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J. Biol. Chem., Vol. 280, Issue 18, 17831-17840, May 6, 2005

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Comparative Agonist/Antagonist Responses in Mutant Human C5a Receptors Define the Ligand Binding Site^*

Adrian Higginbottom, Stuart A. Cain, Trent M. Woodruff¶, Lavinia M. Proctor¶, Praveen K. Madala||, Joel D. A. Tyndall||**, Stephen M. Taylor¶, David P. Fairlie||, and Peter N. Monk

From the Academic Neurology Unit, University of Sheffield Medical School, Sheffield S10 2RX, United Kingdom and ^¶Department of Physiology and Pharmacology and ^||Institute of Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites (Ile¹¹⁶, Arg^175, Arg²⁰⁶, Glu¹⁹⁹, Asp²⁸², and Val²⁸⁶). Agonist peptide 1 (Phe-Lys-Pro-D-cyclohexylalanine-cyclohexylalanine-D-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 (peptide 2). Conversely, mutation of C5aR transmembrane residue Ile¹¹⁶ to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394–3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] (peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile¹¹⁶, the latter is not essential for either activation or antagonism. Arg²⁰⁶ and Arg¹⁷⁵ both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp²⁸² has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu¹⁹⁹ may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.

Received for publication, September 20, 2004 , and in revised form, January 18, 2005.

^* This work is supported by Arthritis Research Campaign Project Grant M0648, Wellcome Trust Project Grant 007521 (to P. N. M.), and Australian Research Council and National Health and Medical Research Council Australian Project Grants DP0210598 (to D. P. F.) and 9937208 (to S. M. T./D. P. F.). 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.

Current address: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.

^** Current address: School of Pharmacy, University of Otago, P. O. Box 913, Dunedin, New Zealand.

These authors contributed equally to this work.

To whom correspondence should be addressed: Academic Neurology Unit, University of Sheffield Medical School, Beech Hill Rd., Sheffield S10 2RX, UK. Tel: 44-114 2261312; Fax: 44-114 2760095; E-mail: p.monk{at}shef.ac.uk.

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