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.