HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lu, D. Articles by Zhu, Z. Search for Related Content PubMed PubMed Citation Articles by Lu, D. Articles by Zhu, Z. Social Bookmarking                      What's this?

J Biol Chem, Vol. 275, Issue 19, 14321-14330, May 12, 2000

Identification of the Residues in the Extracellular Region of KDR Important for Interaction with Vascular Endothelial Growth Factor and Neutralizing Anti-KDR Antibodies^*

Dan Lu, Paul Kussie^§, Bronislaw Pytowski, Kris Persaud, Peter Bohlen^¶, Larry Witte, and Zhenping Zhu

From the Departments of  Molecular and Cell Biology, ^§ Protein Chemistry, and ^¶ Research, ImClone Systems Incorporated, New York, New York 10014

The kinase domain receptor (KDR) of vascular endothelial growth factor (VEGF) is the main human receptor responsible for the angiogenic activity of VEGF. The extracellular region of KDR is comprised of seven immunoglobulin-like domains, of which the first three have been shown to be required for ligand binding. We have previously described antibodies directed against the extracellular region of KDR, including MAB383 and MAB664, which were shown to block the binding of VEGF to the receptor and to inhibit both VEGF-induced mitogenesis of human endothelial cells in vitro and tumor growth in vivo. Here we generated a series of KDR deletion mutants consisting of truncated extracellular regions and mapped out the domain(s) responsible for binding to VEGF and the neutralizing anti-KDR antibodies. All neutralizing antibodies were found to require domain 3 for efficient binding. Alanine-scanning mutagenesis of domain 3 identified two different sets of five residues, Ile²⁵⁶, Asp²⁵⁷, Glu²⁶¹, Leu³¹³, and Thr³¹⁵ and Tyr²⁶², Pro²⁶³, Ser²⁶⁴, Ser²⁶⁵, and Lys²⁶⁶, that were critical for binding to MAB383 and MAB664, respectively. Combination of alanine mutations affecting both MAB383 and MAB664 binding resulted in a variant that also lost binding to VEGF. These results suggest that the residues within this region of domain 3 are critical for VEGF binding. Our studies provide a basis for the mechanism of action of our anti-KDR antibodies and establish a functional foundation for the development of other classes of antagonists to the receptor.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				F. Hilberg, G. J. Roth, M. Krssak, S. Kautschitsch, W. Sommergruber, U. Tontsch-Grunt, P. Garin-Chesa, G. Bader, A. Zoephel, J. Quant, et al. BIBF 1120: Triple Angiokinase Inhibitor with Sustained Receptor Blockade and Good Antitumor Efficacy Cancer Res., June 15, 2008; 68(12): 4774 - 4782. [Abstract] [Full Text] [PDF]

				R.A. Goodlad, A.J. Ryan, S.R. Wedge, I.T. Pyrah, D. Alferez, R. Poulsom, N.R. Smith, N. Mandir, A.J. Watkins, and R.W. Wilkinson Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer Carcinogenesis, October 1, 2006; 27(10): 2133 - 2139. [Abstract] [Full Text] [PDF]

				B. Favier, A. Alam, P. Barron, J. Bonnin, P. Laboudie, P. Fons, M. Mandron, J.-P. Herault, G. Neufeld, P. Savi, et al. Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration Blood, August 15, 2006; 108(4): 1243 - 1250. [Abstract] [Full Text] [PDF]

				J. Shen, M. D. Vil, X. Jimenez, M. Iacolina, H. Zhang, and Z. Zhu Single Variable Domain-IgG Fusion: A NOVEL RECOMBINANT APPROACH TO Fc DOMAIN-CONTAINING BISPECIFIC ANTIBODIES J. Biol. Chem., April 21, 2006; 281(16): 10706 - 10714. [Abstract] [Full Text] [PDF]

				S. R. Wedge, J. Kendrew, L. F. Hennequin, P. J. Valentine, S. T. Barry, S. R. Brave, N. R. Smith, N. H. James, M. Dukes, J. O. Curwen, et al. AZD2171: A Highly Potent, Orally Bioavailable, Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor for the Treatment of Cancer Cancer Res., May 15, 2005; 65(10): 4389 - 4400. [Abstract] [Full Text] [PDF]

				X. Jimenez, D. Lu, L. Brennan, K. Persaud, M. Liu, H. Miao, L. Witte, and Z. Zhu A recombinant, fully human, bispecific antibody neutralizes the biological activities mediated by both vascular endothelial growth factor receptors 2 and 3 Mol. Cancer Ther., March 1, 2005; 4(3): 427 - 434. [Abstract] [Full Text] [PDF]

				K. Persaud, J.-C. Tille, M. Liu, Z. Zhu, X. Jimenez, D. S. Pereira, H.-Q. Miao, L. A. Brennan, L. Witte, M. S. Pepper, et al. Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C J. Cell Sci., June 1, 2004; 117(13): 2745 - 2756. [Abstract] [Full Text] [PDF]

				X. Jiang and J. R. Couchman Perlecan and Tumor Angiogenesis J. Histochem. Cytochem., November 1, 2003; 51(11): 1393 - 1410. [Abstract] [Full Text] [PDF]

				D. Lu, J. Shen, M. D. Vil, H. Zhang, X. Jimenez, P. Bohlen, L. Witte, and Z. Zhu Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 for Enhanced Neutralizing Activity J. Biol. Chem., October 31, 2003; 278(44): 43496 - 43507. [Abstract] [Full Text] [PDF]

				J. Holash, S. Davis, N. Papadopoulos, S. D. Croll, L. Ho, M. Russell, P. Boland, R. Leidich, D. Hylton, E. Burova, et al. VEGF-Trap: A VEGF blocker with potent antitumor effects PNAS, August 20, 2002; 99(17): 11393 - 11398. [Abstract] [Full Text] [PDF]

				T. Matsumoto and L. Claesson-Welsh VEGF Receptor Signal Transduction Sci. Signal., December 11, 2001; 2001(112): re21 - re21. [Abstract] [Full Text] [PDF]

				D. Lu, X. Jimenez, H. Zhang, Y. Wu, P. Bohlen, L. Witte, and Z. Zhu Complete Inhibition of Vascular Endothelial Growth Factor (VEGF) Activities with a Bifunctional Diabody Directed against Both VEGF Kinase Receptors, fms-like Tyrosine Kinase Receptor and Kinase Insert Domain-containing Receptor Cancer Res., October 1, 2001; 61(19): 7002 - 7008. [Abstract] [Full Text] [PDF]

				S. Dias, K. Hattori, B. Heissig, Z. Zhu, Y. Wu, L. Witte, D. J. Hicklin, M. Tateno, P. Bohlen, M. A. S. Moore, et al. Inhibition of both paracrine and autocrine VEGF/ VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias PNAS, September 11, 2001; 98(19): 10857 - 10862. [Abstract] [Full Text] [PDF]

				I. Zachary and G. Gliki Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family Cardiovasc Res, February 16, 2001; 49(3): 568 - 581. [Abstract] [Full Text] [PDF]

				Q. Tao, M. V. Backer, J. M. Backer, and B. I. Terman Kinase Insert Domain Receptor (KDR) Extracellular Immunoglobulin-like Domains 4-7 Contain Structural Features That Block Receptor Dimerization and Vascular Endothelial Growth Factor-induced Signaling J. Biol. Chem., June 8, 2001; 276(24): 21916 - 21923. [Abstract] [Full Text] [PDF]