Endocytosis of Ligand-Activated Sphingosine 1-Phosphate Receptor 1 Mediated by the Clathrin-Pathway

Traffic. 2016 Jan;17(1):40-52. doi: 10.1111/tra.12343. Epub 2015 Dec 9.

Abstract

The sphingosine 1-phosphate receptor 1 (S1PR1) is one of five G protein-coupled receptors activated by the lipid sphingosine 1-phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin-mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin-mediated endocytosis by depleting clathrin or AP-2 or by treating cells with dynasore-OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both β-arrestin 1 and β-arrestin 2, indicating that activated S1PR1 follows the canonical route of endocytosis for G-protein coupled receptor's (GPCR)'s.

Keywords: G-protein coupled receptors; clathrin; downregulation; vesicular traffic.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arrestins / metabolism
  • Clathrin / metabolism*
  • Endocytosis*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Mice
  • Organophosphates / pharmacology
  • Receptors, Lysosphingolipid / agonists
  • Receptors, Lysosphingolipid / metabolism*
  • Sphingosine / analogs & derivatives
  • Sphingosine / pharmacology
  • Sphingosine-1-Phosphate Receptors
  • beta-Arrestin 1
  • beta-Arrestin 2
  • beta-Arrestins

Substances

  • ARRB1 protein, human
  • ARRB2 protein, human
  • Arrb1 protein, mouse
  • Arrb2 protein, mouse
  • Arrestins
  • Clathrin
  • FTY 720P
  • Organophosphates
  • Receptors, Lysosphingolipid
  • S1PR1 protein, human
  • Sphingosine-1-Phosphate Receptors
  • beta-Arrestin 1
  • beta-Arrestin 2
  • beta-Arrestins
  • Sphingosine