Dynasore impairs VEGFR2 signalling in an endocytosis-independent manner.
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.
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.
Dimitris
D
Basagiannis
Institute of Molecular Biology and Biotechnology-Biomedical Research, Foundation for Research and Technology, 45110 Ioannina, Greece.
Sofia
S
Zografou
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.
Katerina
K
Galanopoulou
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.
Savvas
S
Christoforidis
D002118:Calcium / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology*; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008954:Models, Biological; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D002966:Clathrin; D006835:Hydrazones; C511794:N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide; D034741:RNA, Small Interfering; D040301:Vascular Endothelial Growth Factor Receptor-2; D034281:Dynamins; D002118:Calcium