Absorption Characteristics of Vertebrate Non-Visual Opsin, Opn3.
Absorption Characteristics of Vertebrate Non-Visual Opsin, Opn3.
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DOI：
10.1371/journal.pone.0161215
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Terakita A
中科院分区：
文献类型：
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作者：
Sugihara T;Nagata T;Mason B;Koyanagi M;Terakita A
大多数动物都具有多种视蛋白，这些视蛋白感知光以实现视觉和非视觉功能。在这里，我们显示了非视觉视蛋白，脊椎动物Opn 3，这是广泛分布在脊椎动物的光谱特性。我们成功地在哺乳动物培养细胞中表达了斑马鱼Opn 3，并测量了其吸收光谱。当与11-cis retinal孵育时，斑马鱼Opn 3形成了一种蓝色敏感的色素，在465 nm附近具有最大吸收。Opn 3转化为全反式视网膜轴承光产物，其吸收光谱类似于短暂蓝光照射后的黑暗状态。在进一步的照射过程中，光产物经历了显着的蓝移，等吸光点的位置发生了变化。然后，我们使用cAMP依赖性荧光素酶报告基因测定法来研究表达斑马鱼、河豚、变色龙和鸡Opn 3的培养细胞中的光依赖性cAMP反应。野生型视蛋白不产生反应，但表达嵌合体突变体（WT Opn 3，其中第三胞内环被替换为GS偶联水母视蛋白的第三胞内环）的细胞显示cAMP的光依赖性变化。结果表明，Opn 3能够以光依赖性方式激活G蛋白。最后，我们使用该测定来测量表达Opn 3嵌合体的细胞对多种定量匹配刺激的相对波长依赖性响应。推断的斑马鱼Opn 3的光谱灵敏度曲线与测量的吸收光谱准确匹配。我们无法估计小鼠或变色龙Opn 3的光谱敏感性曲线，但与斑马鱼Opn 3一样，鸡和河豚Opn 3-JiL 3嵌合体也形成了蓝色敏感色素。这些发现表明脊椎动物Opn 3可能形成蓝色敏感的G蛋白偶联色素。此外，我们建议，这里描述的方法，结合cAMP依赖性荧光素酶报告基因检测嵌合视蛋白具有第三细胞内环的水母视蛋白，是一种通用的方法，用于估计吸收光谱的视蛋白与未知的信号级联或吸收光谱难以获得。
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.