Expression of dominant negative rab5 in HeLa cells regulates endocytic trafficking distal from the plasma membrane

Exp Cell Res. 2004 Apr 1;294(2):509-22. doi: 10.1016/j.yexcr.2003.12.006.

Abstract

Ligand-mediated endocytosis is an important regulatory mechanism of epidermal growth factor (EGF) receptor (EGFR) signal transduction. Coordinated EGFR internalization and degradation function to regulate the spatial and temporal components of EGFR-effector interactions. In an effort to better understand the molecular mechanisms that control these events, we examined the role of rab5 in the endocytic trafficking of the EGFR. Rab5 is a 25-kDa guanine nucleotide binding protein that has previously been shown to be involved in the early stages of endocytic trafficking. Using adenovirally expressed dominant negative and constitutively active rab5 [rab5(S34N) and rab5(Q79L)] in cells with endogenous EGFRs, we have found that the guanine nucleotide binding state of rab5 has no bearing on the rate of EGFR endocytosis. However, expression of dominant negative rab5 affects downstream endocytic trafficking by slowing the ligand-induced disappearance of total cellular EGFR. Using confocal microscopy to examine EGF/EGFR and rab5 localization indicates that the activity of rab5 governs whether internalized EGF/EGFR and rab5 co-localize. Transferrin, which internalizes via a constitutively internalized cell surface receptor, co-sediments with rab5(WT), but not rab5(S34N) on sucrose gradients. Taken together, these data are consistent with rab5 functioning to regulate intracellular endocytic trafficking distal from the plasma membrane.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Membrane / genetics
  • Cell Membrane / metabolism*
  • Endocytosis / genetics*
  • Endosomes / genetics
  • Endosomes / metabolism*
  • Epidermal Growth Factor / metabolism
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism
  • Genes, Dominant
  • Genetic Vectors / genetics
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Ligands
  • Luminescent Proteins
  • Mutation / genetics
  • Protein Binding / genetics
  • Protein Transport / genetics
  • Receptors, Transferrin / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transferrin / metabolism
  • Transport Vesicles / genetics
  • Transport Vesicles / metabolism*
  • rab5 GTP-Binding Proteins / genetics
  • rab5 GTP-Binding Proteins / metabolism*

Substances

  • Ligands
  • Luminescent Proteins
  • Receptors, Transferrin
  • Recombinant Fusion Proteins
  • Transferrin
  • Green Fluorescent Proteins
  • Epidermal Growth Factor
  • ErbB Receptors
  • rab5 GTP-Binding Proteins