Murdaca Joseph, Treins Caroline, Monthouël-Kartmann Marie-Noëlle, Pontier-Bres Rodolphe, Kumar Sharad, Van Obberghen Emmanuel, Giorgetti-Peraldi Sophie
INSERM U145, Institut Federatif de Recherche 50, Faculte de Medecine, 06107 Nice Cedex 2, France.
J Biol Chem. 2004 Jun 18;279(25):26754-61. doi: 10.1074/jbc.M311802200. Epub 2004 Apr 1.
One of the cellular mechanisms used to prevent continuous and enhanced activation in response to growth factors is the internalization and degradation of their receptors. Little is known about the molecular mechanisms involved in vascular endothelial growth factor receptor-2 (VEGF-R2) degradation. In a previous work, we have shown that the adaptor protein Grb10 is a positive regulator of the VEGF signaling pathway. Indeed, VEGF stimulates Grb10 expression, and Grb10 overexpression induces an increase in the amount and the tyrosine phosphorylation of VEGF-R2. In the present manuscript, we demonstrate that Grb10 stimulates VEGF-R2 expression by inhibiting the Nedd4-mediated VEGF-R2 degradation. First, we show that proteasome inhibition by MG132 induces an increase in VEGF-R2 amount, and that VEGF-R2 is ubiquitinated in response to VEGF. Expression of Nedd4, a HECT domain-containing ubiquitin ligase, induces the disappearance of VEGF-R2 in cells, suggesting that Nedd4 is involved in VEGF-R2 degradation. To determine whether Nedd4 directly ubiquitinates VEGF-R2, we expressed a ubiquitin ligase-deficient mutant Nedd4C854S. In the presence of Nedd4C854S, VEGF-R2 is expressed and ubiquitinated. These results suggest that VEGF-R2 is ubiquitinated but that Nedd4 is not involved in this process. Finally, we show that Grb10 constitutively associates with Nedd4. Co-expression of Nedd4 and Grb10 restores the expression of VEGF-R2, suggesting that Grb10 inhibits the Nedd4-mediated degradation of VEGF-R2. In this study, we show that Grb10 acts as a positive regulator in VEGF-R2 signaling and protects VEGF-R2 from degradation by interacting with Nedd4, a component of the endocytic machinery.
用于防止因生长因子而持续和增强激活的细胞机制之一是其受体的内化和降解。关于血管内皮生长因子受体2（VEGF-R2）降解所涉及的分子机制知之甚少。在先前的工作中，我们已经表明衔接蛋白Grb10是VEGF信号通路的正向调节因子。事实上，VEGF刺激Grb10表达，并且Grb10过表达导致VEGF-R2的量和酪氨酸磷酸化增加。在本论文中，我们证明Grb10通过抑制Nedd4介导的VEGF-R2降解来刺激VEGF-R2表达。首先，我们表明MG132抑制蛋白酶体可导致VEGF-R2量增加，并且VEGF-R2在VEGF作用下会发生泛素化。含HECT结构域的泛素连接酶Nedd4的表达会导致细胞中VEGF-R2消失，这表明Nedd4参与VEGF-R2降解。为了确定Nedd4是否直接使VEGF-R2泛素化，我们表达了泛素连接酶缺陷型突变体Nedd4C854S。在存在Nedd4C854S的情况下，VEGF-R2表达并发生泛素化。这些结果表明VEGF-R2会发生泛素化，但Nedd4不参与此过程。最后，我们表明Grb10与Nedd4持续结合。Nedd4和Grb10共表达可恢复VEGF-R2的表达，这表明Grb10抑制Nedd4介导的VEGF-R2降解。在本研究中，我们表明Grb10在VEGF-R2信号传导中起正向调节作用，并通过与内吞机制的一个组分Nedd4相互作用来保护VEGF-R2不被降解。
