Raikwar Nandita S, Liu Kang Z, Thomas Christie P
Departments of Internal Medicine, University of Iowa Carver College, United States.
Departments of Internal Medicine, University of Iowa Carver College, United States; Departments of Pediatrics, University of Iowa Carver College, United States; Departments of Obstetrics, University of Iowa Carver College, United States; Program in Molecular and Cellular Biology, University of Iowa Carver College of Medicine, United States; Departments of Veterans Affairs Medical Center, Iowa City, IA, United States.
Exp Cell Res. 2016 May 15;344(1):103-111. doi: 10.1016/j.yexcr.2016.03.020. Epub 2016 Mar 23.
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 (767V) 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.
FLT1是一种细胞表面VEGF受体，它被切割后释放出一个N端胞外结构域，该结构域可结合VEGF和PlGF，并能在细胞外环境中拮抗VEGF的作用。为了进一步评估FLT1的加工过程，我们在HEK293和COS7细胞中表达了带有标签的FLT1构建体，通过缺失定位，我们证明切割位点紧邻跨膜结构域（TMD），位于第759和763位氨基酸残基之间。切割可相互调节条件培养基中游离VEGF的水平，并且我们发现切割位点也可转移至另一种跨膜受体。胞外结构域切割下游的第二次切割事件释放出一个胞质C端FLT1片段，FLT1的这种细胞内切割不受上游胞外结构域切割的催化或调节，因为消除胞外结构域切割对下游切割事件没有影响。下游切割事件对γ-分泌酶抑制剂不敏感，γ-分泌酶的催化亚基早老素1的过表达也未改变下游细胞内切割事件。此外，这种切割并非通过先前报道的FLT1跨膜结构域中的缬氨酸残基（767V）发生，这表明存在另一种切割途径。我们测试了胞外结构域切割对p44/42 MAP激酶激活的影响，结果表明与野生型FLT1相比，抗切割的FLT1构建体无法刺激p44/42 MAP激酶的激活。我们的结果表明，FLT1胞外结构域切割不仅调节细胞外环境中游离VEGF的可用性，还通过ERK激酶途径调节细胞信号传导。
