Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane

Mol Cell Biol. 2004 Aug;24(15):6799-810. doi: 10.1128/MCB.24.15.6799-6810.2004.

Abstract

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • COS Cells
  • Cell Membrane / metabolism*
  • Cricetinae
  • Cytosol / metabolism
  • Immunohistochemistry
  • Lipids / chemistry
  • Membrane Microdomains / chemistry
  • Microscopy, Electron
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins p21(ras) / chemistry*
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Sequence Homology, Amino Acid
  • Subcellular Fractions
  • Time Factors

Substances

  • Lipids
  • Proto-Oncogene Proteins p21(ras)