Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
J Biol Chem. 2010 Apr 23;285(17):12595-603. doi: 10.1074/jbc.M109.008581. Epub 2010 Feb 22.
Urokinase plasminogen activator receptor (u-PAR) binds urokinase plasminogen activator (u-PA) and participates in plasminogen activation in addition to modulating several cellular processes such as adhesion, proliferation, and migration. u-PAR is susceptible to proteolysis by its cognate ligand and several other proteases. To elucidate the biological significance of receptor cleavage by u-PA, we engineered and expressed a two-chain urokinase plasminogen activator (tcu-PA) cleavage-resistant u-PAR (cr-u-PAR). This mutated receptor was similar to wild-type u-PAR in binding u-PA and initiating plasminogen activation. However, cr-u-PAR exhibited accelerated internalization and resurfacing due to direct association with the endocytic receptor alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein in the absence of the enzyme x inhibitor complex of tcu-PA and plasminogen activator inhibitor-1 (tcu-PA.PAI-1). cr-u-PAR-expressing cells had enhanced migration compared with wild-type u-PAR-expressing cells, and cr-u-PAR was less sensitive to chymotrypsin cleavage as compared with wt u-PAR. Our studies suggest that these mutations in the linker region result in a rearrangement within the cr-u-PAR structure that makes it resemble its ligand-bound form. This constitutively active variant may mimic highly glycosylated cleavage-resistant u-PAR expressed in certain highly malignant cancer-cells.
尿激酶型纤溶酶原激活物受体(u-PAR)结合尿激酶型纤溶酶原激活物(u-PA),并参与纤溶酶原的激活,除此之外还调节几种细胞过程,如黏附、增殖和迁移。u-PAR 易受其同源配体和几种其他蛋白酶的蛋白水解。为了阐明 u-PA 对受体切割的生物学意义,我们设计并表达了一种两链尿激酶纤溶酶原激活物(tcu-PA)切割抗性 u-PAR(cr-u-PAR)。这种突变受体在结合 u-PA 和启动纤溶酶原激活方面与野生型 u-PAR 相似。然而,由于与内吞受体 α2-巨球蛋白受体/低密度脂蛋白受体相关蛋白的直接关联,cr-u-PAR 表现出加速的内化和再循环,而没有 tcu-PA 和纤溶酶原激活物抑制剂-1(tcu-PA.PAI-1)的酶 x 抑制剂复合物。与表达野生型 u-PAR 的细胞相比,表达 cr-u-PAR 的细胞具有增强的迁移能力,并且与 wt u-PAR 相比,cr-u-PAR 对糜蛋白酶切割的敏感性较低。我们的研究表明,这些连接区的突变导致 cr-u-PAR 结构内的重排,使其类似于其配体结合形式。这种组成型激活的变体可能模拟某些高度恶性癌细胞中表达的高度糖基化的切割抗性 u-PAR。