Institute of Biotechnology, Program in Cellular Biotechnology, PO Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland
* Author for correspondence (e-mail: johan.peranen{at}helsinki.fi)
Accepted 28 May 2003
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Summary |
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Key words: GTPase, R-Ras, Targeting, Focal adhesion
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Introduction |
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R-Ras shows 55% identity with Ras, and its minimal effector region is
identical to that of Ras, but R-Ras contains a 26 amino acid (aa) extension in
its N-terminus. Many Ras effectors and some exchange factors interact with
R-Ras, but R-Ras shows a much lower ability to transform cells compared with
Ras (Cox et al., 1994).
However, R-Ras regulates cell adhesion, spreading and phagocytosis by
activating integrins (Zhang et al.,
1996
; Berrier et al.,
2000
; Self et al.,
2001
), and it has also been shown to antagonize Ras/Raf-initiated
integrin suppression (Sethi et al.,
1999
). How R-Ras activates integrins is not known, but its
effector loop and prenylation site, as well as the proline-rich sequence in
the hypervariable region of RRas, are essential for this activation process
(Oertli et al., 2000
;
Wang et al., 2000
). The
proline-rich region has been shown to bind the adaptor protein Nck, which is
known to interact with proteins accumulating in focal adhesions
(Wang et al., 2000
).
Furthermore, the Eph receptor tyrosine kinase, EphB2, phosphorylates a
tyrosine residue in the effector region of RRas, leading to suppression of
R-Ras-mediated adhesion (Zou et al.,
1999
). A similar relationship has also been found to exist between
activated Src and R-Ras (Zou et al.,
2002
).
Focal adhesions (FA) are specialized signalling platforms on the cell
surface that mediate cell-matrix interactions via integrins, which are
associated with different cytoskeletal proteins
(Sastry and Burridge, 2000).
FAs are dynamic structures that assemble and disassemble when cells migrate or
divide. This assembly/disassembly process is regulated by the Rho GTPases
(Kaibuchi et al., 1999
).
Activation of RhoA promotes the assembly of large focal adhesions through
increased contractility, whereas Rac1 induces the formation of small adhesions
called focal complexes at the leading edge of migrating cells
(Nobes and Hall, 1995
). The
turnover of FAs is regulated by Ras (Nobes
and Hall, 1999
). However, FAs may not be the only platforms that
mediate cell signalling. Recent studies indicate that microdomains called
lipid rafts may also participate in signal transduction
(Simons and Toomre, 2000
).
Whether the lipid rafts have a direct role in processes mediating cell
adhesion is still uncertain (Pande,
2000
).
To better understand the role of R-Ras in cell adhesion we decided to study its targeting to the cell surface. We show here that R-Ras is preferentially targeted to focal adhesions and that this targeting process is dependent on the nucleotide state of R-Ras. Only GTP-bound R-Ras is associated with focal adhesions, whereas the GDP form is excluded from these structures. The hypervariable region of R-Ras was sufficient in targeting another -Ras protein to focal adhesions, indicating that this region contains the targeting signal. Finally, we show that the targeting of R-Ras and the integrity of focal adhesions is dependent on the cholesterol content of the plasma membrane. Our data underscore the importance of specific localization of Ras molecules on the plasma membrane in mediating cell signalling.
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Materials and Methods |
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Western blot
For western blot analysis HeLa cells were grown overnight on two 6 cm
plates and transiently transfected with pEGFP-R-Raswt, pEGFPR-Ras38V or
pEGFP-R-Ras43N using Fugene 6 according to the manufacturer (Roche
Diagnostics, Mannheim, Germany). After 20 hours the cells, which were about
80% confluent, were lysed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide
gel electrophoresis) sample buffer and the DNA was disrupted by passing it
through a needle. The presence of R-Ras was detected by western blot using
anti-R-Ras (Santa Cruz Biotechnology, Santa Cruz, CA) as previously described
(Peränen et al.,
1996).
