Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R) IDENTIFICATION OF A FUNCTIONAL LIGAND BINDING SITE
Pigment Epithelium-derived Factor (PEDF) Prevents Retinal Cell Death via PEDF Receptor (PEDF-R) IDENTIFICATION OF A FUNCTIONAL LIGAND BINDING SITE
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DOI：
10.1074/jbc.m113.487884
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发表时间：
2013-08-16
影响因子：
4.8
通讯作者：
Becerra, S. Patricia
中科院分区：
文献类型：
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作者：
Subramanian, Preeti;Locatelli-Hoops, Silvia;Becerra, S. Patricia
细胞外色素上皮衍生因子（PEDF）通过与细胞表面受体蛋白相互作用显示视网膜存活活性。我们以前报道过PEDF结合并刺激PEDF受体（PEDF- r），一种跨膜磷脂酶。然而，PEDF- r的PEDF结合位点及其与生存活性的关系尚未确定。这项工作的目的是在PEDF-R上确定一个生物学上相关的配体结合位点。PEDF结合PEDF- r外结构域L4 (Leu(159)-Met(325))，其亲和力与全长PEDF- r (Met（1)-Leu(504)）相似。结合实验表明，PEDF选择性地结合了E5b (Ile（193)-Leu(232)）和P1 (Thr（210)-Leu(249)）肽。重组c端截断的PEDF-R4 (Met（1)-Leu(232)）和内部截断的PEDF-R和PEDF-R4 (Delta His（203)-Leu(232)）保留了全长PEDF-R的磷脂酶活性。然而，没有His(203)-Leu（232）区域的PEDF- r多肽失去了PEDF亲和力，从而刺激了它们的酶活性。细胞表面标记表明，PEDF-R存在于视网膜细胞的质膜中。利用siRNA选择性地敲除视网膜细胞中的PEDF-R，我们证明了PEDF-R对pedf介导的细胞存活和抗凋亡活性至关重要。此外，PEDF与P1和E5b肽的预孵育可以阻断PEDF。PEDF- r介导视网膜细胞存活活性，这意味着肽与PEDF的结合排除了细胞表面配体与受体的相互作用。我们的研究结果表明，PEDF在视网膜细胞上的存活和抗凋亡作用需要PEDF- r，并且PEDF在其L4外畴内结合的决定因素对酶促刺激至关重要。
The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu(159)-Met(325)) with affinity similar to the full-length PEDF-R (Met(1)-Leu(504)). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile(193)-Leu(232)) and P1 (Thr(210)-Leu(249)) peptides. Recombinant C-terminal truncated PEDF-R4 (Met(1)-Leu(232)) and internally truncated PEDF-R and PEDF-R4 (Delta His(203)-Leu(232)) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His(203)-Leu(232) region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF.PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.