The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization
The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization
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DOI：
10.1074/jbc.m208456200
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发表时间：
2003-03-21
影响因子：
4.8
通讯作者：
Artemyev, NO
中科院分区：
文献类型：
--
作者：
Muradov, KG;Boyd, KK;Artemyev, NO
视网膜杆cGMP磷酸二酯酶（PDE 6家族）是脊椎动物视觉信号转导级联反应中的效应酶。与形成催化同二聚体的其他已知PDE不同，棒状PDE 6催化核心是由α和β亚基组成的异二聚体。用于杆PDE 6的有效表达的系统不可用。因此，为了阐明杆PDE 6特异性二聚化的结构基础，我们构建了一系列PDE 6 α和PDE 5之间的嵌合蛋白，其含有杆酶的N-末端GAFa/GAFb结构域或其部分。这些嵌合体在Sf 9细胞中以各种组合共表达为His-、myc-或FLAG-标记的蛋白。使用凝胶过滤和蔗糖梯度离心评估嵌合PDE的二聚化。用Western印迹和免疫沉淀法分析形成的二聚体酶的组成。与体内PDE 6二聚化的选择性一致，在PDE 6 α和PDE 6 β的GAF区之间观察到有效的异源二聚化，而没有显著的同源二聚化。此外，PDE 6 α能够与锥PDE 6 α '亚基形成二聚体。此外，我们的分析表明，PDE 6 GAFa结构域包含PDE 6催化亚基二聚化的亲和力和选择性的主要结构决定因素。PDE 6的关键二聚化选择性模块已定位于GAFa结构域PDE 6 α-59-74/PDE6 β-57-72内的小片段。这项研究提供了工具，用于生成同源二聚体的α-和β-酶，这将使我们能够解决杆PDE 6的独特异源二聚化的功能意义的问题。
Retinal rod cGMP phosphodiesterase (PDE6 family) is the effector enzyme in the vertebrate visual transduction cascade. Unlike other known PDEs that form catalytic homodimers, the rod PDE6 catalytic core is a heterodimer composed of alpha and beta subunits. A system for efficient expression of rod PDE6 is not available. Therefore, to elucidate the structural basis for specific dimerization of rod PDE6, we constructed a series of chimeric proteins between PDE6alphabeta and PDE5, which contain the N-terminal GAFa/GAFb domains, or portions thereof, of the rod enzyme. These chimeras were co-expressed in Sf9 cells in various combinations as His-, myc-, or FLAG-tagged proteins. Dimerization of chimeric PDEs was assessed using gel filtration and sucrose gradient centrifugation. The composition of formed dimeric enzymes was analyzed with Western blotting and immunoprecipitation. Consistent with the selectivity of PDE6 dimerization in vivo, efficient heterodimerization was observed between the GAF regions of PDE6alpha and PDE6beta with no significant homodimerization. In addition, PDE6alpha was able to form dimers with the cone PDE6alpha' subunit. Furthermore, our analysis indicated that the PDE6 GAFa domains contain major structural determinants for the affinity and selectivity of dimerization of PDE6 catalytic subunits. The key dimerization selectivity module of PDE6 has been localized to a small segment within the GAFa domains, PDE6alpha-59-74/PDE6beta-57-72. This study provides tools for the generation of the homodimeric alphaalpha and betabeta enzymes that will allow us to address the question of functional significance of the unique heterodimerization of rod PDE6.