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J. Biol. Chem., Vol. 281, Issue 28, 19260-19272, July 14, 2006

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Characterization of the Functional Epitope on the Urokinase Receptor

COMPLETE ALANINE SCANNING MUTAGENESIS SUPPLEMENTED BY CHEMICAL CROSS-LINKING^*

Henrik Gårdsvoll, Bernard Gilquin, Marie Hélène Le Du, Andre Ménèz, Thomas J. D. Jørgensen^¶, and Michael Ploug¹

From the Finsen Laboratory, Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen Ø, Denmark, the Département d'Ingénierie et d'Etudes des Protéines, Commissariat à l'Energie Atomique, CE Saclay, 91191 Gif-sur-Yvette, France, and the ^¶Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

The high affinity interaction between the serine protease urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) represents one of the key regulatory steps in cell surface-associated plasminogen activation. On the basis on our crystal structure solved for uPAR in complex with a peptide antagonist, we recently proposed a model for the corresponding complex with the growth factor-like domain of uPA (Llinas et al. (2005) EMBO J. 24, 1655-1663). In the present study, we provide experimental evidence that consolidates and further develops this model using data from a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were determined by surface plasmon resonance. This complete alanine scanning of uPAR highlighted the involvement of 20 surface-exposed side chains in this interaction. Mutations causing G 1 kcal/mol for the uPA interaction are all located within or at the rim of the central cavity uniquely formed by the assembly of all three domains in uPAR, whereas none are found outside this crevice. Identification of specific cross-linking sites in uPAR and pro-uPA enabled us to build a model of the uPAR·uPA complex in which the kringle domain of uPA was positioned by the constraints established by the range of these cross-linkers. The nature of this interaction is predominantly hydrophobic and highly asymmetric, thus emphasizing the importance of the shape and size of the central cavity when designing low molecular mass antagonists of the uPAR/uPA interaction.

Received for publication, December 21, 2005 , and in revised form, April 7, 2006.

^* This work was supported by grants from the John and Birthe Meyer Foundation, the Lundbeck Foundation, the Danish Cancer Society, the Dansk Kræftforskningsfond, the Carlsberg Foundation, the Kong Christian IX og Dronning Louises Jubilæumslegat, the Danish Instrument Biotechnology Center, and the Copenhagen Hospital Corp. (H:S) and by European Union Contract LSHC-CT2003-503297. 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 Figs. S1-S3 and Table SI.

¹ To whom correspondence should be addressed. Tel.: 45-3545-5706; Fax: 45-3538-5450; E-mail: m-ploug{at}finsenlab.dk.

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