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. 2020 Jun 30;36:101629. doi: 10.1016/j.redox.2020.101629

Fig. 6.

Fig. 6

ELA-32-induced cAMP levels are not affected by APJ-S335 mutations, but this mutation can impair the interaction of receptors with AP2 and β-arrestin.

A, B ELA-32 and ELA-21 induced a cAMP response for WT and APJ mutants (S335A, S339A, S345A, S348A, S369A, only pcDNA3.1(+)) measured with an EPAC-based BRET biosensor. Concentration–response curves of intracellular cAMP levels with various concentrations of ELA-32 (Fig. 6, A) or ELA-21 (Fig. 6, B) (0.001–1000 nM) in HEK293 cells expressing WT or APJ mutants. Data are expressed as means ± S.E. from five independent experiments. C, D Kinetic-response curves of interactions between β-arrestin1 and AP2 or β-arrestin 2 and AP2 induced by APJ and APJ mutants upon activation by the ELA-32 agonist, obtained from real-time dynamic BRET determination. Data are expressed as means ± S.E. from five independent experiments. **P < 0.01 APJ + β-arrestin1-Rluc + AP2-YFP vs APJ335 + β-arrestin1-Rluc + AP2-YFP, **P < 0.01APJ + β-arrestin2-Rluc + AP2-YFP vs APJ335 + β-arrestin2-Rluc + AP2-YFP. E, F Kinetic-response curves of interactions between β-arrestin1 or β-arrestin 2 induced by APJ and APJ mutants upon activation by the ELA-32 agonist, obtained from real-time dynamic BRET determination. Data are expressed as means ± S.E. from five independent experiments. APJ-Rluc + β-arrestin1-EGFP vs APJ335-Rluc + β-arrestin1-EGFP, ***P < 0.001 APJ-Rluc + β-arrestin2-EGFP vs APJ335-Rluc + β-arrestin2-EGFP.