Ectodomain cleavage of FLT1 regulates receptor activation and function and is not required for its downstream intracellular cleavage.
Ectodomain cleavage of FLT1 regulates receptor activation and function and is not required for its downstream intracellular cleavage.
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FLT1 的胞外域裂解调节受体的激活和功能，并且不是其下游细胞内裂解所必需的。
DOI：
10.1016/j.yexcr.2016.03.020
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发表时间：
2016
影响因子：
3.7
通讯作者：
Thomas,ChristieP
中科院分区：
文献类型：
--
作者：
Raikwar,NanditaS;Liu,KangZ;Thomas,ChristieP
FLT1 是一种细胞表面 VEGF 受体，被裂解后释放 N 末端胞外域，该胞外域结合 VEGF 和 PlGF，并可以拮抗 VEGF 在细胞外环境中的作用。为了进一步评估 FLT1 加工，我们在 HEK293 和 COS7 细胞中表达标记的 FLT1 构建体，通过删除图谱证明，切割位点紧邻残基 759 和 763 之间的跨膜结构域 (TMD)。切割相互调节条件培养基中的游离 VEGF，并且我们表明切割位点也可转移到另一个跨膜受体。胞外域裂解下游的第二次裂解事件释放胞质 C 端 FLT1 片段，并且 FLT1 的这种细胞内裂解不受上游胞外域裂解的催化或调节，因为胞外域裂解的消除对下游裂解事件没有影响。下游裂解事件对γ-分泌酶抑制剂和早老素1的过度表达不敏感，γ-分泌酶的催化亚基不改变下游细胞内裂解事件。此外，这种裂解并不是通过先前发表的 FLT1 TMD 中的缬氨酸残基 (767 V) 发生的，表明存在另一种裂解途径。我们测试了胞外域切割对 p44/42 MAP 激酶激活的影响，并证明与野生型 FLT1 相比，抗切割 FLT1 构建体无法刺激 p44/42 MAP 激酶激活。我们的结果表明，FLT1 胞外域裂解不仅调节细胞外环境中游离 VEGF 的可用性，而且还通过 ERK 激酶途径调节细胞信号传导。
FLT1 is a cell surface VEGF receptor which is cleaved to release an N-terminal ectodomain which binds VEGF and PlGF and can antagonize the effects of VEGF in the extracellular milieu. To further evaluate FLT1 processing we expressed tagged FLT1 constructs in HEK293 and COS7 cells where we demonstrate, by deletion mapping, that the cleavage site is immediately adjacent to the transmembrane domain (TMD) between residues 759 and 763. Cleavage reciprocally regulates free VEGF in conditioned media and we show that the cleavage site is also transferable to another transmembrane receptor. A second cleavage event downstream of the ectodomain cleavage releases a cytosolic C-terminal FLT1 fragment and this intracellular cleavage of FLT1 is not catalyzed or regulated by the upstream ectodomain cleavage since abolition of the ectodomain cleavage has no impact on the downstream cleavage event. The downstream cleavage event is not susceptible to γ-secretase inhibitors and overexpression of presenilin 1, the catalytic subunit of γ-secretase did not change the downstream intracellular cleavage event. Furthermore, this cleavage did not occur via a previously published valine residue (767 V) in the TMD of FLT1, indicating the existence of another cleavage pathway. We tested the impact of the ectodomain cleavage on p44/42 MAP kinase activation and demonstrate that compared to wild type FLT1, cleavage resistant FLT1 constructs failed to stimulate p44/42 MAP kinase activation. Our results indicate that FLT1 ectodomain cleavage not only regulates the availability of free VEGF in the extracellular milieu but also regulates cellular signaling via the ERK kinase pathway.