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 K d 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.Ras signaling | Ras assay

Stabilization of C-RAF:KSR1 complex by DiRas3 reduces availability of C-RAF for dimerization with B-RAF

“…It is well established that numerous GTPases are regulated by dimerization [24,[39][40]. Also our previous experiments revealed that DiRas3 is able to heterodimerize with active H-Ras [27].…”

Plasma membrane regulates Ras signaling networks

“…Computer simulations suggest that lipid anchors in the HVR of H-Ras and N-Ras are capable of forming dimers and nanoclusters. 71,73,83 In-cell studies of the K-Ras lipid anchor using Homo-FRET and electron microscopy also demonstrate that the lipid anchor is sufficient for dimerization and clustering on the membrane. 84 How can the HVR, -that is not involved in nucleotide binding or hydrolysis -affect dimer and nanocluster formation in a nucleotide dependent manner?…”

“…H-Ras dimerization in lipid bilayers has been demonstrated by time-resolved fluorescence spectroscopy and microscopy, however no higher order oligomers of H-Ras were observed. 73 Variations in membrane composition had no effect on H-Ras dimerization. N-Ras also dimerizes in the GDP-bound inactive state in POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayers.…”

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

“…5). An allosteric level of control over dimerization could be an important regulator of Ras interactions with other signaling proteins (63), begging the question of whether the dimers of each isoform should be treated as discrete entities with overlapping yet unique functional and structural characteristics.…”