KDR stimulates endothelial cell migration through heterotrimeric G protein Gq/11-mediated activation of a small GTPase RhoA
Authors: Dezheng Zhao
Huiyan Zeng
Debabrata Mukhopadhyay
Journal: Journal of Biological Chemistry (2002)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Vascular permeability factor; VEGF; receptor tyrosine kinases; Flt-1; KDR
Affiliations: Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

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. 

Retracted: No

PubPeer comments:
 There are several similarities among the Western bolt bands in FIG.6 and FIG.7. The Red Boxes A and B shared the similar two bands, while Red Box C was the horizontal rollover of Red Box A or B (Green arrowheads refer to the obvious feature). The Blue Box D was similar to the Blue Box E, with the same feature as the Orange arrowheads shown. ![file] , 
 #1 Thanks very much for your comments and appreciate for your time to review our work more close to two decade ago. We will be happy to review the work with the authors and get back to you. Just keep in mind that due the COVID situation and lock down and also two decades old work, it might take little time that usual. Nonetheless, we will do proper review and follow up accordingly. , 
 Dear Colleague: First of all, sorry for the delayed response of your note due to several issues, obviously for pandemic related lockdown and also the nature of the question, which was close to two decades back. If fact, you correctly noted that those bands as referred above in your statement are the same. Actually, first two control lanes of both Fig 6a and Fig 7b (Rho activation and Rho cellular extracts) and Fig 6b and Fig 7c (Rac activation and total Rac cellular extracts) are same. Originally, we ran those experiments, Fig 6a and 7b and also Fig 6b and 7c in respective gets with same controls lanes and should be all in one Figure (Fig 6). However, due to some reason (which we couldn t remember), most likely for better presentation and easier for explanation to the readers, we separated two figures next to each other. Hence, in Fig 7b and c we used the original control lanes as of Fig 6a and b respectively. Also, another point we would like to mention that we used primary HUVEC cells where the same passage of cells were utilized to complete one set of experiments. Furthermore, the Rho and Rac activation methods we performed not using the commercial kits, rather we had to make those reagents at the same day to complete the all the experiments. Those days, we couldn t get the commercial kit as we can get easily now. Hence, our goal was to do all the experiments in one set of passage of cells and one set of preparation of the GTPase activation reagents at the same day. So, we ran all the treatments group in the same time in same controls to minimize the sample sizes. Now all those kits are available and the test methods are much simpler. Again, thanks very much for your inquires and hope you can now understand why all those control lanes were some due to same set of experiments. With best regards ,