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

J. Biol. Chem., Vol. 282, Issue 35, 25698-25707, August 31, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/35/25698    most recent M702942200v1 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 Geretti, E. Articles by Klagsbrun, M. Search for Related Content PubMed PubMed Citation Articles by Geretti, E. Articles by Klagsbrun, M. Social Bookmarking                      What's this?

Site-directed Mutagenesis in the B-Neuropilin-2 Domain Selectively Enhances Its Affinity to VEGF₁₆₅, but Not to Semaphorin 3F^*

Elena Geretti, Akio Shimizu, Peter Kurschat¹, and Michael Klagsbrun²

From the Departments of Surgery and Pathology, Vascular Biology Program, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF₁₆₅ and SEMA3F. Cell binding studies reveal that VEGF₁₆₅ and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nM, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF₁₆₅ binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF₁₆₅ binding, point mutations were introduced by site-directed mutagenesis. VEGF₁₆₅ is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280–290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF₁₆₅ affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF₁₆₅ affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF₁₆₅ affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF₁₆₅ binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF₁₆₅/NRP2 interactions.

Received for publication, April 6, 2007 , and in revised form, June 4, 2007.

^* This work was supported by National Institutes of Health NCI Grants CA37392 and CA45548 (to M. K.) and Deutsche Forschungsgemeinschaft DFG Grant KU 1497/1-1 (to P. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Dermatology, University of Cologne, Kerpener Str. 62, 50924 Cologne, Germany.

² To whom correspondence should be addressed: Karp Bldg. 12210, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-919-2157; Fax: 617-730-0233; E-mail: michael.klagsbrun{at}childrens.harvard.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				E. Sulpice, J. Plouet, M. Berge, D. Allanic, G. Tobelem, and T. Merkulova-Rainon Neuropilin-1 and neuropilin-2 act as coreceptors, potentiating proangiogenic activity Blood, February 15, 2008; 111(4): 2036 - 2045. [Abstract] [Full Text] [PDF]