Cells transfection, labelling and cholesterol depletion
Hela cells were grown overnight on collagen-coated (30 µg/ml) coverslips
and transiently transfected with constructs expressing RRas, Rac and their
corresponding mutants by Fugene 6 according to the manufacturer (Roche)
(Peränen and Furuhjelm,
2001). Equal molarity of constructs was used in double
transfection studies. Labelling of pEGFP-H-Ras61L-, pEGFP-R-Ras38V- and
pEGFP-Rras38V/213A-transfected HeLa cells with
[9,10(n)-3H]palmitic acid (Amersham Biosciences Europe) at
200 µCi/ml in modified Eagle's medium plus 5% dialysed bovine serum for 4
hours. Immunoprecipitation of the indicated Ras molecules from the labelled
cells was done by H-Ras- and R-Ras-specific antibodies as described earlier
(Peränen and Furuhjelm,
2001
). The immunoprecipates were analysed by SDS-PAGE and
fluorography. Cholesterol depletion of HeLa cells with 0.5-1%
ß-methylcyclodextrin (MßCD) for 30-60 minutes was carried out as
described previously (Parton and Hancock,
2001
). Cholesterol/MßCD inclusion complexes were added to
cholesterol-depleted cells or to cells grown in serum
(Parton and Hancock,
2001
).
Cell spreading
Subconfluent Hela cells were transfected overnight with indicated
EGFP-R-Ras constructs. They were then harvested with 0.5 mM EDTA in
phosphate-buffered saline (PBS), resuspended in MEM containing fatty-acid-free
bovine serum albumin (5 mg/ml), and the cells were counted. An appropriate
number of cells were then plated onto collagen-(30 µg/ml) coated cover
slips. After 60 minutes at 37oC cells were fixed with
paraformaldehyde and stained for vinculin. Spread versus unspread
EGFP-positive cells were counted.
Immunocytochemistry
Transfected C2C12 or HeLa cells were processed for immunofluorescence or
confocal microscopy as previously described
(Peränen et al., 1996;
Peränen and Furuhjelm,
2001
). Antibodies used were anti-Arf6 (NeoMarkers, Fremont, CA),
anti-caveolin (BD Biosciences, San Diego, CA), anti-ß1
integrin (BRL/GIBCO, Gaitersburg, MD), anti-paxillin (Trans lab),
anti-phospho-caveolin (Trans lab), anti-p115 (Trans lab), anti-R-Ras (Santa
Cruz), anti-talin (Sigma), anti-vinculin (Sigma). Goat anti-rabbit
IgG-lissamine and goat anti-mouse IgG lissamine were from Jackson
Immunoresearch.
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Results |
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|
GTP-dependent targeting of R-Ras to focal adhesions
The plasma membrane is considered to be the main platform for Ras-mediated
signalling, where R-Ras is thought to regulate adhesion and integrin
activation (Zhang et al.,
1996; Sethi et al.,
1999
). Interestingly, both EGFP-R-Raswt and EGFP-R-Ras38V were
localized to focal adhesion-like structures in HeLa cells, whereas
EGFP-R-Ras43N was evenly distributed on the plasma membrane
(Fig. 2A-F). EGFP-RRas38V
showed the strongest staining intensity in focal adhesions. Quantification of
cells harbouring the abovementioned constructs showed that 84% of the
R-Ras38V-expressing cells contained R-Ras38V in focal adhesions, whereas the
value for EGFP-R-Raswt was 40% (Fig.
2I). By contrast, only 0.5% of cells expressing EGFP-R-Ras43N
contained R-Ras-positive focal adhesions, suggesting that the GTP-bound form
of R-Ras is preferentially targeted to focal adhesions. When R-Ras was
expressed in excess its localization to the focal adhesions was more difficult
to observe due to strong staining of the surrounding plasma membrane, possibly
indicating that the binding of R-Ras to focal adhesions was saturated (data
not shown). Identical results were obtained with untagged R-Ras constructs in
combination with anti-R-Ras antibodies
(Fig. 3). R-Ras-38V was also
observed in C2C12 cells (Fig.
2G,H), therefore its localization to focal adhesions was not
restricted to HeLa cells. However, in the HT1080 fibrosarcoma cell line and
COS-7 cells EGFP-R-Ras38V was not associated with focal adhesions (not shown).
