significant structural differences between oRK and mammalian RK, these results provide i further evidence of the importance of singly phosphorylated species of Rho* in the generation of arrestin binding sites.