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J. Biol. Chem., Vol. 280, Issue 24, 22951-22961, June 17, 2005

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Receptor Epitope Usage by an Interleukin-5 Mimetic Peptide^*

Tetsuya Ishino, Cecilia Urbina, Madhushree Bhattacharya, Dominick Panarello, and Irwin Chaiken

From the Department of Biochemistry and Molecular Biology and the A. J. Drexel Institute of Basic and Applied Protein Science, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102

The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor (IL5R) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556–568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5R interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide·receptor complex is comparable with that of the cytokine·receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5R, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5R by alanine substitution of Asp⁵⁵, Asp⁵⁶, Glu⁵⁸, Lys¹⁸⁶, Arg¹⁸⁸, and Arg²⁹⁷ of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5R, in particular Asp⁵⁵, Arg¹⁸⁸, and Arg²⁹⁷ in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system.

Received for publication, March 2, 2005 , and in revised form, April 4, 2005.

^* This work was supported by National Institutes of Health Grants GM55648 and AI40462. 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.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Drexel University College of Medicine, 11102 New College Bldg., 245 N. 15th St., Philadelphia, PA 19102. Tel.: 215-762-4197; Fax: 215-762-4452; E-mail: imc23{at}drexel.edu.

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This article has been cited by other articles:

				M. Zaks-Zilberman, A. E. Harrington, T. Ishino, and I. M. Chaiken Interleukin-5 Receptor Subunit Oligomerization and Rearrangement Revealed by Fluorescence Resonance Energy Transfer Imaging J. Biol. Chem., May 9, 2008; 283(19): 13398 - 13406. [Abstract] [Full Text] [PDF]