title: Identification of serine 348 on the apelin receptor as a novel regulatory phosphorylation site in apelin-13-induced G protein-independent biased signaling.
abstract: Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway.
authors.affiliation: From the Department of Physiology, Shandong University School of Medicine, Jinan, Shandong 250012, People's Republic of China, the Department of physiology, Taishan Medical College, Taian, Shandong 271000, People's Republic of China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Chen
authors.affiliation: the Neurobiology Institute, Jining Medical University, Jining, Shandong 272067, People's Republic of China, bbai@mail.jnmc.edu.cn.
authors.forename: Bo
authors.initials: B
authors.lastname: Bai
authors.affiliation: the Neurobiology Institute, Jining Medical University, Jining, Shandong 272067, People's Republic of China.
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Tian
authors.affiliation: the Department of physiology, Taishan Medical College, Taian, Shandong 271000, People's Republic of China.
authors.forename: Hui
authors.initials: H
authors.lastname: Du
authors.affiliation: the Neurobiology Institute, Jining Medical University, Jining, Shandong 272067, People's Republic of China, the Division of Translational and Systems Medicine, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom, and Jing.Chen@warwick.ac.uk.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000075244:Apelin Receptors; D002118:Calcium / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D006984:Hypertrophy; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry*; D015398:Signal Transduction
chemical_list: C104575:APLNR protein, human; D000075244:Apelin Receptors; D036341:Intercellular Signaling Peptides and Proteins; D043562:Receptors, G-Protein-Coupled; C495199:apelin-13 peptide; D012694:Serine; D002118:Calcium
keywords: 7-Helix Receptor; Apelin Receptor (APJ); Biased Signal Pathway; Bioluminescence Resonance Energy Transfer (BRET); G Protein-coupled Receptor (GPCR); Protein Phosphorylation; Signal Transduction