Dynasore impairs VEGFR2 signalling in an endocytosis-independent manner

Abstract

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.

A highly-sensitive high throughput assay for dynamin's basal GTPase activity

“…Moreover, the reported IC 50 values we measured for Dynasore and Dyngo-4a NT-stimulated GTPase activity performed at physiological salt concentrations using 100 nM dynamin were much higher than those reported for assays performed in the presence of sonicated PS liposomes, under low salt conditions with 20 nM dynamin (0.4 μM and 12 μM, respectively). Given the reported off-target effects of Dynasore and Dyngo-4a [ 16 – 18 ] and their uncertain mechanism of dynamin inhibition, a more robust and specific inhibitor of dynamin would be of immense value.…”

“…These off-target effects on endocytosis may reflect their reported ability to perturb plasma membrane cholesterol levels [ 17 ] and destabilize actin filaments [ 16 ]. Recently, Dynasore was shown to impair VEGFR2 signaling in an endocytosis-independent manner [ 18 ]. Based on the clear evidence for dynamin-independent, off-target effects of these compounds, there remains a need to develop more specific and robust dynamin inhibitors.…”

Intracellular transport and regulation of transcytosis across the blood–brain barrier

“…Coated pits characteristic of CME can be observed in BECs in brain tissue by electron microscopy [28]. Furthermore, multiple receptors that undergo RMT in BECs require CME for internalization, such as TfR [35, 36] and insulin receptor [37]. Despite the prevalence of CME, other clathrin-independent pathway(s) may also regulate RMT across the BBB.…”

Clathrin-mediated endocytosis regulates fMLP-mediated neutrophil polarization

“…Due to the potential off-target effects of Pitstop2 that has been reported to block clathrin-independent endocytosis [ 17 , 18 ], results obtained from using Pitstop2 are included in supplemental material. It is worth noting that there have also been non-specific effects reported for Dynasore [ 19 , 20 ]. In order to evaluate the effects of endocytosis inhibitors on neutrophil polarization in an objective manner, we used automated imaging and analysis of fixed cells labeling microtubule, actin, phospho-myosin II (pMyosinII) and DAPI ( Fig.…”

“…Taken together, perturbation of CME by Pitstop2 and Dynasore caused disruption of neutrophil polarization. Even though we cannot rule out off target effects of these small molecules on the cells [ 18 , 19 , 20 , 24 ], the finding that both small molecule inhibitors targeting endocytosis abrogated neutrophil polarization suggest that CME plays a crucial role in neutrophil polarization.…”