Differential phosphorylation signals control endocytosis of GPR15

• Published in: Molecular biology of the cell 2017-08, Vol.28 (17), p.2267-2281
• Main Authors: Okamoto, Yukari, Shikano, Sojin
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; beta-Arrestins - metabolism; Cell Membrane - metabolism; Clathrin - metabolism; Endocytosis - physiology; HEK293 Cells; Humans; Ligands; Mutation; Phosphorylation; Protein Binding; Protein Domains; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Receptors, Peptide - genetics; Receptors, Peptide - metabolism; Signal Transduction
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E16-09-0627

GPR15 is an orphan G protein-coupled receptor (GPCR) that serves for an HIV coreceptor and was also recently found as a novel homing receptor for T-cells implicated in colitis. We show that GPR15 undergoes a constitutive endocytosis in the absence of ligand. The endocytosis was clathrin dependent and partially dependent on β-arrestin in HEK293 cells, and nearly half of the internalized GPR15 receptors were recycled to the plasma membrane. An Ala mutation of the distal C-terminal Arg-354 or Ser-357, which forms a consensus phosphorylation site for basophilic kinases, markedly reduced the endocytosis, whereas phosphomimetic mutation of Ser-357 to Asp did not. Ser-357 was phosphorylated in vitro by multiple kinases, including PKA and PKC, and pharmacological activation of these kinases enhanced both phosphorylation of Ser-357 and endocytosis of GPR15. These results suggested that Ser-357 phosphorylation critically controls the ligand-independent endocytosis of GPR15. The functional role of Ser-357 in endocytosis was distinct from that of a conserved Ser/Thr cluster in the more proximal C-terminus, which was responsible for the β-arrestin- and GPCR kinase-dependent endocytosis of GPR15. Thus phosphorylation signals may differentially control cell surface density of GPR15 through endocytosis.