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J. Biol. Chem., Vol. 282, Issue 1, 124-131, January 5, 2007

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Identification of Ligand Binding Site of Phytosulfokine Receptor by On-column Photoaffinity Labeling^*

From the Graduate School of Bio-agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Phytosulfokine (PSK), an endogenous 5-amino-acid-secreted peptide in plants, affects cellular potential for growth via binding to PSKR1, a member of the leucine-rich repeat receptor kinase (LRR-RK) family. PSK interacts with PSKR1 in a highly specific manner with a nanomolar dissociation constant. However, it is not known which residues in the PSKR1 extracellular domain constitute the ligand binding pocket. Here, we have identified the PSK binding domain of carrot PSKR1 (DcPSKR1) by photoaffinity labeling. We cross-linked the photoactivatable PSK analog [¹²⁵I]-[N-(4-azidosalicyl)Lys⁵]PSK with DcPSKR1 using UV irradiation and mapped the cross-linked region using chemical and enzymatic fragmentation. We also established a novel "on-column photoaffinity labeling" methodology that allows repeated incorporation of the photoaffinity label to increase the efficiency of the photoaffinity cross-linking reactions. We purified a labeled DcPSKR1 tryptic fragment using anti-PSK antibodies and identified a peptide fragment that corresponds to the 15-amino-acid Glu⁵⁰³-Lys⁵¹⁷ region of DcPSKR1 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Deletion of Glu⁵⁰³-Lys⁵¹⁷ completely abolishes the ligand binding activity of DcPSKR1. This region is in the island domain flanked by extracellular LRRs, indicating that this domain forms a ligand binding pocket that directly interacts with PSK.

Received for publication, May 12, 2006 , and in revised form, November 8, 2006.

^* This work was supported by the 21st Century Center of Excellence Program Grant 14COEA02) and by Grants-in-aid for Scientific Research for Priority Areas (14036214) and Young Scientists (18687003). 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 To whom correspondence should be addressed: Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan. Tel.: 81-52-789-4117; Fax: 81-52-789-4118; E-mail: matsu{at}agr.nagoya-u.ac.jp.

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