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J. Biol. Chem., Vol. 281, Issue 17, 12010-12019, April 28, 2006

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Genetic Analysis of the First and Third Extracellular Loops of the C5a Receptor Reveals an Essential WXFG Motif in the First Loop^*

Jeffery M. Klco, Gregory V. Nikiforovich, and Thomas J. Baranski¹

From the Departments of Medicine and Molecular Biology and Pharmacology and the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.

Received for publication, January 18, 2006 , and in revised form, February 27, 2006.

^* This work was supported by an award from the American Heart Association (to J. M. K.), by American Cancer Society Grant IRG-58-010-43 (to T. J. B.), the Culpeper Award, Rockefeller Brothers Fund (to T. J. B.), and National Institutes of Health Grants GM63720-01 (to T. J. B.) and GM068460 (to G. V. N.). 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: Depts. of Medicine and MolecularBiology and Pharmacology, Washington University School of Medicine, Campus Box 8127, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-747-3997; Fax: 314-362-7641; E-mail: baranski{at}wustl.edu.

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