Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis.
Identification of polycystin-1 and Gα12 binding regions necessary for regulation of apoptosis.
复制标题
鉴定调节细胞凋亡所需的多囊蛋白-1 和Gα12 结合区域。
DOI：
10.1016/j.cellsig.2010.09.005
复制
发表时间：
2011
影响因子：
4.8
通讯作者：
Denker,BradleyM
中科院分区：
文献类型：
--
作者：
Yu,Wanfeng;Ritchie,BenjaminJ;Su,Xuefeng;Zhou,Jing;Meigs,ThomasE;Denker,BradleyM
大多数常染色体显性遗传性多囊肾病（ADPKD）患者携带PKD 1突变，PKD 1是多囊蛋白-1（PC 1）的基因，PC 1是一种具有细胞质C末端的跨膜蛋白，可与多种信号分子（包括Gα12）相互作用。PC 1的功能和囊肿发展导致肾功能衰竭的机制是复杂的。最近，我们报道了PC 1表达水平调节Gα12刺激的细胞凋亡的活性（Yu et al.，2010 285（14）：10243-51）。在此，对Gα12和PC 1进行突变分析，以鉴定它们相互作用所需的区域和调节凋亡的能力。在GST下拉中，检测了一组用NAAIRS系统性替换6个氨基酸的Gα12突变与PC 1 C-末端的结合。此外，检查了PC 1 C末端内的一系列缺失与Gα12的结合。我们在Gα12中鉴定了3个NAAIRS取代，完全废除了结合，并鉴定了先前描述的在PC 1 C-末端的74个氨基酸Gαi/o结合结构域，其为Gα12相互作用所必需的。在细胞凋亡试验中，使用具有诱导型PC 1过表达的HEK 293细胞研究了PC 1/Gα12结合解偶联的功能后果。PC 1结合缺陷的Gα12突变体对PC 1抑制Gα12刺激的细胞凋亡不敏感。同样地，从PC 1胞质结构域中删除Gα12相互作用序列也消除了其对Gα12刺激的细胞凋亡的抑制。基于Gα12的晶体结构，PC 1相互作用位点可能位于G蛋白螺旋结构域内的暴露区域。这些结构细节应有助于设计试剂来解偶联ADPKD中的PC 1/Gα12信号传导。
Most patients with autosomal dominant polycystic kidney disease (ADPKD) harbor mutations in PKD1, the gene for polycystin-1 (PC1), a transmembrane protein with a cytoplasmic C-terminus that interacts with numerous signaling molecules, including Gα12. The functions of PC1 and the mechanisms of cyst development leading to renal failure are complex. Recently, we reported that PC1 expression levels modulate activity of Gα12-stimulated apoptosis (Yu et al., J. Biol. Chem. 2010 285(14):10243-51). Herein, a mutational analysis of Gα12 and PC1 was undertaken to identify regions required for their interaction and ability to modulate apoptosis. A set of Gα12 mutations with systematic replacement of six amino acids with NAAIRS was tested for binding to the PC1 C-terminus in GST pulldowns. Additionally, a series of deletions within the PC1 C-terminus was examined for binding to Gα12. We identified 3 NAAIRS substitutions in Gα12 that completely abrogated binding, and identified a previously described 74 amino acid Gαi/o binding domain in the PC1 C-terminus as necessary for Gα12 interaction. The functional consequences of uncoupling PC1/Gα12 binding were studied in apoptosis assays utilizing HEK293 cells with inducible PC1 overexpression. Gα12 mutants deficient in PC1 binding were refractory to PC1 inhibition of Gα12-stimulated apoptosis. Likewise, deletion of the Gα12-interacting sequence from the PC1 cytoplasmic domain abrogated its inhibition of Gα12-stimulated apoptosis. Based on the crystal structure of Gα12, the PC1 interaction sites are likely to reside on exposed regions within the G protein helical domain. These structural details should facilitate the design of reagents to uncouple PC1/Gα12 signaling in ADPKD.