Institute of Molecular Biology and Biotechnology-Biomedical Research, Foundation for Research and Technology, 45110 Ioannina, Greece.
Laboratory of Biological Chemistry, Department of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, Greece.
Sci Rep. 2017 Mar 22;7:45035. doi: 10.1038/srep45035.
VEGFR2 is a critical angiogenic receptor playing a key role in vascular homeostasis. Upon activation by VEGF, VEGFR2 becomes endocytosed. Internalisation of VEGFR2 is facilitated, in part, through clathrin mediated endocytosis (CME), the role of which in VEGFR2 function is debated. Here, we confirm the contribution of CME in VEGFR2 uptake. However, curiously, we find that different approaches of inhibition of CME exert contradictory effects on VEGF signalling; knockdown of clathrin, or of dynamin, or overexpression of dynamin K44A, do not affect VEGF-induced phosphorylation of ERK1/2, while dynasore causes strong inhibition. We resolve this discrepancy by showing that although dynasore inhibits CME of VEGFR2, its inhibitory action in ERK1/2 phosphorylation is not related to attenuation of VEGFR2 endocytosis; it is rather due to an off-target effect of the drug. Dynasore inhibits VEGF-induced calcium release, a signalling event that lies upstream of ERK1/2, which implies that this effect could be responsible, at least in part, for the inhibitory action of the drug on VEGF-to-ERK1/2 signalling. These results raise caution that although dynasore is specific in inhibiting clathrin- and dynamin-mediated endocytosis, it may also exert off-target effects on signalling molecules, hence influencing the interpretation of the role of endocytosis in signalling.
VEGFR2 是一种关键的血管生成受体，在血管稳态中发挥着关键作用。VEGF 激活 VEGFR2 后，VEGFR2 被内吞。网格蛋白介导的内吞作用（CME）部分促进了 VEGFR2 的内化，其在 VEGFR2 功能中的作用存在争议。在这里，我们证实了 CME 在 VEGFR2 摄取中的作用。然而，奇怪的是，我们发现 CME 抑制的不同方法对 VEGF 信号产生相反的影响；网格蛋白、动力蛋白的敲低或动力蛋白 K44A 的过表达都不会影响 VEGF 诱导的 ERK1/2 磷酸化，而 dynasore 则会强烈抑制。我们通过显示 dynasore 虽然抑制了 VEGFR2 的 CME，但它在 ERK1/2 磷酸化中的抑制作用与 VEGFR2 内吞作用的衰减无关，而是由于药物的脱靶效应。Dynasore 抑制了 VEGF 诱导的钙释放，这是一个位于 ERK1/2 上游的信号事件，这意味着这种效应至少部分解释了药物对 VEGF-ERK1/2 信号的抑制作用。这些结果提醒人们注意，尽管 dynasore 特异性地抑制网格蛋白和动力蛋白介导的内吞作用，但它也可能对信号分子产生脱靶效应，从而影响对内吞作用在信号中的作用的解释。
