title: Absorption Characteristics of Vertebrate Non-Visual Opsin, Opn3.
abstract: Most animals possess multiple opsins which sense light for visual and non-visual functions. Here, we show spectral characteristics of non-visual opsins, vertebrate Opn3s, which are widely distributed among vertebrates. We successfully expressed zebrafish Opn3 in mammalian cultured cells and measured its absorption spectrum spectroscopically. When incubated with 11-cis retinal, zebrafish Opn3 formed a blue-sensitive photopigment with an absorption maximum around 465 nm. The Opn3 converts to an all-trans retinal-bearing photoproduct with an absorption spectrum similar to the dark state following brief blue-light irradiation. The photoproduct experienced a remarkable blue-shift, with changes in position of the isosbestic point, during further irradiation. We then used a cAMP-dependent luciferase reporter assay to investigate light-dependent cAMP responses in cultured cells expressing zebrafish, pufferfish, anole and chicken Opn3. The wild type opsins did not produce responses, but cells expressing chimera mutants (WT Opn3s in which the third intracellular loops were replaced with the third intracellular loop of a Gs-coupled jellyfish opsin) displayed light-dependent changes in cAMP. The results suggest that Opn3 is capable of activating G protein(s) in a light-dependent manner. Finally, we used this assay to measure the relative wavelength-dependent response of cells expressing Opn3 chimeras to multiple quantally-matched stimuli. The inferred spectral sensitivity curve of zebrafish Opn3 accurately matched the measured absorption spectrum. We were unable to estimate the spectral sensitivity curve of mouse or anole Opn3, but, like zebrafish Opn3, the chicken and pufferfish Opn3-JiL3 chimeras also formed blue-sensitive pigments. These findings suggest that vertebrate Opn3s may form blue-sensitive G protein-coupled pigments. Further, we suggest that the method described here, combining a cAMP-dependent luciferase reporter assay with chimeric opsins possessing the third intracellular loop of jellyfish opsin, is a versatile approach for estimating absorption spectra of opsins with unknown signaling cascades or for which absorption spectra are difficult to obtain.
authors.affiliation: Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sugihara
authors.affiliation: Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagata
authors.affiliation: Department of Genetics, Stanford School of Medicine, Stanford University, Stanford, California, United States of America.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mason
authors.affiliation: Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka, Japan.|Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Kawaguchi, Saitama, Japan.
authors.forename: Mitsumasa
authors.initials: M
authors.lastname: Koyanagi
authors.affiliation: Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka, Japan.
authors.forename: Akihisa
authors.initials: A
authors.lastname: Terakita
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D000242:Cyclic AMP / Q000737:chemistry; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D012172:Retinaldehyde / Q000737:chemistry; D017299:Rod Opsins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D037401:Scyphozoa; D013053:Spectrophotometry; D023721:Tetraodontiformes; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*
chemical_list: D029941:Fish Proteins; C000621715:Opn3 protein, zebrafish; D017299:Rod Opsins; D029961:Zebrafish Proteins; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D012172:Retinaldehyde