HT1080 cells contain a mutant N-ras allele that mediates a transformed
phenotype, including disorganized actin and poor adherence, and these features
may counteract the targeting of R-Ras to focal adhesions
(Marshall et al., 1982
). This
indicates that the localization of R-Ras to focal adhesions is typical for
strongly adherent cells, like HeLa and C2C12. Furthermore, we also showed that
endogenous R-Ras in HeLa cells is localized to focal adhesions that are
positive for vinculin (Fig.
2E,F). EGFP alone did not localize to focal adhesions, but was
preferentially found in the nucleus (Fig.
2D).
|
To characterize the identity of R-Ras-containing focal adhesions we stained
EGFP-Ras38V-expressing HeLa cells with known focal adhesion markers. Vinculin
colocalized nicely with EGFP-Ras38V when analysed by confocal microscopy
(Fig. 3). This was also true
for ß1-integrin, talin and paxillin (data not shown). By
contrast, EGFP-R-Ras43N did not colocalize with these markers
(Fig. 3). It has recently been
shown that Rac12V colocalizes with p21-activated protein kinase, -PAK,
to focal adhesions of HeLa cells (Manser
et al., 1997
). Likewise, we found that EGFP-Rac12V localized with
R-Ras38V in focal adhesions, indicating that there may be a functional link
between Rac1 and R-Ras (Fig.
3).
Focal adhesions formation is linked to R-Ras activity
R-Ras is known to regulate cell adhesion. Thus, we next studied whether
R-Ras influences the structure and number of focal adhesions in HeLa cells.
Cells were transfected overnight with constructs encoding EGFP-R-Raswt,
EGFP-R-Ras38V or EGFP-R-Ras43N (Fig
4). Cells expressing EGFP-R-Raswt were often elongated and
contained focal adhesions in the distal regions of the cell. When cells
expressed EGFP-R-Ras38 they were symmetrical and spread, and they contained
large focal adhesions that were located nearer the center of the cell. By
contrast, cells expressing EGFP-R-Ras43N were contracted and often contained
filopodia-like structures. Moreover, the focal adhesions located distally and
were very small compared with those found in EGFP-R-Ras38-expressing cells. We
next quantified the number of focal adhesions in cells expressing these
EGFP-R-Ras constructs (Fig.
4J). We found that RRas38V-expressing cells contained more focal
adhesions than did R-Raswt, whereas R-Ras43-expressing cells had fewer
adhesions than R-Raswt. This suggests that active R-Ras promotes the formation
of focal adhesions, whereas R-R-Ras inhibits their formation. The same
EGFP-R-Ras constructs were also used to test cell spreading
(Fig. 4K). EGFP-R-Raswt and
EGFP-R-Ras38V cells spread with an equal efficiency, whereas EGFP-R-Ras43N
expression led to fewer spread cells. The difference in cell spreading
correlated with the formation of focal adhesions. EGFP-R-Ras38N cells were
symmetrical with large focal adhesions, whereas EGFP-R-Raswt cells had
adhesions more peripherally. By contrast, EGFP-R-Ras43N-expressing cells
contained very few and tiny focal adhesions. In summary, our results show that
R-Ras is essential for the formation of focal adhesions, and confirm previous
studies that R-Ras controls cell spreading
(Berrier et al., 2000;
Zhang et al., 1996
).
|
Potential targeting signals in R-Ras
R-Ras contains an extended N-terminus compared with other Ras molecules. We
deleted this N-terminal region (1-28 aa) and expressed the molecule,
EGFP-R-Ras38V-NT, in HeLa cells. Because EGFP-R-Ras38V-NT was nicely localized
to focal adhesions it is unlikely that this region is important for targeting
(Fig. 6A,B). The hypervariable
region of small GTPases has been implicated in membrane-specific targeting
processes (Choy et al., 1999;
Chavrier et al., 1991
;
Michaelson et al., 2001
). This
raised the possibility that the hypervariable region, including the outermost
C-terminus, is responsible for targeting R-Ras to focal adhesions. To test
this hypothesis we fused the hypervariable region (HVR: 191-218 aa) of R-Ras
to EGFP (Fig. 5A). This
EGFP-HVR was efficiently transported to the plasma membrane via Golgi.
