C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution

BBRC Biochemical and Biophysical Research Communications 311 (2003) 829–838 www.elsevier.com/locate/ybbrc

C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution Malene Hansen,a,b,* Ian A. Prior,c,1 Paul E. Hughes,a Beat Oertli,a Fan-Li Chou,a Berthe M. Willumsen,b John F. Hancock,c and Mark H. Ginsberga a Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA Department of Molecular Cell Biology, Copenhagen University, Oester Farimagsgade 2A, DK-1353 Copenhagen K, Denmark Institute for Molecular Bioscience and Department of Molecular and Cellular Pathology, University of Queensland, 4072, Australia b

c

Received 8 October 2003

Abstract The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175–203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype. Ó 2003 Elsevier Inc. All rights reserved. Keywords: R-Ras; H-Ras; Integrin affinity modulation; Subcellular localization

The founder members of the Ras family of small GTP-binding proteins, N-, K-, and H-Ras, which function as molecular switches in the transduction of extracellular signals involved in cell growth and proliferation [1]. All Ras proteins share a very similar core effector domain and bind to many of the same effectors and some exchange factors; however, there are often striking differences in the biological responses to individual isoforms. For instance, H-Ras and the related R-Ras share 55% overall identity but they show several distinct biological properties, e.g., they transform fibroblasts to different *

Corresponding author. Present address: Department of Biochemistry and Biophysics, University of California at San Francisco, Genentech Hall, San Francisco, CA 94143-2200, USA. Fax: 1-415-5144145. E-mail address: [email protected] (M. Hansen). 1 Present address: The Physiological Laboratory, University of Liverpool, UK. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.074

degrees [2–4] and they have contrasting roles in cell adhesion [5–7], focal adhesion formation [8,9], and in the regulation of integrin function [7,10–12]. Integrins are cell adhesion receptors that mediate transmembrane signaling and a characteristic feature of many integrins is their ability to alter their affinity for ligands in response to intracellular signals, a process termed ‘actitivation’ [13]. The interaction of integrins with their extracellular ligands is important for cell migration, growth, and survival [14]. The exact mechanism of integrin activation is poorly understood, but a role for Ras GTPases has been suggested. H-Ras has been found to suppress integrin activation in CHO cells [15] and R-Ras antagonizes, or rescues, this effect without interfering with the capacity of H-Ras to activate downstream effectors suck as ERK MAP kinase [12]. Structure–function studies of H-Ras and R-Ras have suggested a role for the highly divergent C-terminal hypervariable region (HVR) of both of these small

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GTPases in their effect on integrins. Amino acids 148– 171 of H-Ras are required for suppression of integrin affinity [16] and sequences in the C-terminal HVR of RRas (amino acids 193–218, equivalent to H-Ras amino acids 166–189) are involved in integrin regulation [17]. Interestingly, the R-Ras HVR has also been reported to contain a focal adhesion targeting signal [9]. The effectors involved in the integrin response to R-Ras in fibroblasts are currently not known; mutations in the effector domain of R-Ras have been found to abolish the rescue phenotype in these cells but the R-Ras effector PI3-kinase does not seem to be involved [18]. Attachment of Ras proteins to the plasma membrane is essential for their biological activity [19]. Signals for correct plasma membrane localization are present in the C-terminal HVR (amino acids 166–189) and include: (i) attachment of a C15 farnesyl moiety to the C-terminal CAAX box, common to all three Ras proteins, plus (ii) one or two palmitoylated cysteine residues in N- and H-Ras, respectively, or a polybasic stretch of six contiguous lysines in K-Ras [20,21]. H-Ras and K-Ras localize differently in the plasma membrane. H-Ras localizes to cholesterol-rich microdomains or lipid rafts whereas K-Ras does not [22–24]. Furthermore, H-Ras translocates from lipid rafts into non-raft plasma membrane microdomains upon activation [25]. The mechanism for this translocation is unknown but requires the linker domain (amino acids 166–179) of the H-Ras HVR and raft disassociation correlates closely with the biological activity of H-Ras [25,26]. R-Ras localizes to the plasma membrane but is posttranslationally modified by a different prenyl moiety than H-Ras, since the C-terminal amino acid CVLL directs the attachment of a C20 geranyl-geranyl group rather than a farnesyl group [27]. In addition, R-Ras is palmitoylated on position C213 [9]. Membrane association of R-Ras is essential for the rescue of H-Rasinduced integrin suppression [18] and for targeting of R-Ras to focal adhesions [9]. Interestingly, the R-Ras HVR contains a proline-rich PXXP domain to which the second SH3 domain of the adaptor protein Nck binds; mutation of this domain reduces R-Ras-mediated adhesion of mouse 32D monocytic cells [28]. A C-terminal PXXP motif is also present in the HVR of the R-Ras homolog TC21/R-Ras2, another member of the Ras superfamily of small GTP-binding proteins that, like Ras, has been implicated in the regulation of growth-stimulating pathways. Overall, TC21 shows 70% homology to R-Ras and 55% homology to Ras but seems to share more functional similarity to H-Ras than to R-Ras in that the transforming activity resembles that of H-Ras [29,30]. Both R-Ras and TC21 have, however, been found to promote integrin-mediated migration of breast epithelial cells [31]. To better understand the mechanism by which H-Ras and R-Ras exert opposing biological effects on integrin

