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First published online 30 July 2003
doi: 10.1242/jcs.00689

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The C-terminal end of R-Ras contains a focal adhesion targeting signal

Institute of Biotechnology, Program in Cellular Biotechnology, PO Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland

View larger version (50K): [in a new window]   Fig. 1. R-Ras uses endomembranes to reach the plasma membrane. Hela (A-C) and Cos-7 (D-F) cells were transfected with vectors pEGFP-R-Raswt (A,B) and pEGFP-R-Ras43N (C,D), or double transfected with pEGFP-R-Ras43N and pcDNA4/TO-Arf6-27N (E,F). EGFP-R-Raswt colocalized with a p115 Golgi marker (arrow) and was also seen on the nuclear envelope (arrowhead). In addition to these organelles, EGFP-R-Ras43N was found on vesicular (C) and vacuolar (D) structures, which colocalized with Arf6-27N (arrowheads) (E,F). HeLa cells were transfected with pEGFP-RRaswt (wt), pEGFP-R-Ras38V (38V), pEGFP-R-Ras43N (43N) and pEGFP-C1A, and total cell extracts were analysed by western blot (G). The positions of molecular size markers are indicated.

View larger version (45K): [in a new window]   Fig. 2. Plasma membrane localization of R-Ras molecules. HeLa cells transiently expressing EGFP-R-Raswt (A), EGFP-R-Ras38V (B), EGFP-R-Ras43N (C) and EGFP (D) were fixed and analysed by fluorescence microscopy. Note that EGFP-R-Raswt and EGFP-RRas38V are found in adhesion-like structures, whereas EGFP-RRas43N and EGFP are not. Localization of endogenous R-Ras (E) to vinculin (F) containing focal adhesions in untransfected HeLa were seen using specific antibodies against R-Ras and vinculin. EGFP-RRas38V was also detected in focal adhesions in C2C12 cells (G,H). Arrowheads indicate focal adhesion-like localization. Bars, 10 µm. (I) The percentage of cells containing R-Ras in focal adhesions was calculated for R-Raswt, R-Ras38V and R-Ras43N. Values are means ± s.d. from three independent experiments in which >100 cells were scored per condition.

View larger version (111K): [in a new window]   Fig. 3. R-Ras in focal adhesions. Cells were transfected with pEGFP-R-Ras38V (R-Ras38V), and pEGFP-R-Ras43N (R-Ras43N) fixed, stained for vinculin and analysed by confocal microscopy. Note that R-Ras43N does not colocalize with vinculin, whereas R-Ras38V shows a strong colocalization. In R-Ras-38V/Rac1-12V the pCDNA4-TO-R-Ras38V vector was cotransfected with pEGFP-Rac12V and stained with an antibody against R-Ras. Arrowheads indicate colocalization of R-Ras and EGFP-Rac1 to focal adhesions. Bars, 10 µm.

View larger version (51K): [in a new window]   Fig. 4. R-Ras modulates focal adhesion formation and cell spreading. Hela cells grown on collagen-coated glass coverslips were transfected overnight with pEGFP-Raswt (A,D), pEGFP-R-Ras38V (B, E) and pEGFP-RRas43N (C,F). Note that the R-Raswt cell contains distal focal adhesions, the R-Ras38V cell contains numerous large focal adhesions and the R-Ras43V cell contains few small adhesions. R-Raswt cells were often elongated (D), RRas38V cells (E) well spread and R-Ras43N cells (F) retracted with filopodia-like structures. The number of focal adhesions was calculated from above transfected cells, 15 cells/construct/experiment (J). Results shown are the mean ± s.e.m. of three experiments. Cells expressing EGFP-Raswt, EGFP-R-Ras38V and pEGFP-RRas43N were also plated on collagen-coated coverslips for 60 minutes for estimation of cell spreading (K). 30 cells were counted for each construct and experiment. Results shown are the mean ± s.e.m. of three experiments. Typical spread cells are shown for corresponding construct in G, H and I. Note the numerous large focal adhesions in EGFP-R-Ras38V cells and the few small in EGFP-R-Ras43N cells. Bars, 10 µm.

View larger version (47K): [in a new window]   Fig. 6. Determinants affecting R-Ras targeting. HeLa cells were transiently transfected with pEGFP-R-Ras38V-NT (A,B), pEGFPHVR (C,D), pEGFP-R-Ras38V-dHVR (E,F), pEGFP-R-Ras38V-2PA (G,H), pEGFP-R-Ras38V-CA (I,J), pEGFP-R-Ras38V-SP (K,L) and pEGFP-R-Ras38V-dC (M,N), fixed and stained with anti-vinculin. Observe the absence of EGFP-HVR from vinculin-containing adhesions. Note the accumulation of R-Ras38V-dHVR, R-Ras38VCA in Golgi and R-Ras38V-SP in ER. EGFP-R-Ras38V-dC that lacks signals for lipid modifications is found in the nucleus.

