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

Abstract

R-Ras promotes cell adhesion and activation of integrins through a process that is yet unknown. We show here that active R-Ras (38V) promotes the formation of focal adhesions and a spread cell shape. By contrast, the dominant-negative mutant of R-Ras (43N) reduces the number of focal adhesions, leading to the formation of refractile cells. In adherent cells wild-type R-Ras, activated (38V) R-Ras and endogeous R-Ras were preferentially targeted to focal adhesions, whereas the dominant-negative mutant (43N) of R-Ras was excluded from these structures. Activated mutants of H-Ras and K-Ras were not found in focal adhesions. We dissected R-Ras to find out the determinants that are important for the targeting process. The outermost region in the N-terminus of R-Ras, as well as the intact proline-rich sequence in the C-terminus of RRas that mediates binding to Nck, were not essential. Mutating the potential palmitoylation site (C213A) of RRas results in depalmitoylation and accumulation of R-Ras in the Golgi. Using H-Ras/R-Ras, R-Ras/H-Ras and RRas/K-Ras hybrid molecules we showed that the C-termini (175-218 amino acids) of R-Ras contains the signal for focal adhesions targeting. Exchanging the hypervariable region of H-Ras to R-Ras inhibited the targeting of R-Ras to focal adhesions, whereas H-Ras obtained the ability to localize to focal adhesions after receiving the hypervariable region of R-Ras. This indicates that R-Ras targeting is mediated both by the nucleotide binding status as well as through a specific region in the C-terminus of R-Ras. These results indicate that targeting and activation of R-Ras are linked processes in the formation of focal adhesions.

Eph receptors inactivate R-Ras through different mechanisms to achieve cell repulsion

“…Furthermore, activated R-Ras enhances the formation of focal adhesions in some cells (Kwong et al, 2003). Although R-Ras has not been detected in the focal adhesions of COS cells (Furuhjelm and Peranen, 2003), we observed that activated R-Ras enhances COS cell spreading, similar to manganese treatment. Furthermore, RRas causes spreading and enlargement of neuronal growth cones.…”

“…R-Ras is also known to positively regulate integrin function in many cell types (Holly et al, 2005;Hughes et al, 2001;Keely et al, 1999;Oertli et al, 2000;Sethi et al, 1999;Zhang et al, 1996;Zou et al, 1999) and activated R-Ras reportedly localizes to focal adhesions through the hypervariable region at the carboxyl terminus, which is also the region important for integrin activation (Furuhjelm and Peranen, 2003;Hansen et al, 2002;Oertli et al, 2000). Furthermore, activated R-Ras enhances the formation of focal adhesions in some cells (Kwong et al, 2003).…”

Characterization of the Rab8-specific membrane traffic route linked to protrusion formation

“…Our results support a model where Rab8 activity is needed to rapidly take in membrane from the cell surface (ruffles) regions that no longer support adhesion. This membrane that contains structural (adhesion receptors) and regulatory (Arf6, Rho and Ras) components is then recycled back to the plasma membrane via a recycling compartment for the formation of new protrusions (Radhakrishna and Donaldson, 1997;Ng et al, 1999;Furuhjelm and Peränen, 2003;Deretic et al, 2004;Powelka et al, 2004;Schlunck et al, 2004). According to this, a shift from exocytosis to endocytosis (macropinocytosis) of membranes would stop protrusive activity and result in retraction of cell surface domains (tracking tail).…”

Identification of Endosomal Epidermal Growth Factor Receptor Signaling Targets by Functional Organelle Proteomics

“…Transfection-pEGFP-R-Ras wild type construct was described previously (25). Transfection was performed using 1 g of plasmid DNA, Lipofectamine 2000 (Invitrogen), and Opti-MEM medium (Invitrogen) according to the manufacturer's instructions.…”