regulation, we have investigated the sequence requirements for the R-Ras-specific reversal of H-Ras-induced integrin affinity modulation. By using a previously described set of chimeras [16], we have now found that amino acids 175–203 of R-Ras are required for reversal of H-Ras-initiated integrin suppression. We found that mutation of the proline-rich PXXP site in this region did not impair the ability of R-Ras to reverse H-Ras suppression. In addition, we found that the small GTPase TC21 rescues H-Ras-induced integrin suppression to the same level as R-Ras. Furthermore, we observed distinct localization patterns of activated forms of R-Ras and HRas in the plasma membrane; however, this difference in subcellular localization did not account for the contrasting effects of these GTPases on integrins. These findings provide new insight into the function of the R-Ras C-terminus and provide further support to a proposed functional role of this region in directing R-Ras biological specificity.

Materials and methods Antibodies and reagents. The activation-dependent anti-aIIb b3 monoclonal antibodies PAC1 [32] and anti-LIBS6 have been described previously [33]. PAC1 was a generous gift from Dr. Sandy Shattil, The Scripps Research Institute, La Jolla, CA. The hybridoma cell line (HB8784) producing the anti-Tac antibody 7G7B6 was obtained from the American Tissue Culture Collection (ATCC, Rockville, MD); the 7G7B6 antibody was biotinylated with biotin-N -hydroxysuccinimide (Sigma, St. Louis, MO) according to the manufacturer’s directions. The aIIb a6 b3 b1 -specific peptidomimetic inhibitor Ro43-5054 [34] was a generous gift of Dr. Beat Steiner, Hoffmann-LaRoche, Basel, Switzerland. Rabbit anti-caveolin-1 was obtained from BD Transduction Laboratories (San Jose, CA) and monoclonal anti-GFP was from Boehringer–Mannheim/Roche (Indianapolis, IN). cDNA constructs, cell lines, and transfection. The mammalian expression vectors encoding H-Ras(G12V), R-Ras(G38V), and HAERK2 have been described previously [12,15]; the H-Ras/R-Ras chimeras (Chimeras A–F in Fig. 2A) were described in [16]. H-Ras(PPSP) was constructed using PCR mutagenesis with pDCR-H-Ras(G12V). The H-Ras C-terminal chimeras, H-Ras(165)R-Ras(193–218) and H-Ras(180)R-Ras(208–218) (Chimeras H and I, respectively, in Fig. 2A), were described as HR166 and HR181, respectively, in [17]. Constructs expressing green fluorescent protein (GFP) fusions were made by cloning either wildtype R-Ras or R-Ras 87L into a linkermodified pEGFP-C1 vector (Clontech), thereby creating fusions to the C-terminus of GFP. Wildtype R-Ras and R-Ras 87L were generated from pMH15/R-Ras 38V [17] using oligonucleotide directed copying of deoxyuridine containing M13 derived template with minor modifications [35] of the method described earlier [36]. The pSG5 plasmids encoding the R-Ras proline to alanine mutants were constructed by using the Stratagene Quick Change kit (Stratagene, La Jolla, CA). All chimeras and mutant constructs were verified by DNA sequencing prior to further analysis. The plasmid pDCR-H-Ras(G12V) was a gift from Dr. M.H. Wigler (Cold Spring Harbor Laboratory, USA), pSG5R-Ras(G38V) was provided by Dr. Julian Downward (Signal Transduction Laboratory, ICRF, London, UK). GFP-R-Ras 87L was assayed in the PAC assay and found to rescue H-Ras-induced integrin suppression (data not shown); in addition, RRas 87L and R-Ras 38V rescue integrin suppression to largely the same degree (data not shown). The vH-Ras and H-Ras 12V constructs

