VEGF-A selectively inhibits FLT1 ectodomain shedding independent of receptor activation and receptor endocytosis.
VEGF-A selectively inhibits FLT1 ectodomain shedding independent of receptor activation and receptor endocytosis.
复制标题
VEGF-A 选择性抑制 FLT1 胞外域脱落，与受体激活和受体内吞作用无关。
DOI：
10.1152/ajpcell.00247.2017
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Thomas,ChristieP
中科院分区：
文献类型：
--
作者：
Raikwar,NanditaS;Shibuya,Masabumi;Thomas,ChristieP
胞外结构域的脱落和受调控的胞内蛋白分解可以决定细胞表面蛋白的命运或功能。FMS相关酪氨酸激酶(Flt)或血管内皮生长因子受体1是一种高亲和力的细胞表面血管内皮生长因子A受体酪氨酸激酶，它被结构性地切割释放出NH2末端的血管内皮生长因子A结合的胞外结构域，一旦脱落，就可以拮抗血管内皮生长因子A在细胞外环境中的作用。我们评估了血管内皮生长因子-A在天然细胞和瞬时和稳定表达系统中对Flt1裂解的影响。我们证明，VEGF-A以时间和剂量依赖的方式抑制Flt1胞外区域的切割，而在HEK293细胞中敲除VEGF-A会增加胞外区域的脱落。尽管与Flt1类似，KDR或VEGF受体2也可被细胞外和细胞内切割，但VEGF-A并不抑制KDR的切割。VEGF-A对Flt1裂解的抑制不依赖于Flt1的酪氨酸激酶活性或细胞内的Flt1残基。蛋白酶体抑制剂N-乙酰亮氨酰去甲亮氨酸(ALLN)、内体酸化抑制剂巴菲霉素A和动力蛋白抑制剂DATORE均增加了Flt1的丰度和脱落的胞外结构域，表明Flt1受动力蛋白介导的内吞作用的影响，并对蛋白酶体和溶酶体的降解敏感。血管内皮生长因子-A对切割的抑制不能被ALLN、巴菲霉素A或王朝逆转。然而，细胞外免疫球蛋白7结构域30个氨基酸的缺失导致Flt1的切割增强，从而显著降低了对血管内皮生长因子的抑制作用。我们的结果表明，VEGF-A对Flt1胞外区切割的抑制既不依赖于受体的激活，也不依赖于内化，也不是受体降解的结果，可能是对受体切割的直接抑制作用。
Ectodomain shedding and regulated intracellular proteolysis can determine the fate or function of cell surface proteins. Fms-related tyrosine kinase (FLT) or VEGF receptor 1 is a high-affinity cell surface VEGF-A receptor tyrosine kinase that is constitutively cleaved to release an NH2-terminal VEGF-A binding ectodomain that, once shed, can antagonize the effects of VEGF-A in the extracellular milieu. We evaluated the effect of VEGF-A on FLT1 cleavage in native cells and in transient and stable expression systems. We demonstrate that VEGF-A inhibits FLT1 ectodomain cleavage in a time- and dose-dependent manner, whereas VEGF-A knockdown in HEK293 cells increases ectodomain shedding. Although kinase insert domain receptor (KDR) or VEGF receptor 2, analogous to FLT1, is also subject to extracellular and intracellular cleavage, VEGF-A does not inhibit KDR cleavage. VEGF-A inhibition of FLT1 cleavage is not dependent on FLT1 tyrosine kinase activity or the intracellular FLT1 residues. N-acetylleucylleucylnorleucinal (ALLN), a proteasomal inhibitor; bafilomycin A, an inhibitor of endosomal acidification; and dynasore, a dynamin inhibitor, all increase the abundance of FLT1 and the shed ectodomain, indicating that FLT1 is subject to dynamin-mediated endocytosis and susceptible to proteasomal and lysosomal degradation. VEGF-A inhibition of cleavage is not reversed by ALLN, bafilomycin A, or dynasore. However, a 30 AA deletion in the extracellular immunoglobulin 7 domain leads to enhanced cleavage of Flt1 with a significant reduction of the VEGF inhibitory effect. Our results indicate that the inhibition of FLT1 ectodomain cleavage by VEGF-A is dependent neither on receptor activation nor on internalization nor a consequence of receptor degradation and likely represents a direct inhibitory effect on receptor cleavage.