Figure 8.

Sequence alignment of C5aR2 with C5aR1, βarr2 recruitment, and ERK1/2 phosphorylation. A, the sequences of human C5aR1 and C5aR2 were retrieved from Uniprot and aligned on M-coffee server with default parameters. Alignment reliability was assessed by core/TCS and generated alignment was visualized using Espript 3. Specific mutations in the DRY and NPXXY motif are highlighted. B, HEK-293 cells expressing C5aR2-Venus and βarr2-Rluc8 constructs were first incubated with luciferase-substrate for 2 h. Subsequently, the cells were stimulated with respective ligands, and BRET signals were monitored using a dose-response curve. The data represent averages ± S.E. of three independent experiments, and the EC₅₀ values are compared using unpaired t test. ***, p < 0.001. C, HEK-293 cells expressing C5aR1 or C5aR2 were stimulated with C5a (100 nm) for indicated time points followed by detection of ERK1/2 phosphorylation using Western blotting. D, densitometry-based quantification of data presented in C normalized with C5a response for C5aR1 (treated as 100%) and analyzed using two-way ANOVA. ***, p < 0.001.