The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes

J Biol Chem. 2005 Jun 3;280(22):21661-6. doi: 10.1074/jbc.M503099200. Epub 2005 Mar 31.

Abstract

The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factor 6
  • ADP-Ribosylation Factors / metabolism
  • ADP-Ribosylation Factors / physiology*
  • Animals
  • Brain / metabolism
  • Cell Membrane / metabolism
  • Clathrin / chemistry
  • Cricetinae
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Endocytosis
  • Glutathione Transferase / metabolism
  • Guanosine Triphosphate / chemistry
  • Guanosine Triphosphate / metabolism
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Kinetics
  • Liposomes / chemistry
  • Liposomes / metabolism
  • Microscopy, Confocal
  • Mutation
  • Phosphatidylinositols / chemistry
  • Protein Binding
  • Protein Transport
  • Receptors, Transferrin / metabolism
  • Time Factors
  • Transcription Factor AP-2
  • Transcription Factors / chemistry*
  • Transcription Factors / metabolism
  • Transfection
  • Transferrin / metabolism

Substances

  • ADP-Ribosylation Factor 6
  • Clathrin
  • DNA-Binding Proteins
  • Liposomes
  • Phosphatidylinositols
  • Receptors, Transferrin
  • Transcription Factor AP-2
  • Transcription Factors
  • Transferrin
  • Guanosine Triphosphate
  • Glutathione Transferase
  • ADP-Ribosylation Factors
  • ARF6 protein, human