Ras plasma membrane signalling platforms

Abstract

The plasma membrane is a complex, dynamic structure that provides platforms for the assembly of many signal transduction pathways. These platforms have the capacity to impose an additional level of regulation on cell signalling networks. In this review, we will consider specifically how Ras proteins interact with the plasma membrane. The focus will be on recent studies that provide novel spatial and dynamic insights into the micro-environments that different Ras proteins utilize for signal transduction. We will correlate these recent studies suggesting Ras proteins might operate within a heterogeneous plasma membrane with earlier biochemical work on Ras signal transduction.

Figure 1. Membrane anchors of Ras proteins

The fully processed C-terminal membrane anchors of H-, N- and K-ras are depicted in the upper panel. The C-terminal cysteine residue of all three Ras proteins is farnesylated and carboxylmethylated (shown in red) as a result of the triplet of modifications directed by the C-terminal CAAX motif, i.e. farnesylation, removal of the AAX sequence and methylesterification. The anchor of N- and H-ras is completed by palmitoylation (shown in blue) of one or two cysteine residues, and of K-ras by a sequence of lysine residues (shown in blue). The anchor (shown in grey in the lower panel) is connected to the G-domain by the linker sequence of the HVR (shown in black). The linker comprises residues 166–180 in H- and N-ras, and residues 166–174 in K-ras. Structures of H-ras linker and N-ras anchor have recently been reported and are discussed in the text.

Figure 2. EM mapping of Ras proteins

(A) Example of a plasma membrane sheet prepared from BHK (baby hamster kidney) cells expressing GFP–H-rasG12V. The sheet is labelled with anti-GFP antibody coupled to 5 nm gold and imaged at 100000× magnification in an electron microscope. (B) Statistical analysis of multiple sheets is used to determine the extent of clustering and the radius of the clusters. In this type of analysis, if the K-function [L(r)−r] curve leaves the confidence interval (99% CI: broken black lines), it indicates that the gold pattern is significantly clustered. The analysis shows that (i) H-rasG12V (red line) is clustered, (ii) knockdown of galectin-1 expression (blue line) causes a loss of H-rasG12V clustering, and (iii) ectopic expression of galectin-1 (green line) increases the radius of H-rasG12V clusters by 8 nm.

Figure 3. SPT of Ras proteins

Trajectories of Ras proteins monitored by SPT (reproduced with permission from Murakoshi, H., Iino, R., Kobayashi, T., Fujiwara, T., Ohshima, C., Yoshimura, A. and Kusumi, A. Single-molecule imaging analysis of Ras activation in living cells. Proc. Natl. Acad. Sci. U.S.A. 101, 7317–7322 © 2004 National Academy of Sciences, U.S.A.). (A) Inactive (GDP-loaded) YFP–H-ras and YFP–K-ras single-molecule trajectories were recorded at video rate (30 Hz) and followed for 1 s on the cell membrane. After EGF stimulation, trajectories of single-activated Ras molecules are followed by FRET between YFP–Ras and BODIPY®-TR–GTP (where BODIPY® is 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene and TR is Texas Red). Using this elegant technique, Kusumi and colleagues demonstrate that some activated Ras trajectories exhibit immobile periods. This is illustrated further in (B), where trajectories of YFP–Ras molecules are followed until they bind BODIPY®-TR–GTP, and the resulting FRET signal (red) is then tracked; three of the trajectories exhibiting FRET are immobile.

Figure 4. Model of Ras protein organization on the plasma membrane

The upper panel shows the theoretical outcome of ‘simultaneously’ analysing the plasma membrane of a cell expressing GFP–H-rasG12V by SPT and EM. In the SPT experiment, four Ras molecule trajectories are followed, two of these exhibit an immobile period. EM provides a snapshot of the positions of all of the GFP–H-rasG12V proteins present on the membrane when the imaging by SPT is terminated. In the overlay of the two experiments, note that the two immobile trajectories result from GFP–H-rasG12V proteins forming nanoclusters, whereas EM detects the two mobile GFP–H-rasG12V proteins as non-clustered random particles. The lower panel summarizes the characteristics of the two types of H-ras nanocluster discussed in the text. Activated H-ras forms a nanocluster that is stabilized by scaffolds, such as galectin-1 and Sur-8. The nanocluster becomes trapped by the actin cytoskeleton and immobilized. The cholesterol-dependent nanocluster, formed by inactive H-ras, is more transient than the activated H-ras nanocluster.