FIGURE 3.

Engineering C5aR to a chemokine-type receptor; signaling profile of mutant C5aR receptors, co-expressed with C5a in yeast. Left panel, canonical receptor topology for C5aR. The N119S mutation on TM3 is a novel constitutively active mutant identified for C5aR in this study. Asp²⁷, Ser³⁰, and Ser²⁷², highlighted in white with black background, have been mutated to cysteine for engineering a possible extra disulfide linkage between the N terminus and EC3 loop of C5aR. Middle and right panel, respectively, illustrate the effect of S30C, S272C, S30C/S272C, and D27C, S272C, D27C/S272C on C5a-stimulated C5aR signaling. Vector represents the β-galactosidase activity of the engineered yeast in the absence of both receptor and ligand. Each bar represents means ± S.D. of signaling activity for three independent transformants, and data are representative of at least two independent experiments.