Rac1 and calmodulin interactions modulate dynamics of ARF6-dependent endocytosis

Traffic. 2011 Dec;12(12):1879-96. doi: 10.1111/j.1600-0854.2011.01274.x. Epub 2011 Sep 21.

Abstract

The main cellular Ca(2+) sensor, calmodulin (CaM), interacts with and regulates several small GTPases, including Rac1. The present study revealed high binding affinity of Rac1 for CaM and uncovered two new essential binding domains in Rac1: the polybasic region, important for phosphatidylinositol-4-phosphate 5-kinase (PIP5K) interaction, and the adjacent prenyl group. CaM inhibition increased Rac1 binding to PIP5K and induced an extensive phosphatidylinositol 4,5-bisphosphate (PI4,5P(2) )-positive tubular membrane network. Immunofluorescence demonstrated that the tubules were plasma membrane invaginations resulting from an ADP-ribosylation factor 6 (ARF6)-dependent and clathrin-independent pathway. The role of Rac1 in this endocytic route was analyzed by expressing constitutively active and inactive mutants. While active Rac1 impaired tubulation, the inactive mutant enhanced it. Intriguingly, inactive mutant expression elicited tubulation by recruiting PIP5K and inhibiting Rac1 at the plasma membrane. Accordingly, CaM inhibition inactivated Rac1 and increased Rac1/PIP5K interaction. Therefore, our findings highlight an important new role for Rac1 and CaM in controlling clathrin-independent endocytosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP-Ribosylation Factors / metabolism*
  • Animals
  • COS Cells
  • Calmodulin / metabolism*
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Chlorocebus aethiops
  • Clathrin / metabolism
  • Endocytosis / physiology*
  • Mice
  • NIH 3T3 Cells
  • Phosphatidylinositol 4,5-Diphosphate / metabolism
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Structure, Tertiary
  • Vero Cells
  • rac1 GTP-Binding Protein / metabolism*

Substances

  • Calmodulin
  • Clathrin
  • Phosphatidylinositol 4,5-Diphosphate
  • Phosphotransferases (Alcohol Group Acceptor)
  • 1-phosphatidylinositol-4-phosphate 5-kinase
  • ADP-Ribosylation Factors
  • ADP-ribosylation factor 6
  • rac1 GTP-Binding Protein