Melanopsin, an atypical vertebrate visual pigment, mediates non-image-forming light responses including circadian photoentrainment and pupillary light reflexes and contrast detection for image formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells are characterized by sluggish activation and deactivation of their light responses. The molecular determinants of mouse melanopsin's deactivation have been characterized (i.e., C-terminal phosphorylation and β-arrestin binding), but a detailed analysis of melanopsin's activation is lacking. We propose that an extended third cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the inactive conformation, which is stabilized by the ionic interaction of these two regions. This model is supported by site-directed spin labeling and electron paramagnetic resonance spectroscopy of melanopsin, the results of which suggests a high degree of steric freedom at the third cytoplasmic loop, which is increased upon C-terminus truncation, supporting the idea that these two regions are close in three-dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin's activation rate. Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo melanopsin-which has substitutions of various potential phosphorylation sites and a substitution of the conserved tyrosine-indicates that substitution of these potential phosphorylation sites and the tyrosine residue result in dramatically slower activation kinetics, a finding that also supports the role of phosphorylation in signaling activation. We therefore propose that melanopsin's C-terminus is proximal to intracellular loop 3, and C-terminal phosphorylation permits the ionic interaction between these two regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling.

中文翻译：

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C 末端和第三细胞质环协同激活小鼠黑视蛋白光转导

黑视蛋白是一种非典型脊椎动物视觉色素，介导非图像形成光反应，包括昼夜节律光诱导和瞳孔光反射以及图像形成的对比度检测。表达黑视蛋白的本质光敏视网膜神经节细胞的特征是其光反应的激活和失活缓慢。小鼠黑视蛋白失活的分子决定因素已得到表征（即 C 末端磷酸化和 β-arrestin 结合），但缺乏对黑视蛋白激活的详细分析。我们提出，延伸的第三个细胞质环与处于非活性构象的小鼠黑视蛋白的近端C端区域相邻，该区域通过这两个区域的离子相互作用而稳定。该模型得到了黑视蛋白定点自旋标记和电子顺磁共振波谱的支持，其结果表明第三个细胞质环具有高度的空间自由度，并且在 C 末端截断后增加，支持了这两个观点野生型黑视蛋白的三维空间区域很接近。为了测试功能关键的 C 端构象，对黑视蛋白突变体进行钙成像，包括近端 C 端截短（残基 365）和该近端区域的脯氨酸突变（H377P、L380P、Y382P）延迟了黑视蛋白的激活速率。所有潜在的磷酸化位点（包括高度保守的酪氨酸残基（Y382））突变为丙氨酸也延迟了激活速率。小鼠黑视蛋白与犰狳黑视蛋白（具有各种潜在磷酸化位点的取代和保守酪氨酸的取代）的比较表明，这些潜在磷酸化位点和酪氨酸残基的取代导致激活动力学显着减慢，这一发现也支持了磷酸化在信号激活中的作用。因此，我们认为黑视蛋白的 C 末端靠近细胞内环 3，并且 C 末端磷酸化允许这两个区域之间的离子相互作用，从而形成稳定的结构构象，这对于启动 G 蛋白信号传导至关重要。