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Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor

Published online by Cambridge University Press:  01 November 1999

JIANZHONG SUN
Affiliation:
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
JULIA A. EMBER
Affiliation:
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
TA-HSIANG CHAO
Affiliation:
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
YOSHIHIRO FUKUOKA
Affiliation:
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 Present address: Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106.
RICHARD D. YE
Affiliation:
Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois 60612
TONY E. HUGLI
Affiliation:
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037
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Abstract

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane α-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.

Type
Research Article
Copyright
© 1999 The Protein Society

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