Comparative Study of Elabela and Apelin on Apelin Receptor Activation Through β-Arrestin Recruitment

Abstract

Apelin receptor (APJ) ligands elabela (ELA) and apelin have divergent distributions and function differently in vitro and in vivo. Whether differences exist in their capacity of recruitment of β-arrestins (ARRBs) to APJ remains unknown. The aim of the current study was to investigate the different effects of ELA and apelin on the interaction between APJ and ARRBs in live cells by NanoBiT®. NanoBiT® system is a new technology for studying protein-protein interaction in real-time in live cells, based on the emission of luminescence when two split components of NanoLuc luciferase, large Bit (LgBit) and small Bit (SmBit), complement each other to form an enzymatically active entity. We tagged the APJ and ARRBs with LgBit or SmBit and then evaluated their interactions in transiently transfected HEK293T cells, and determined the signal strength yielded as a result of the interaction. We also investigated the concentration-dependent response of the APJ-ARRB interaction in response to ELA and apelin. Finally, we assessed the effect of F13A, an APJ antagonist which is structurally very similar to apelin-13, on ELA- and apelin-mediated APJ-ARRB interactions. The NanoLuc® luciferase signal was highest in the pair of APJ-LgBit with SmBit-ARRB1 or SmBit-ARRB2. NanoLuc® luciferase signal increased in a concentration-dependent manner from 0.1 nM to 10 μM in response to ELA or apelin. Interestingly, ELA elicited weaker APJ-ARRB interaction signals than apelin. Pre-treatment with F13A potently reduced the APJ-ARRB interaction in response to both ELA and apelin. Our results demonstrated that both ELA and apelin promoted the interaction of APJ and ARRBs in a concentration-dependent manner, and ELA is less efficacious than apelin in inducing the recruitment of ARRBs to APJ, providing a biased functional aspect of ELA vs. apelin at the receptor signaling level. Additionally, ELA and apelin may share the same binding site(s) or pocket(s) at the APJ level.

Keywords: ARRBs; Apelin; Elebela/Toddler; NanoBiT®; The apelin receptor (APJ).

Fig. 1

Schematic structures of NanoBiT®‐tagged constructs used in this study. APJ and ARRBs are fused to Lgbit or Smbit of NanoLuc® luciferase. When apelin or ELA binds to APJ, APJ recruits ARRBs in response to apelin or ELA, and the interaction yields a bright, luminescent signal in the presence of a substrate

Fig. 2

Pairing optimization of NanoBiT®-tagged APJ and ARRBs. a–d. NanoBiT®-tagged APJ- and ARRB1/2-expressing HEK293T cells were stimulated by apelin (1 μM) or PBS. e–h. NanoBiT®-tagged APJ- and ARRB1/2-expressing HEK293T cells were stimulated by ELA (1 μM) or PBS. Each value represents the mean ± SEM from 4 independent experiments. ^***P < 0.001 and ^**P < 0.01 vs. the corresponding control group; ^###P < 0.001 and ^##P < 0.01 vs. the corresponding APJ-LgBit/SmBit-ARRBs group. NS: no statistical significance

Fig. 3

Concentration–response relationship of the interaction of APJ and ARRBs in response to ELA and apelin. a, indicated concentrations of apelin were added to living HEK293T cells transiently overexpressing APJ-LgBit/SmBit-ARRB1. b, indicated concentrations of ELA were added to live HEK293T cells transiently overexpressing APJ-LgBit/SmBit-ARRB2. EC₅₀ was calculated from the concentration–response curve. Data are expressed as the mean ± SE (n = 6) and fitted to sigmoidal curves using GraphPad Prism 6 software

Fig. 4

Inhibition of the APJ-ARRB interaction by F13A. a, b, Luciferase activities were measured in the cells pretreated with F13A (1uM) for 5 min, before the addition apelin or ELA. Each value represents the mean ± SEM from 6 independent experiments. ***P < 0.001. Control vectors SmBit-PRKACA and LgBit-PRKAR2A (Promega) were used as the negative control of no protein–protein interaction