Fig. 6: Interfering with the GPRC5B/EP2 interaction: in silico docking and mutation of GPRC5B.

A The refined predicted orientation of the GPRC5B-EP2 complex (GPRC5B in green, EP2 in cyan). B Three sets of residues within GPRC5B considered for site-directed mutagenesis. C Exemplary photomicrographs showing cellular distribution of FLAG/Myc-tagged wild type GRC5B (G5B-wt, top) and GPRC5B carrying 7 alanine mutations (G5B-mut7, bottom) in permeabilized HEK cells (WGA as membrane staining). D Western blot detection of HA and FLAG signals in lysates of HEK cells expressing HA-tagged EP2 in combination with empty vector (EV), FLAG-tagged human G5B-wt or G5B-mut7: “input” shows lysates before, “pulldown HA” after immunoprecipitation with anti-HA beads. E, Butaprost-induced cAMP production in HEK cells transfected with cAMP GloSensor plasmid, HA-EP2, and wild type or mutant GPRC5B as indicated (n = 4). F Western blot detection of HA and FLAG signals in lysates of HEK cells expressing HA-tagged EP2 in combination with empty vector (EV) or FLAG-tagged human wild type or mutant GPRC5B as indicated. G, Butaprost-induced cAMP production in HEK cells transfected with cAMP GloSensor plasmid, HA-EP2, and wild type or mutant GPRC5B as indicated (n = 10). H, I HEK cells were transfected with HA-EP2 and wild type or mutant GPRC5B as indicated and subjected to plasma membrane staining with WGA, followed by permeabilization and staining with anti-HA antibodies: exemplary photomicrographs (H) and statistical evaluation of the HA-EP2 signal within WGA (I) (n = 36/26/31/37/35/30 cells). Data are means ± SEM; comparisons with empty vector-transfected samples (E) or G5B-wt-transfected samples (G, I) were performed using Kruskal-Wallis test with Dunn’s correction for multiple testing. EV, empty vector; n, number of independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are provided as a Source Data file.