Fig. 5. The CD177/GPR97/PAR2/CD16b receptor complex is required for efficient GPR97-mPR3 interaction that induces PAR2 activation.
a–c Flow cytometry analyses of GPR97-mPR3 interaction in HEK-293T cells expressing various receptors as indicated. a Surface levels of membrane-bound PR3 (green), GPR97E-mFc (red), and mFc (blue) were detected using appropriate Abs. b, c The percentage of transfectants displaying the positive GPR97E-mFc binding signal in the ligand-binding assay. n = 3 b, 4 c independent experiments. The underlined receptor in c indicates the substituted component of the core receptor complex. Data are means ± SEM and p value was determined by two-sided unpaired student’s t-test. d The PLA analyses showed the interaction signals of specific receptor pairs of the CD177-associated complex in resting neutrophils. Scale bar, 10 μm. Experiments were repeated 3 times with similar results. e, f Flow cytometry analyses of PAR2 proteolysis in neutrophils e and HEK-293T cells expressing the CD177-associated receptor complex f. Trypsin-treated neutrophils and mFc were included as a positive and a negative control, respectively. n = 5 independent experiments. Data are means ± SEM and p value was determined by one-way ANOVA. g ELISA analyses of IL-8 secreted by neutrophils incubated with the indicated reagents. n = 4 independent experiments. Data are means ± SEM and p value was determined by two-way ANOVA. ns, non-significant. h Schematic diagram of the PR3/CD177/GPR97/PAR2/CD16b complex in neutrophils. CD16, a green-colored receptor with two connected ovals; GPR97, a brown-colored 7TM receptor with two extracellular ovals; PAR2, a blue-colored 7TM receptor; PR3, a purple-colored soluble molecule; CD177, a red-colored receptor with two diamonds. Source data are provided in the Source Data file.