Heterodimerization with vascular endothelial growth factor receptor-2 (VEGFR-2) is necessary for VEGFR-3 activity

doi:10.1016/j.bbrc.2004.08.237

Biochemical and Biophysical Research Communications

Volume 324, Issue 2, 12 November 2004, Pages 909-915

https://doi.org/10.1016/j.bbrc.2004.08.237 Get rights and content

Abstract

VEGFR-3 is essential for vascular development and maintenance of lymphatic vessel’s integrity. Little is known about its cooperative effect with other receptors of the same family. Contrary to VEGFR-2, stimulation of VEGFR-3 by VEGF-C and -D failed to enhance its phosphorylation either in HEK293T or in PAE cells. These ligands were unable to induce angiogenesis of PAEC expressing VEGFR-3 alone. In the presence of VEGFR-2, VEGF-C and -D induced heterodimerization of VEGFR-3 with VEGFR-2. This heterodimerization was associated with enhanced VEGFR-3 phosphorylation and subsequent cellular responses as evidenced by the formation of capillary-like structures in PAE cells and proliferation of primary human endothelial cells expressing both receptors. Taken together, these results show for the first time that VEGFR-3 needs to be associated to VEGFR-2 to induce ligand-dependent cellular responses.

Section snippets

Materials and methods

Cells, antisera, and other reagents. Polyclonal antiserum to Flk-1/KDR (VEGFR-2) was from Santa Cruz Biotechnology (Heidelberg, Germany). Recombinant human VEGF-A and VEGF-D were obtained from R&D Systems (Lille, France) and VEGF-C was from ReliaTech (Braunschweig, Germany). The horseradish peroxidase (HRP)-conjugated monoclonal antibody anti-phosphotyrosine PY20 was from Zymed, San Francisco, CA. Agarose-conjugated monoclonal anti-HA antibody and the HRP-conjugated anti-HA were from Santa Cruz

Angiogenesis of PAEC expressing VEGFR-2 or VEGFR-3

PAEC stably expressing either VEGFR-2 or VEGFR-3 were subjected to VEGF-A, -C or -D in the Matrigel model. Morphology of the cells in Matrigel is illustrated in Fig. 1A and the quantification of the total tubules length is shown in Fig. 1B. The results indicate that in PAEC expressing VEGFR-2, VEGF-A induced a potent angiogenic response, whereas VEGF-C had a less pronounced effect and VEGF-D was totally inactive. In VEGFR-3-expressing PAEC, none of the three ligands were able to induce tubule

Discussion

In this paper, we have demonstrated for the first time that VEGF-C and VEGF-D are unable to enhance the basal level of VEGFR-3 phosphorylation unless it is co-expressed with VEGFR-2. Our results indicate that the VEGFR-3 kinase function is not defective per se but that the receptor alone did not seem to be able to couple to one or several cellular signal transduction molecules. Using “wild-type” VEGFR-3 constructs, phosphorylation of the receptor was undetectable unless the cells were

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Heterodimerization with vascular endothelial growth factor receptor-2 (VEGFR-2) is necessary for VEGFR-3 activity

Abstract

Section snippets

Materials and methods

Angiogenesis of PAEC expressing VEGFR-2 or VEGFR-3

Discussion

Blood

J. Biol. Chem.

J. Biol. Chem.

Lab. Invest.

J. Biol. Chem.

J. Biol. Chem.

Blood

Int. J. Biochem. Cell Biol.

Mechanisms of angiogenesis

Nature

The biology of VEGF and its receptors

Nat. Med.

VEGF receptor signal transduction

Sci. STKE

Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development

Proc. Natl. Acad. Sci. USA

VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development

Development

Signalling properties of FLT4, a proteolytically processed receptor tyrosine kinase related to two VEGF receptors

Oncogene

Evidence for heterotypic interaction between the receptor tyrosine kinases TIE-1 and TIE-2

J. Biol. Chem.