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.