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J. Biol. Chem., Vol. 281, Issue 52, 40033-40040, December 29, 2006

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Negative Constraints Underlie the ErbB Specificity of Epidermal Growth Factor-like Ligands^*

Sebastian P. van der Woning¹, Walter van Rotterdam, Sander B. Nabuurs, Hanka Venselaar, Saskia Jacobs-Oomen, Miriam Wingens, Gert Vriend, Catelijne Stortelers², and Everardus J. J. van Zoelen

From the Department of Cell Biology and Centre for Molecular and Biomolecular Informatics, Radboud University Nijmegen, Faculty of Science, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands

Epidermal growth factor (EGF)-like growth factors bind their ErbB receptors in a highly selective manner, but the molecular basis for this specificity is poorly understood. We have previously shown that certain residues in human EGF (Ser²-Asp³) and TGF (Glu²⁶) are not essential for their binding to ErbB1 but prevent binding to ErbB3 and ErbB4. In the present study, we have used a phage display approach to affinity-optimize the C-terminal linear region of EGF-like growth factors for binding to each ErbB receptor and thereby shown that Arg⁴⁵ in EGF impairs binding to both ErbB3 and ErbB4. By omitting all these so-called negative constraints from EGF, we designed a ligand designated panerbin that binds ErbB1, ErbB3, and ErbB4 with similarly high affinity as their wild-type ligands. Homology models, based on the known crystal structure of TGF-bound ErbB1, showed that panerbin is able to bind ErbB1, ErbB3, and ErbB4 in a highly similar manner with respect to position and number of interaction sites. Upon in silico introduction of the experimentally known negative constraints into panerbin, we found that Arg⁴⁵ induced local charge repulsion and Glu²⁶ induced steric hindrance in a receptor-specific manner, whereas Ser²-Asp³ impaired binding due to a disordered conformation. Furthermore, radiolabeled panerbin was used to quantify the level of all three receptors on human breast cancer cells in a single radioreceptor assay. It is concluded that the ErbB specificity of EGF-like growth factors primarily results from the presence of a limited number of residues that impair the unintended interaction with other ErbB receptors.

Received for publication, April 3, 2006 , and in revised form, August 4, 2006.

^* The present work was supported by grants from the Netherlands Organization for Scientific Research, The Netherlands Cancer Society, and the Stichting Bergh in het Zadel (Beek, The Netherlands). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Figs. 1 and 2.

² Present address: Division of Cellular Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

¹ To whom correspondence should be addressed. Tel.: 31-24-3652522; Fax: 31-24-3652999; E-mail: b.vanderwoning{at}science.ru.nl.

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