Frizzled-7 turnover at the plasma membrane is regulated by cell density and the Ca(2+) -dependent protease calpain-1.
Frizzled-7 turnover at the plasma membrane is regulated by cell density and the Ca(2+) -dependent protease calpain-1.
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DOI：
10.1016/j.yexcr.2007.07.012
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发表时间：
2007-10
影响因子：
3.7
通讯作者：
I. Struewing;Corey D. Barnett;Wei Zhang;S. Yadav;Catherine D. Mao
中科院分区：
文献类型：
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作者：
I. Struewing;Corey D. Barnett;Wei Zhang;S. Yadav;Catherine D. Mao
卷曲的是七个跨膜结构域g蛋白偶联受体，参与细胞极性和Wnt信号传导。调节它们在质膜上转换的机制尚不清楚。我们通过异位表达N-和c -末端标记的frizzled7蛋白，在内皮细胞中发现了frizzled7的c -末端切割。这种特异性的切割产生了一个10 kDa的c端片段，该片段仍然与细胞内囊泡相关，并通过n端测序和靶向诱变定位于第三胞质内环。卷曲-7突变形式显示c端切割减少，在质膜上也存在dvl2易位缺陷。PMA是PKC和内吞作用的激活剂，而Wnt13A和Wnt5A则不是，PMA增加了含c末端的frizzled7囊泡的外观和frizzled7的切割。Concanavalin-A是一种受体内化抑制剂，可降低组成型和pma诱导的frizzled7切割，而Δ95-295-Eps15显性阴性抑制内吞途径仅能阻止pma诱导的frizzled7切割。Frizzled-7 c -末端的分裂随着细胞密度和Ca2+离子载体离子霉素的作用而增加，而通过特异性calpain抑制剂、DN-calpain-1的表达和sirna对calpain-1水平的下调而减少。总之，我们的研究结果明确了calpain-1是质膜上frizzled7转换的调节因子，并揭示了frizzled7切割与其活性之间的联系。
Frizzled are seven-transmembrane domain G-protein coupled receptors involved in cell polarity and Wnt signaling. The mechanisms regulating their turnover at the plasma membrane remain unclear. We have identified a regulated C-terminus cleavage of Frizzled-7 in endothelial cells using ectopic expression of N- and C-termini-tagged Frizzled-7 proteins. This specific cleavage produced a 10 kDa C-terminus fragment that remained associated with intracellular vesicles and was localized within the 3rd intracytoplasmic loop using N-terminal sequencing and targeted mutagenesis. Frizzled-7 mutated forms displaying reduced C-terminus cleavage were also defective for dvl2 translocation at the plasma membrane. PMA, an activator of PKC and endocytosis, but not Wnt13A and Wnt5A, increased the appearance of Frizzled-7 C-terminus-containing vesicles and Frizzled-7 cleavage. Concanavalin-A, an inhibitor of receptor internalization decreased both constitutive and PMA-induced Frizzled-7 cleavage, while inhibition of the endocytic pathway with Δ95–295-Eps15 dominant-negative prevented only PMA-induced Frizzled-7 cleavage. Frizzled-7 C-terminus cleavage was increased with cell density and by the Ca2+ionophore ionomycin and was decreased by specific calpain inhibitors, by the expression of DN-calpain-1 and the down-regulation of calpain-1 levels by siRNAs. Altogether, our findings pinpoint calpain-1 as a regulator of Frizzled-7 turnover at the plasma membrane and reveal a link between Frizzled-7 cleavage and its activity.