WDR36 acts as a scaffold protein tethering a G-protein-coupled receptor, Gαq and phospholipase Cβ in a signalling complex
WDR36 acts as a scaffold protein tethering a G-protein-coupled receptor, Gαq and phospholipase Cβ in a signalling complex
复制标题
DOI：
10.1242/jcs.085795
复制
发表时间：
2011-10-01
影响因子：
4
通讯作者：
Parent, Jean-Luc
中科院分区：
文献类型：
--
作者：
Cartier, Andreane;Parent, Audrey;Parent, Jean-Luc
我们通过酵母双杂交筛选鉴定了含有WD重复序列的蛋白质WDR 36，它是血栓素A（2）受体β亚型（TP β）的相互作用伴侣。我们证明，WDR 36直接相互作用的C-末端和第一个细胞内环的TP β在体外GST-下拉测定。通过免疫共沉淀观察到细胞背景下的相互作用，其受到TP β刺激的积极影响。在未刺激的HEK 293细胞中，通过共聚焦显微镜在质膜处检测到TP β-WDR 36共定位，但在受体刺激后，复合物易位到细胞内囊泡。共表达WDR 36及其siRNA介导的敲除分别增加和抑制TP β诱导的G α q信号传导。有趣的是，WDR 36与G α q共免疫沉淀，并促进TP β-G α q相互作用。WDR 36还与磷脂酶C β（PLC β）相关，并增加G α q和PLC β之间的相互作用，但阻止GRK 2对激活的G α q的螯合。此外，WDR 36的表达增强了PLC β免疫沉淀物中TP β的存在。最后，WDR 36的疾病相关变体影响其通过TP β调节G α q介导的信号传导的能力。我们报告说，WDR 36作为一种新的支架蛋白拴在一个信号复合物的G蛋白偶联受体，G α q和PLC β。
We identified the WD-repeat-containing protein, WDR36, as an interacting partner of the beta isoform of thromboxane A(2) receptor (TP beta) by yeast two-hybrid screening. We demonstrated that WDR36 directly interacts with the C-terminus and the first intracellular loop of TP beta by in vitro GST-pulldown assays. The interaction in a cellular context was observed by co-immunoprecipitation, which was positively affected by TP beta stimulation. TP beta-WDR36 colocalization was detected by confocal microscopy at the plasma membrane in non-stimulated HEK293 cells but the complex translocated to intracellular vesicles following receptor stimulation. Coexpression of WDR36 and its siRNA-mediated knockdown, respectively, increased and inhibited TP beta-induced G alpha q signalling. Interestingly, WDR36 co-immunoprecipitated with G alpha q, and promoted TP beta-G alpha q interaction. WDR36 also associated with phospholipase C beta (PLC beta) and increased the interaction between G alpha q and PLC beta, but prevented sequestration of activated G alpha q by GRK2. In addition, the presence of TP beta in PLC beta immunoprecipitates was augmented by expression of WDR36. Finally, disease-associated variants of WDR36 affected its ability to modulate G alpha q-mediated signalling by TP beta. We report that WDR36 acts as a new scaffold protein tethering a G-protein-coupled receptor, G alpha q and PLC beta in a signalling complex.