title: H-Ras forms dimers on membrane surfaces via a protein-protein interface.
abstract: The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein-protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization Kd is measured to be ∼1 × 10(3) molecules/µm(2), and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.
authors: Howard Hughes Medical Institute and Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, CA 94720.
authors: Wan-Chen
authors: WC
authors: Lin
authors: Lars
authors: L
authors: Iversen
authors: Hsiung-Lin
authors: HL
authors: Tu
authors: Christopher
authors: C
authors: Rhodes
authors: Sune M
authors: SM
authors: Christensen
authors: Jeffrey S
authors: JS
authors: Iwig
authors: Scott D
authors: SD
authors: Hansen
authors: William Y C
authors: WY
authors: Huang
authors: Jay T
authors: JT
authors: Groves
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D019281:Dimerization; D005454:Fluorescence Polarization; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D018631:ras Proteins
keywords: Ras assay; Ras signaling