However, it was not targeted to focal adhesions; instead, it showed a uniform
distribution at the plasma membrane, indicating that this part of the
hypervariable region is essential for membrane targeting but insufficient for
localization of R-Ras to focal adhesions
(Fig. 6C,D). We also made a
construct encoding a protein (EGFP-R-Ras38V-dHVR) that lacked the whole HVR
(175-212 aa) but which contained the six outermost amino acids needed for
lipid modification. This protein was trapped in the Golgi and on small
vesicles, indicating that HVR is essential for proper transport to the plasma
membrane (Fig. 6E,F). The
proline-rich sequence in the hypervariable region binds a SH3 domain of Nck,
indicating that it might contribute to the targeting mechanism of R-Ras
(Wang et al., 2000
). However,
the introduction of mutations (P202A, P203A) in the proline-rich sequence of
R-Ras38V did not inhibit targeting of RRas38V to focal adhesions
(Fig. 5A;
Fig. 6G,H).
|
|
R-Ras is palmitoylated and contains a potential palmitoylation site (C213)
upstream of the CAAX box (Lowe and
Goeddel, 1987; Schmittberger
and Waldmann, 1999
). We showed that EGFP-R-Ras38V is labelled with
palmitate, whereas EGFP-R-Ras38V/C213A, which contains a mutation in the
potential palmitoylation site did not incorporate palmitate, suggesting that
cysteine 213 is the target for palmitoylation in R-Ras
(Fig. 5A,B). Moreover,
EGFP-RRas38V/C213A showed an increased accumulation in Golgi and consequently
a decreased appearance on the plasma membrane when compared with the other
R-Ras mutants (Fig. 6I,J). Such
a Golgi retention has also been observed when the palmitoylation sites of
H-Ras and N-Ras are mutated (Choy et al.,
1999
). EGFP-R-Ras38V incorporates far less palmitate than
EGFP-H-Ras61L; this can not be explained simply by the fact that H-Ras
contains two palmitoylation sites but R-Ras contains only one
(Fig. 5B). Both are labelled
equally well by methionine when analysed by immunoprecipitation (not shown).
One possibility is that the turnover rate of palmitate is different for these
two Ras molecules. The C213A mutation did not inhibit R-Ras38V association
with focal adhesions (not shown). The introduction of an additional mutation
(S173P), which probably destabilizes nucleotide binding, into
EGFP-RRas38V/C213A led to retention of R-Ras in the endoplasmic reticulum
(ER), suggesting that proper nucleotide binding or protein folding is needed
for transport from the ER (Fig.
5A; Fig. 6K,L).
Finally, deletion of both the CAAX box and the palmitoylation site resulted in
the accumulation of the molecule in the cytoplasm and nucleus, suggesting that
R-Ras can not associate with focal adhesions when it is not bound to the
membrane (Fig. 5A;
Fig. 6M,N). Taken together, our
results show that the hypervariable region of R-Ras is crucial for membrane
targeting and transport.
Dissecting the R-Ras-specific targeting signal by using
R-Ras/H-ras/K-Ras hybrid molecules
H-Ras and K-Ras are known to localize to different subdomains on the plasma
membrane (Prior and Hancock,
2001). We fused H-Ras61L and K-Ras12V to EGFP, and expressed them
in HeLa cells. In contrast to EGFP-R-Ras, neither EGFP-H-Ras61L nor
EGFP-K-Ras12V localized to focal adhesions
(Fig. 7B). However, both
proteins induced the formation of lamellipodia and ruffles, which are typical
for transformed cells. Moreover, their expression resulted in a decrease in
the number and size of the focal adhesions. To see whether the HVR of R-Ras is
essential for the targeting process, we replaced the 175-218 aa region from
R-Ras38V with the corresponding regions (148-189 aa) from H-Ras or from K-Ras
(147-188 aa) (Fig. 7A). The
hybrid proteins EGFP-R-Ras38V/H-RasC and EGFP-R-Ras38V were expressed in HeLa
cells. Neither proteins were localized to focal adhesions but were uniformly
distributed at the plasma membrane (Fig.
