Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane.
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.
Rotblat
Barak
B
Institute for Molecular Bioscience, 306 Carmody Road, University of Queensland, Brisbane 4072, Australia.
Prior
Ian A
IA
Muncke
Cornelia
C
Parton
Robert G
RG
Kloog
Yoel
Y
Henis
Yoav I
YI
Hancock
John F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D007150:Immunohistochemistry; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013997:Time Factors
D008055:Lipids; D016283:Proto-Oncogene Proteins p21(ras)