VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

Gareth W Fearnley,Stephen B Wheatcroft,Michael A Harrison,Mark T Kearney,Sreenivasan Ponnambalam,Gina A Smith,Nadira Yuldasheva,Izma Abdul-Zani,Ian C Zachary,Nadeem A Mughal,Shervanthi Homer-Vanniasinkam,Darren C Tomlinson

doi:10.1242/bio.017434

Gareth W Fearnley, Stephen B Wheatcroft + Show 10 more

Open Access

https://doi.org/10.1242/bio.017434

Copy DOI

Abstract

ABSTRACTVascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes.

Highlights

Vascular endothelial growth factor A (VEGF-A) is a soluble ligand that is essential for mammalian development and function (Carmeliet et al, 1996; Ferrara et al, 1996; Koch et al, 2011)
To test the idea that VEGF-A isoforms trigger differential VEGFR2 activation, we monitored the presence of such VEGFR2 phosphotyrosine-epitopes in human umbilical vein endothelial cells (HUVECs) in response to stimulation with different VEGF-A isoforms (1.25 nM; 020 min) using immunoblot analysis (Fig. 1A)
Three different VEGFA isoforms can bind with similar affinity to the extracellular domain of this receptor tyrosine kinase (RTK) (VEGFR2) but differentially program the cytoplasmic domain to acquire post-translational modifications, leading to specific patterns of trafficking and proteolysis (Fig. 6)

Summary

Introduction

Vascular endothelial growth factor A (VEGF-A) is a soluble ligand that is essential for mammalian development and function (Carmeliet et al, 1996; Ferrara et al, 1996; Koch et al, 2011). The underlying mechanism(s) by which VEGF-A isoforms act are still unclear, VEGF-A isoform-specific binding is implicated in recruiting a co-receptor called neuropilin 1 (NRP1) (Ballmer-Hofer et al, 2011; Harper and Bates, 2008; Herzog et al, 2011; Kawamura et al, 2008a,b; Pan et al, 2007; Tillo et al, 2015) This membrane receptor can bind both VEGFA165 and VEGF-A121 but only VEGF-A165 is believed to form a trimeric complex with VEGFR2 and NRP1 (Koch et al, 2011)

Methods

Results

Conclusion