Katadae Maiko, Hagiwara Ken'ichi, Wada Akimori, Ito Masayoshi, Umeda Masato, Casey Patrick J, Fukada Yoshitaka
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Biochemistry. 2008 Aug 12;47(32):8424-33. doi: 10.1021/bi800359h. Epub 2008 Jul 18.
G protein gamma-subunits are isoprenylated and carboxyl methylated at the C-terminal cysteine residue, and the set of the posttranslational modifications is indispensable for the function of the photoreceptor G protein transducin (Talpha/Tbetagamma). To explore farnesyl-mediated molecular interactions, we investigated molecular targets of a Tbetagamma analogue that was engineered to have a photoreactive farnesyl analogue, (3-azidophenoxy)geranyl (POG), covalently bound to the C-terminal cysteine of Tgamma. POG-modified Tbetagamma was further subjected to modification by methylation at the C-terminal carboxyl group, which copies a complete set of the known posttranscriptional modifications of Tbetagamma. Photoaffinity labeling experiment with the photoreactive Tbetagamma analogue in its free form indicated that the POG moiety of Tgamma interacted with Tbeta. In the trimeric Talpha/Tbetagamma complex, the POG moiety was cross-linked with Talpha in addition to concurrent affinity labeling of Tbeta. When photoreactive Tbetagamma was reconstituted with Talpha and light-activated rhodopsin (Rh*) in rod outer segment (ROS) membranes, the POG moiety interacted with not only Talpha and Tbeta but also Rh* and membrane phospholipids. The cross-linked phospholipid species was analyzed by ELISA employing a variety of lipid-binding probes, which revealed phosphatidylethanolamine (PE) and phosphatidylserine (PS) as selective phospholipids for POG interaction in the ROS membranes. These results demonstrate that the modifying group of Tgamma plays an active role in protein-protein and protein-membrane interactions and suggest that the farnesyl-PE/PS interaction may support the efficient formation of the signaling ternary complex between transducin and Rh*.
G蛋白γ亚基在C末端半胱氨酸残基处发生异戊二烯化和羧基甲基化，这一系列翻译后修饰对于光感受器G蛋白转导素（Tα/Tβγ）的功能不可或缺。为了探究法尼基介导的分子相互作用，我们研究了一种Tβγ类似物的分子靶点，该类似物经工程改造后，使其具有一个与Tγ的C末端半胱氨酸共价结合的光反应性法尼基类似物（3-叠氮苯氧基）香叶基（POG）。POG修饰的Tβγ进一步在C末端羧基处进行甲基化修饰，从而复制了Tβγ已知的完整转录后修饰。对游离形式的光反应性Tβγ类似物进行光亲和标记实验表明，Tγ的POG部分与Tβ相互作用。在三聚体Tα/Tβγ复合物中，除了对Tβ进行同时的亲和标记外，POG部分还与Tα发生交联。当光反应性Tβγ与Tα和杆状外段（ROS）膜中的光激活视紫红质（Rh*）重组时，POG部分不仅与Tα和Tβ相互作用，还与Rh和膜磷脂相互作用。通过使用各种脂质结合探针的酶联免疫吸附测定（ELISA）分析交联的磷脂种类，结果显示磷脂酰乙醇胺（PE）和磷脂酰丝氨酸（PS）是ROS膜中POG相互作用的选择性磷脂。这些结果表明，Tγ的修饰基团在蛋白质-蛋白质和蛋白质-膜相互作用中发挥着积极作用，并表明法尼基-PE/PS相互作用可能支持转导素和Rh之间信号三元复合物的有效形成。
