Intersectin-2L regulates caveola endocytosis secondary to Cdc42-mediated actin polymerization

J Biol Chem. 2009 Sep 18;284(38):25953-61. doi: 10.1074/jbc.M109.035071. Epub 2009 Jul 21.

Abstract

Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH(ITSN-2L)) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The "catalytically dead" DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH(ITSN-2L)-transfected cells compared with control and 1 microM jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane.

Publication types

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

MeSH terms

  • Actin-Related Protein 2-3 Complex / genetics
  • Actin-Related Protein 2-3 Complex / metabolism
  • Actins / genetics
  • Actins / metabolism*
  • Adaptor Proteins, Vesicular Transport / antagonists & inhibitors
  • Adaptor Proteins, Vesicular Transport / genetics
  • Adaptor Proteins, Vesicular Transport / metabolism*
  • Caveolae / metabolism*
  • Endocytosis / physiology*
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism*
  • Enzyme Activation / physiology
  • Humans
  • Protein Structure, Tertiary / physiology
  • Pseudopodia / genetics
  • Pseudopodia / metabolism
  • RNA, Small Interfering / genetics
  • Wiskott-Aldrich Syndrome Protein / genetics
  • Wiskott-Aldrich Syndrome Protein / metabolism
  • cdc42 GTP-Binding Protein / genetics
  • cdc42 GTP-Binding Protein / metabolism*

Substances

  • Actin-Related Protein 2-3 Complex
  • Actins
  • Adaptor Proteins, Vesicular Transport
  • ITSN2 protein, human
  • RNA, Small Interfering
  • WAS protein, human
  • Wiskott-Aldrich Syndrome Protein
  • cdc42 GTP-Binding Protein