7B). In addition, both had a phenotype that more resembled
H-Ras61L and K-Ras12V. Because the 175-218 aa region of R-Ras seemed to be
essential for focal adhesion targeting we reasoned that this region could also
function in H-Ras as a targeting signal. Thus, we made an HRas61L/R-RasC
chimera, which was expressed in HeLa cells
(Fig. 7A). This chimera, which
is mainly composed of H-Ras, was localized to vinculin-containing focal
adhesions, indicating that the 175-218 aa of R-Ras contains the essential
elements for focal adhesion targeting (Fig.
7B).
|
Focal adhesions are sensitive to cholesterol depletion
It has been shown recently that H-Ras resides in cholesterol-rich lipid
rafts and caveolae on the plasma membrane, and that activation of H-Ras leads
to its segregation from rafts (Prior et
al., 2001; Prior and Hancock,
2001
). K-Ras, by contrast, is localized predominantly to the
disordered plasma membrane (Prior et al.,
2001
). Caveolin, the main component of caveolae, is known to
participate in integrin-mediated adhesion and Ras signalling
(Parton and Hancock, 2001
;
Wei et al., 1999
;
Roy et al., 1999
). Thus, we
stained EGFP-R-Ras38V-expressing cells with anti-caveolin and
anti-phospho-caveolin recognizing caveolin phosphorylated on tyrosine 14
(Lee et al., 2000
). Caveolin
was found in patches over the cell surface and along the cell margins, but
there was no colocalization with EGFPR-Ras38V (data not shown). By contrast,
phospho-caveolin colocalized nicely with EGFP-R-Ras38V in focal adhesions
(Fig. 8A,B). To investigate
whether the localization of RRas38V to focal adhesions is dependent on
cholesterol-rich subdomains, we depleted serum-starved cells expressing
EGFP-R-Ras-38V with 0.5-1% ß-methylcyclodextrin for 30 minutes. This
resulted in the smooth distribution of EGFP-RRas-38V on the plasma membrane
and redistribution of phospho-caveolin to small dot-like structures that no
longer colocalized with EGFP-R-Ras38V (Fig.
8E,F). This was associated with a simultaneous decrease in the
size and number of focal adhesions. Repletion of cholesterol depleted cells
with cholesterol/CD inclusion complexes for 30-60 minutes resulted in
reformation of focal adhesions and retargeting of both EGFP-R-Ras38V and
phospho-caveolin (Fig. 8G,H).
Finally, cholesterol replenishment on cells that had not been serum starved
showed normal focal localization for both EGFP-RRas-38V and phospho-caveolin
(Fig. 8C,D). We conclude that
the integrity of focal adhesions and the localization of EGFPR-Ras38V and
phospo-caveolin to adhesions are dependent on the cholesterol content of the
plasma membrane.
|
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Discussion |
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To unravel the targeting signal of R-Ras we constructed different R-Ras
mutants. We showed that the N-terminus present in R-Ras is not necessary for
the targeting process. Although mutations in the proline-rich sequence of
R-Ras38V suppresses cell attachment, the mutants are still more potent than
R-Raswt in inducing cell attachment (Wang
et al., 2000). Because the mutations in this sequence did not
affect R-Ras targeting, the sequence must have other functions that are
related to the binding of Nck (Wang et
al., 2000
). One possibility is that Nck could mediate a cross-talk
between RRas and Rac1, because it binds PAK, which is known to interact with
Rac1 (Manser et al., 1994
;
Manser et al., 1997
).
Moreover, we showed that R-Ras and Rac1 colocalize in focal adhesions. Such a
cross-talk could be important in amplifying signals that mediate cell adhesion
and spreading.
We showed that lipid modification is essential for R-Ras-specific targeting, because R-Ras lacking these signals is found in the cytoplasma and nucleus. In addition, we showed for the first time that amino acid C213 of R-Ras is the most probable attachment site for palmitic acid. When this site was mutated, R-Ras accumulated in the Golgi. This was also the case when the hypervariable region was deleted. Together, this suggests that palmitoylation and the HVR region are important for the transport of R-Ras by endomembranes to the plasma membrane.