M. Hansen et al. / Biochemical and Biophysical Research Communications 311 (2003) 829–838 were also compared in the integrin assay and found to have comparable suppressive integrin activity (data not shown). Chinese hamster ovary (CHO)-K1 cells were obtained from the ATCC (American Type Culture Collection). The generation of CHO ab-py cells has been described previously [37]. These cells stably express the polyoma large T antigen and bear a recombinant chimeric integrin that has the extracellular and transmembrane domains of integrin aIIb b3 joined to the cytoplasmic domains of integrin a6A b1A ðaIIb a6 b3 b1 Þ. Cells were cultured in DMEM (BioWhittaker, Walkersville, MD) containing 10% fetal calf serum (Gibco-BRL), 1% non-essential amino acids, 2 mM glutamine (Sigma), 100 U/ml penicillin, and 100 lg/ml streptomycin (Gibco-BRL). CHO cells were transiently transfected using Lipofectamine (Gibco-BRL) as described previously [38], using 2 lg of each Ras construct and 1 lg Tac-a5 construct per 60–70% confluent 10-cm dish. BHK cells were cultured at 37 °C in DMEM supplemented with 10% heat-inactivated serum supreme (BioWhittaker, Walkersville, MD) and 2 mM glutamine (Gibco-BRL). Cells were plated on coverslips at 30–40% confluence, or 10-cm plates at 50% confluence, and transfected using Lipofectamine (Gibco-BRL) with 2–15 lg of each expression plasmid. After overnight incubation, the cells were processed for electron microscopy, confocal or biochemical analysis as indicated. Flow cytometry. PAC1 binding was measured by two-color flow cytometry as described previously [15]. Briefly, ab-py cells were transiently transfected with 2 lg of each Ras construct and 1 lg Tac-a5 vector (encodes the extracellular domain of the interleukin-2 receptor [39] and serves as transfection marker) using Lipofectamine (GibcoBRL). Forty-eight hours after transfection cells were harvested by a brief trypsination and washed in DMEM/1% BSA. The 5
105 cells were incubated with 0.1% PAC1 ascites in the presence of the competitive inhibitor Ro43-5054 at 1 lM or aIIb b3 -activating antibody anti-LIBS6 ascites. After a 30 min incubation period at room temperature, cells were washed with cold DMEM/1% BSA and incubated with the biotinylated anti-Tac antibody 7G7B6 for 30 min on ice. After washing, cells were incubated with 10% FITC-conjugated goat antimouse IgM (TAGO) and 4% phycoerythrin–streptavidin (Molecular Probes, chromophore-conjugated molecule) for another 30 min on ice. Cells were washed in ice-cold PBS and resuspended in PBS. Cells were analyzed on a FACScan (Becton–Dickinson) flow cytometer as described [15] and the collected data were analyzed using CellQuest software (Becton–Dickinson). To obtain quantitative estimates of integrin activation, an activation index (AI), defined as 100
ðFo  Fr Þ=ðFo LIBS6  Fr Þ, was calculated where Fo is the median fluorescence intensity (MFI) of PAC1 binding; Fr is the MFI of binding in the presence of competitive inhibitor (Ro43-5054, 1 lM); and Fo LIBS6 is the MFI of binding in the presence of aIIb b3 -activating antibody (anti-LIBS6, 2 lM). The percentage suppression was calculated as 100(AIo ) AI)/AIo , where AIo is the activation index in the absence of the co-transfected test cDNA and AI is the activation index in its presence. The percentage rescue was calculated as 100(AIxþH-Ras ) AIH-Ras )/(AI0 ) AIH-Ras ), where AIo is the activation index in the absence of any co-transfected test cDNA, AIH-Ras is the activation index in the presence of H-Ras 12V, and AIxþH-Ras is the activation index in the presence of H-Ras 12V and co-transfected cDNA. Nck pull-down assay. CHO cells were transfected using Lipofectamine with 2 lg each of HA-PAK1 and Myc-R-Ras plasmids as described [38]. HA-PAK1 was a gift from Dr. Martin Schwartz, The Scripps Research Institute, La Jolla, CA. Forty-hours after transfection the cells were placed on ice and washed three times with ice-cold PBS. The cells were then treated with ice-cold lysis buffer (50 mM Tris– HCl, pH 7.5, 50 mM NaCl, 0.5% Triton X-100, 10% glycerol, and 0.1% BSA), plus protease inhibitors (Complete, Roche, Nutley, NJ). Finally, the lysates were clarified by centrifugation for 10 min at high speed. To assess the interactions of R-Ras with the GST-Nck fusion proteins the cell lysates were incubated with 5 lg GST-Nck SH3B precoupled to

