Fig. 5.

APJ-S339A mutation impairs the receptor's interactions with GRK2 and β-arrestins via apelin-36 stimulation.

A-C, Concentration–response curves of the recruitment of GRK2 or β-arrestin1/2 to WT and APJ mutates, measured by BRET assay. HEK293 cells were transiently transfected with Rluc-tagged receptors and Venus-tagged GRK2 (A), Rluc-tagged receptors and EGFP-tagged β-arrestin1 (B), or Rluc-tagged receptors and EGFP-tagged β-arrestin2 (C) and then treated with increasing concentration of apelin-36. The BRET signals between receptor and GRK2 were recorded at 10 min after agonist stimulation, the BRET signals between receptor and β-arrestin1/2 were recorded at 15 min after agonist stimulation. Data are expressed as means ± S.E. from five independent experiments.

D-F, Kinetic-response curves of the recruitment of GRK2 or β-arrestin1/2 to WT and APJ mutants following apelin-36 stimulation, measured by BRET in living cells. Real-time analysis of the interaction between WT or mutant APJ and GRK2 (D), WT or mutant APJ and β-arrestin1 (E), and WT or mutant APJ and β-arrestin2 (F). Coelenterazine H was added to transiently co-transfected HEK293 cells and then the BRET signal was recorded after apelin-36 stimulation. Data are expressed as means ± S.E. from five independent experiments.