Heterotrimeric Gαq/Gα11 Proteins Function Upstream of Vascular Endothelial Growth Factor (VEGF) Receptor-2 (KDR) Phosphorylation in Vascular Permeability Factor/VEGF Signaling

Huiyan Zeng,Dezheng Zhao,Suping Yang,Kaustubh Datta,Debabrata Mukhopadhyay

doi:10.1074/jbc.m209712200

Abstract

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor-tyrosine kinases, Flt-1 (VEGF receptor (VEGFR)-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell proliferation and migration, whereas Flt-1 down-modulates KDR-mediated endothelial cell proliferation. Our most recent works show that pertussis toxin-sensitive G proteins and Gbetagamma subunits are required for Flt-1-mediated down-regulation of human umbilical vein endothelial cell (HUVEC) proliferation and that Gq/11 proteins are required for KDR-mediated RhoA activation and HUVEC migration. In this study, we demonstrate that Gq/11 proteins are also required for VPF/VEGF-stimulated HUVEC proliferation. Our results further indicate that Gq/11 proteins specifically mediate KDR signaling such as intracellular Ca2+ mobilization rather than Flt-1-induced CDC42 activation and that a Gq/11 antisense oligonucleotide completely inhibits MAPK phosphorylation induced by KDR but has no effect on Flt-1-induced MAPK activation. More importantly, we demonstrate that Gq/11 proteins interact with KDR in vivo, and the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation. Surprisingly, the Gq/11 antisense oligonucleotide completely inhibits VPF/VEGF-stimulated KDR phosphorylation. Expression of a constitutively active mutant of G11 but not Gq can cause phosphorylation of KDR and MAPK. In addition, a Gbetagamma minigene, hbetaARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Taken together, this study demonstrates that Gq/11 proteins mediate KDR tyrosine phosphorylation and KDR-mediated HUVEC proliferation through interaction with KDR.

Highlights

Kines and growth factors, such as vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF),1 basic fibroblast growth factor, platelet-derived growth factor (PDGF), and transforming growth factor-␤, have an angiogenic activity [1,2,3]
We show for the first time that inhibition of Gq/11 protein expression by a Gq/11specific antisense oligonucleotide blocked VPF/VEGF-stimulated human umbilical vein endothelial cell (HUVEC) proliferation and activation of signaling molecules in VPF/VEGF-stimulated HUVEC that are mediated by KDR, not by Flt-1
These activities include induction of microvascular hyperpermeability, stimulation of proliferation and migration, reprogramming of gene expression, endothelial cell survival, and prevention of senescence. All of these functions are thought to be mediated by two receptor tyrosine kinases, KDR and Flt-1, that are expressed on the vascular endothelium and are up-regulated at sites of VPF/VEGF overexpression as in the case of tumors, healing wounds, chronic inflammation, etc

Summary

Introduction

Kines and growth factors, such as vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF),1 basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor-␤, have an angiogenic activity [1,2,3]. Expression of the G␤␥ minigene, h␤ARK1(495), inhibits VPF/VEGFstimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2ϩ mobilization but has no effect on KDR To do this, serumstarved HUVEC that were transfected with ODN-Gq/11 or nontransfected were stimulated with VPF/VEGF for 1 min, and cellular extracts were subjected to a CDC42 activation assay as described under “Experimental Procedures.” The data indicate that ODN-Gq/11 has no effect on CDC42 activation (Fig. 2b).

Results

Conclusion