Chemical Inhibitors of Dynamin Exert Differential Effects in VEGF Signaling

Abstract

VEGFR2 is the main receptor and mediator of the vasculogenic and angiogenic activity of VEGF. Activated VEGFR2 internalizes through clathrin-mediated endocytosis and macropinocytosis. As dynamin is a key regulator of the clathrin pathway, chemical inhibitors of dynamin are commonly used to assess the role of the clathrin route in receptor signaling. However, drugs may also exert off-target effects. Here, we compare the effects of three dynamin inhibitors, dynasore, dyngo 4a and dynole, on VEGFR2 internalization and signaling. Although these drugs consistently inhibit clathrin-mediated endocytosis of both transferrin (a typical cargo of this route) and VEGFR2, surprisingly, they exert contradictory effects in receptor signaling. Thus, while dynasore has no effect on phosphorylation of VEGFR2, the other two drugs are strong inhibitors. Furthermore, although dyngo does not interfere with phosphorylation of Akt, dynasore and dynole have a strong inhibitory effect. These inconsistent effects suggest that the above dynamin blockers, besides inhibiting dynamin-dependent endocytosis of VEGFR2, exert additional inhibitory effects on signaling that are independent of endocytosis; i.e., they are due to off-target effects. Using a recently developed protocol, we comparatively validate the specificity of two endocytic inhibitors, dynasore and EIPA. Our findings highlight the importance of assessing whether the effect of an endocytic drug on signaling is specifically due to its interference with endocytosis or due to off-targets.

A universal method to analyze cellular internalization mechanisms via endocytosis without non‐specific cross‐effects

“…However, in many cases, the results were discussed without considering the cross‐effects of inhibitors. Thus, the specificity of these inhibitors and their potential unexpected effect on other cellular functions were not enough documented, although some published works indicated off‐target effects of endocytosis inhibitors 45–48 . We previously observed that well‐known endocytosis inhibitors (Table S1) had negligible inhibitory effects, lacked specificity on the cellular uptake of endocytosis markers (Tf, CTB, and dextran), or reduced cell viability 14 .…”

“…Thus, the specificity of these inhibitors and their potential unexpected effect on other cellular functions were not enough documented, although some published works indicated off-target effects of endocytosis inhibitors. [45][46][47][48] We previously observed that well-known endocytosis inhibitors (Table S1) had negligible inhibitory effects, lacked specificity on the cellular uptake of endocytosis markers (Tf, CTB, and dextran), or reduced cell viability. 14 For example, sucrose, a clathrin-mediated endocytosis inhibitor, completely abolished the cellular uptake of all endocytosis markers.…”

CRHR1-mediated Akt activation involves endocytosis and soluble adenylyl cyclase activity

“…Recent work showed that chemical inhibitors of dynamin may exert additional inhibitory effects on signaling independent of the blockage of its target dynamin. These off-target functions were evidenced when genetic approaches to block dynamin gave opposite results (Basagiannis et al, 2017; Basagiannis et al, 2021; Park et al, 2013; Persaud et al, 2018). We have previously demonstrated that both Dyngo-4a treatment and DynK44A overexpression block CRHR1 endocytosis after ligand stimulation causing a diminished cAMP response and a blockage of the sustained ERK1/2 activation phase in HT22-CRHR1 cells (Bonfiglio et al, 2013; Inda et al, 2016).…”

Adenomatous polyposis coli (APC) regulates internalization and signaling of the chemorepellent receptor, Roundabout (ROBO) 1

“…In Drosophila, Slit-induced endocytosis of Robo and subsequent axonal repulsion have been shown to be dynamin-and clathrin-dependent, and to require AP2-binding motifs present in the cytoplasmic domain of ROBO-1 (Chance and Bashaw, 2015). We, therefore, used Dyngo-4a, a potent dynamin inhibitor (McCluskey et al, 2013, Basagiannis et al, 2021 Similar to Robo in Drosophila (Chance and Bashaw, 2015), we found that human ROBO-1 also contains two AP2-binding motifs (Yxxφ) (Collins, 2002)…”