Individual phosphorylation sites at the C-terminus of the apelin receptor play different roles in signal transduction

Redox Biology

Sheng Li¹  Xiaoyu Chen²  Yunlu Jiang³  Jing Chen⁴  Rumin Zhang⁴  Xin Cai⁴  Maocai Yan⁴  Huiling Mao⁴  Bingyuan Ji⁴  Chunmei Wang⁵ 

[1] Corresponding author. Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, United Kingdom. Tel.: [phone].;Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, United Kingdom;Department of Physiology, Shandong First Medical University (Shandong Academy of Medical Sciences), Taian, Shandong, China;Neurobiology Institute, Jining Medical University, Jining, Shandong, China;School of Pharmacy, Jining Medical University, Shandong, China;

关键词: Apelin receptor (APJ);    Phosphorylation;    Mass spectrometry;    Signal transduction;    Bioluminescence resonance energy transfer (BRET);    Internalization;   

DOI  :  

来源: DOAJ

【 摘 要 】

The apelin and Elabela proteins constitute a spatiotemporal double-ligand system that controls apelin receptor (APJ) signal transduction. Phosphorylation of multiple sites within the C-terminus of APJ is essential for the recruitment of β-arrestins. We sought to determine the precise mechanisms by which apelin and Elabela promote APJ phosphorylation, and to elucidate the influence of β-arrestin phosphorylation on G-protein-coupled receptor (GPCR)/β-arrestin-dependent signaling. We used techniques including mass spectrometry (MS), mutation analysis, and bioluminescence resonance energy transfer (BRET) to evaluate the role of phosphorylation sites in APJ-mediated G-protein-dependent and β-dependent signaling. Phosphorylation of APJ occurred at five serine residues in the C-terminal region (Ser335, Ser339, Ser345, Ser348 and Ser369). We also identified two phosphorylation sites in β-arrestin1 and three in β-arrestin2, including three previously identified residues (Ser412, Ser361, and Thr383) and two new sites, Tyr47 in β-arrestin1 and Tyr48 in β-arrestin2. APJ mutations did not affect the phosphorylation of β-arrestins, but it affects the β-arrestin signaling pathway, specifically Ser335 and Ser339. Mutation of Ser335 decreased the ability of the receptor to interact with β-arrestin1/2 and AP2, indicating that APJ affects the β-arrestin signaling pathway by stimulating Elabela. Mutation of Ser339 abolished the capability of the receptor to interact with GRK2 and β-arrestin1/2 upon stimulation with apelin-36, and disrupted receptor internalization and β-arrestin-dependent ERK1/2 activation. Five peptides act on distinct phosphorylation sites at the APJ C-terminus, differentially regulating APJ signal transduction and causing different biological effects. These findings may facilitate screening for drugs to treat cardiovascular and metabolic diseases.

【 授权许可】

Unknown