View larger version (30K): [in a new window]   Fig. 5. R-Ras constructs and R-Ras palmitoylation. (A) GFP constructs used were wild-type R-Ras (pEGFP-R-Raswt), constitutively active R-Ras38V (pEGFP-R-Ras38V), dominant-negative R-Ras43 (pEGFP-R-Ras43N), constitutively active R-Ras deleted of the first 28 amino acids (pEGFP-R-Ras38V-NT), constitutively active R-Ras38V deleted of its last six amino acids (pEGFP-R-Ras38VdC), constitutively active R-Ras38V deleted of its HVR (175-212) but containing the last six amino acids (212-218) (pEGFP-R-Ras38V-dHVR), constitutively active R-Ras38V-2PA with two proline substituted on P202A and P203A (pEGFP-RRas38V-2PA), constitutively active R-Ras38V with a mutated palmitoylation site C213A (pEGFP-R-Ras38V-CA), constitutively active R-Ras38V with both palmitoylation and nucleotide destabilizing mutations C213A/S172P (pEGFP-R-Ras38V-SP) and the hypervariable region encoding 191-218aa of R-Ras (pEGFPHVR). (B) HeLa cells were transfected overnight with the following constructs: pEGFP-H-Ras61L (1), pEGFP-R-Ras38V-CA (2) and pEGFP-R-Ras38V (3). The cells were then labelled with [3H]palmitic acid for 4 hours, immunoprecipitated and analysed by SDS-PAGE. Note that the construct encoding EGFP-R-Ras with a mutated palmitoylation site (C213A; lane 2) is not labelled with palmitate, whereas EGFP-H-Ras61L (lane 1) is strongly labelled, and EGFP-R-Ras38V (lane 3) to a lesser extent.

View larger version (36K): [in a new window]   Fig. 7. Chimeric Ras constructs unravelled the R-Ras-specific focal adhesion targeting signal. (A) Activated forms of H-Ras (61L), K-Ras (12V) and R-Ras (38V), as well as indicated hybrids, were all fused to EGFP. The R-Ras38V/H-RasHVR contained the first 1-174 amino acids from R-Ras38V and the 148-189 amino acids from the C-termini of H-Ras12V. The H-Ras12V/R-RasHVR contained the N-termini 1-147 amino acids from H-Ras61L and the 175-218 amino acids from the C-termini of R-Ras38V. The R-Ras38V/K-RasHVR hybrid contained the 1-174 amino acids from the N-termini of RRas38V and the 148-188 amino acids from the C-termini of KRas12V. (B) Hela cells were transfected overnight with pEGFP-HRas61L (a,b), pEGFP-K-Ras12V (c,d), pEGFP-R-Ras38V/HRasHVR (e,f) pEGFP-R-Ras38V/K-RasHVR (g,h), and pEGFP-HRas61L/R-RasHVR (i,j), fixed and stained for vinculin. Note that neither K-Ras12V nor H-Ras61L was found in focal adhesions. However, both showed typical ruffle and lamellipodia structures and usually a reduced number of focal adhesions. Replacing the R-Ras specific HVR with corresponding regions from H-Ras61L and KRas12V inhibited R-Ras targeting (e-h). By contrast, adding the HVR from R-Ras to H-Ras61L led to focal adhesion targeting (i,j). Arrowheads show H-Ras61L/R-RasHVR in focal adhesions.

View larger version (46K): [in a new window]   Fig. 8. Cholesterol-dependent localisation of R-Ras to focal adhesion. Hela cells transfected with pEGFP-R-Ras38V were grown over night in serum-free (K) medium (A,B). Cholesterol replenishment (CD/Chol) was carried out for 30 minutes on EGFPR-Ras38V expressing cells grown overnight in the presence of serum (C,D). Some serum-starved cells were depleted of cholesterol by adding 0.5% ß-methylcyclodextrin (MßCD) for 30 minutes (E,F). (G,H) Cholesterol replenishment of cholesterol depleted cells (CDCD/Chol) for 30 minutes. EGFP-R-Ras38V expressing cells (A,C,E,G) were fixed with paraformaldehyde, permeabilized with 0.1% Triton-X-100 and stained with an antibody against phosphocaveolin (P-Caveolin) (B,D,F,H). Bars, 10 µm.