Deletions and mutations might have pronounced effects on the function of
different proteins, making it difficult for us to find targeting signals.
R-Ras is closely related to H-Ras and K-Ras, which makes it possible to switch
regions between these molecules without making gross changes in the protein
architecture. However, H-Ras and R-Ras have opposing effects on integrin
activation. R-Ras promotes integrin activation, whereas H-Ras suppresses
integrin activation (Zhang et al.,
1996; Hughes et al.,
1997
). When the hypervariable region of R-Ras (aa 175-218) was
replaced by the corresponding region (aa 147-189) of H-Ras the targeting of
R-Ras to focal adhesions was inhibited. Interestingly, an identical
replacement between R-Ras and H-Ras was recently shown to suppress
R-Ras-mediated integrin activation (Hughes
et al., 2002
). The hypervariable region of K-Ras also inhibited
the targeting of R-Ras. Furthermore, when the hypervariable region of H-Ras
(aa 147-189) was replaced by the corresponding region (aa 175-218) of R-Ras,
the H-Ras molecule was targeted to focal adhesions, showeing that the
hypervariable region of R-Ras contains a focal adhesion-specific targeting
signal. Recently, a similar construct was shown to confer R-Ras specificity to
H-Ras (Hansen et al., 2002
).
Taken together, this suggests that the hypervariable region of R-Ras is
important for both focal adhesion targeting and integrin activation, and that
these two processes are closely linked to each other.
What would be the advantages of GTP-dependent targeting of R-Ras to focal
adhesions? First, the coupling of R-Ras activation to targeting would localize
the function of R-Ras to a defined region on the plasma membrane, eliminating
randomized signalling. Second, a localized high concentration of R-Ras
molecules could amplify the signal that mediates cell adhesion. This could
lead to the recruitment of other signal molecules and scaffolding proteins,
thereby building up the focal adhesion. This is supported by the fact that
R-Ras enhances the phosphorylation of focal adhesion kinase (FAK) and
p130cas (Kwong et al.,
2003). Conversely, deactivation of R-Ras would lead to exclusion
of R-Ras from focal adhesions, making it free for a new round of targeting.
Deactivation could be mediated by GTPase-activating proteins (GAPs), or
through phosphorylation. Interestingly, the effector domain of R-Ras is
phosphorylated by an Eph receptor kinase and by the activated Src, leading to
suppression of R-Ras-mediated cell adhesion
(Zou et al., 1999
;
Zou et al., 2002
). Finally, a
specific lipid raft-like composition of the adhesion structure might support
protein-protein-based targeting of R-Ras to focal adhesions. The cholesterol
content especially may have an important role in modulating the rigidity of
focal adhesions (Gopalakrishna et al.,
2000
), as we show here.
Why is H-Ras not localized to focal adhesions? One possibility is that the
time during which H-Ras resides in focal adhesions is more transient. This is
supported by the fact that H-Ras12V has been found to be associated with focal
adhesions at early times of expression and when expressed at relatively low
levels (Nobes and Hall, 1999).
In addition, activated H-Ras has a more suppressive function on integrins,
inducing loss and enhanced turnover of adhesions
(Nobes and Hall, 1999
;
Hughes et al., 1997
), which
might explain why RRas is excluded from HT1080 cells that possess a mutant
N-ras allele.
Both H-Ras and N-Ras use the endomembrane system to reach the plasma
membrane (Choy et al., 1999).
We showed that this is also true for R-Ras. Likewise, palmitoylation is
crucial for the efficient transport of R-Ras from Golgi onwards. Whether R-Ras
proteins are internalized from the plasma membrane is unclear. However, we
observed the GDP form of R-Ras on vesicular structures colocalizing with Arf6,
suggesting that R-Ras undergoes internalization and recycling
(Brown et al., 2001
). An
interesting possibility is that R-Ras deactivation versus activation is
coupled to a membrane-based recycling route that is important for the
regulation of adhesion turnover. Future studies in this direction could be
important in understanding the functions of focal adhesions.
In conclusion, our data on R-Ras further strengthens the importance of specific micro-domains on the plasma membrane as platforms for signalling by small GTPases.
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Acknowledgments |
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