Department of Ophthalmology, Gavin Herbert Eye Institute, University of California, Irvine, CA, United States of America.
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States of America.
PLoS One. 2022 Aug 8;17(8):e0272506. doi: 10.1371/journal.pone.0272506. eCollection 2022.
Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light.
异三聚体 G 蛋白转导蛋白 Gt 是视网膜杆状和锥状光感受器细胞中的关键信号转导和放大器。尽管它们的 Gγ亚基具有相似的亚基组成、接近的氨基酸同一性和相同的翻译后法呢基化,但杆状细胞和锥状细胞分别依赖于独特的 Gγ1(Gngt1)和 Gγc(Gngt2)同工型。唯一其他法尼基化 G 蛋白 γ-亚基 Gγ11(Gng11)在多种组织中表达,但不在视网膜中表达。为了确定 Gγ1 是否独特地调节杆状细胞光转导,我们在 Gγ1 缺陷型小鼠中生成了表达 Gγ1、Gγc 或 Gγ11 的转基因杆状细胞,并分析了它们的特性。免疫组织化学和 Western blot 分析表明,每种转基因 Gγ 在杆状细胞中均有强烈表达,并恢复了 Gαt1 的表达,而 Gαt1 在 Gγ1 缺陷型杆状细胞中大大减少。视网膜电图显示,三种转基因 Gγ1 缺陷型系的视觉功能均得到恢复。从单个转基因杆状细胞的记录中显示,在 Gγ1 缺陷型杆状细胞中受损的光敏性也得到了完全恢复。在所有暗适应的转基因系中,Gαt1 被靶向到外节,逆转了在 Gγ1 缺陷型杆状细胞中发现的弥散定位。在所有三种转基因株中,明亮的光照都会触发 Gαt1 从杆状细胞外节向内节的转位。然而,在 Gγ11 转基因小鼠中,Gαt1 的转位发生在背景光明显更暗的情况下。与这一结果一致的是,在 Gγ11 转基因小鼠的中度背景光照下,经视网膜外视网膜电图记录显示逐渐恢复反应,但在 Gγ1 对照中则没有。因此,虽然法尼基化 Gγ 亚基在支持杆状细胞光转导方面具有功能性和高度可互换性,但由普遍存在的 Gγ11 替代视网膜特异性 Gγ 同工型会影响杆状细胞适应背景光的能力。