KDR Stimulates Endothelial Cell Migration through Heterotrimeric G Protein Gq/11-mediated Activation of a Small GTPase RhoA

Huiyan Zeng,Dezheng Zhao,Debabrata Mukhopadhyay

doi:10.1074/jbc.m206133200

Abstract

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on the primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell (EC) proliferation and migration, whereas Flt-1 down-modulates KDR-mediated EC proliferation. Flt-1 mediates down-regulation of EC proliferation through pertussis toxin-sensitive G proteins, betagamma subunits, small GTPase CDC42, and partly by Rac-1. However, the molecular mechanism by which KDR mediates EC migration is not clear yet. Here we show for the first time that activation of RhoA and Rac1 is fully and partially required for KDR-mediated human umbilical vein endothelial cell (HUVEC) migration, respectively, and that CDC42, however, is not involved. Furthermore, overexpression of the RhoA dominant negative mutant RhoA-19N does not affect VPF/VEGF-stimulated KDR phosphorylation, intracellular Ca(2+) mobilization, and mitogen-activated protein kinase phosphorylation. Utilizing the receptor chimeras (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor (EGFR) was fused to the transmembrane domain and the intracellular domains of KDR and Flt-1, respectively, we demonstrate that RhoA activation is mediated by EGDR, not by EGLT, and that EGDR mediates activation of Rac1, not CDC42. Furthermore, the EGDR-mediated RhoA and Rac1 activation is regulated by G proteins Gq/11, Gbetagamma, and phospholipase C independent of phosphatidylinositol 3-kinase and intracellular Ca(2+) mobilization. Interestingly, the RhoA activation can be partially inhibited by overexpression of Rac1-17N, but overexpression of RhoA-19N has no effect on Rac1 activation. Finally, Gq/11 and Gbetagamma subunits are also required for VPF/VEGF-stimulated HUVEC migration. Taken together, our results indicate that KDR stimulates endothelial cell migration through a heterotrimeric G protein Gq/11 and Gbetagamma-mediated RhoA pathway.

Highlights

VPF/VEGF1 is a multifunctional cytokine that is required for tumorigenesis as well as vasculogenesis [1]
Effect of the Dominant Negative Mutants of Rho Family GTPases on VPF/VEGF-stimulated human umbilical vein endothelial cell (HUVEC) Migration—Recently, we have shown [12] that dominant negative mutants of RhoA, Rac1, and Cdc42 (RhoA-19N, Rac1–17N, and CDC42– 17N) that block their endogenous functions have different effects on VPF/VEGF-stimulated HUVEC proliferation
In this study we further examined whether the dominant negative mutants CDC42– 17N, RhoA-19N, and Rac1–17N have any effects on VPF/ VEGF-stimulated HUVEC migration

Summary

Introduction

VPF/VEGF1 is a multifunctional cytokine that is required for tumorigenesis as well as vasculogenesis [1]. We show for the first time that activation of RhoA and Rac1 is fully and partially required for KDR-mediated human umbilical vein endothelial cell (HUVEC) migration, respectively, and that CDC42, is not involved. We show that Gq/11 proteins and G␤␥ subunits are required for KDR-mediated RhoA and Rac1 activation and for VPF/VEGF-stimulated HUVEC migration.

Results

Conclusion