Dynamin inhibitors impair platelet-derived growth factor β-receptor dimerization and signaling

Abstract

The role of plasma membrane composition and dynamics in the activation process of receptor tyrosine kinases (RTKs) is still poorly understood. In this study we have investigated how signaling via the RTK, platelet-derived growth factor β-receptor (PDGFR-β) is affected by Dynasore or Dyngo-4a, which are commonly used dynamin inhibitors. PDGFR-β preferentially internalizes via clathrin-coated pits and in this pathway, Dynamin II has a major role in the formation and release of vesicles from the plasma membrane by performing the membrane scission. We have found that dynamin inhibitors impedes the activation of PDGFR-β by impairing ligand-induced dimerization of the receptor monomers, which leads to a subsequent lack of phosphorylation and activation both of receptors and downstream effectors, such as ERK1/2 and AKT. In contrast, dynamin inhibitors did not affect epidermal growth factor receptor (EGFR) dimerization and phosphorylation. Our findings suggest that there is a link between plasma membrane dynamics and PDGFR-β activation, and that this link is not shared with the epidermal growth factor receptor.

A method for Boolean analysis of protein interactions at a molecular level

“…Upon ligand-induced activation, the membrane-bound PDGFR-β is mostly internalized via Clathrin-coated pits 34 , 35 , which has an important function in downstream signaling in the early endosomes 36 . It has been previously demonstrated with in situ PLA that upon PDGF-BB stimulation PDGFR-β in fibroblasts shows increased colocalization with Clathrin 37 . We therefore treated BJ h-TERT cells with PDGF-BB for 0 min (“control”) and 15 min (“PDGF-BB treated”) accordingly, and applied MolBoolean to quantify and compare the amounts of free Clathrin and free PDGFR-β, as well as the amount of dual signal under both conditions.…”

“…For the Clathrin–PDGFR-β assay, as described in ref. 37 , BJ-hTERT cells were starved overnight in DMEM starvation medium (0.2% FBS), and then either stimulated with 20 ng/mL PDGF-BB in for 15 min (“treated” condition), or left untreated in the same medium (“control” condition).…”

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

“…We hypothesized that the binding of Nsp3 to clathrin would interfere with clathrin-mediated endocytosis resulting in impaired internalization of activated PDGFRβ. We observed a sharp increase in PLA signal probing for activated PDGFRβ phosphorylated at Tyr751 58 10 min after activation with PDGF-BB in all four experimental setups. Consistent with our hypothesis, the signal decreased after 60 min in non-transfected cells, mock-transfected cells, or in cells transfected with a motif-mutant construct Nsp3 (EEEV) mut but persisted in cells transfected with wild-type Nsp3 (EEEV) (Fig.…”

Large-scale phage-based screening reveals extensive pan-viral mimicry of host short linear motifs

“…We hypothesized that the binding of Nsp3 to clathrin would interfere with clathrin-mediated endocytosis resulting in impaired internalization of activated PDGFRβ. We observed a sharp increase in PLA signal probing for activated PDGFRβ phosphorylated at Tyr751 (Heldin et al, 2019) 10 minutes after activation with PDGF- BB in all four experimental setups. Consistent with our hypothesis, the signal decreased after 60 minutes in non-transfected cells, mock-transfected cells, or in cells transfected with a motif-mutant construct Nsp3 (EEEV) mut but persisted in cells transfected with wild-type Nsp3 (EEEV).…”