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glutathione–glutationine beads. The purified GST-Nck SH3B fusion protein was a gift of Dr. David Schlaepfer, The Scripps Research Institute, La Jolla, CA. Samples were then incubated for 4 h on a tumbler at 4 °C followed by three washes in cell lysis buffer. The beads were boiled in Laemmli sample buffer and subjected to SDS–PAGE and Western blotting to assay for the recovery of the positive control PAK1 or R-Ras. In parallel, PAK1 and R-Ras expression was monitored by fractionating 20 lg of whole-cell lysate on a 4–20% SDS– polyacrylamide gel, followed by transfer to nitrocellulose membranes and immunoblotting with the mixture of the anti-HA antibody, 12CA5, and the anti-myc antibody, 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA). Detection was performed with the enhanced chemiluminescence system according to the supplier’s specifications (Amersham, Piscataway, NJ). Sucrose gradient assay and subcellular fractionation. Lipid raft/caveolar fractions were enriched as described presiously [40]. Briefly, transfected BHK cells were homogenized in 0.5 M sodium carbonate and loaded at the bottom of a continuous 10–40% sucrose gradient. Following centrifugation at for 6 h at 200,000gav ten equal fractions were removed, diluted, and pelleted at 100,000gav for 30 min. The membrane pellets were resuspended in 50 ll of Laemmli sample buffer and 20 ll was used for immunoblotting. Subcellular fractionation was performed according to [41]. On a 12% polyacrylamide gel either 5 lg of whole-cell lysate/lane, 5 lg of S100/lane, and equivalent proportion of P100 pellet (when total volumes of P100 and S100 are compared) were loaded, followed by transfer to nitrocellulose membranes and immunoblotting with a GFPspecific antibody (Boehringer–Mannheim/Roche, Indianapolis, IN).

Results Residues 175–203 of R-Ras are necessary to antagonize H-Ras-induced integrin suppression The two small GTPases H-Ras and R-Ras have contrasting phenotypes in the integrin modulation assay (Fig. 1A). H-Ras suppresses integrin activation compared to the empty vector control as assessed by reduction in the binding of the activation-specific monoclonal antibody to CHO cells expressing the active chimeric integrin aIIb a6 b3 b1 [15]. These cells are referred to as ab-py cells [37]. In contrast, activated R-Ras influences integrin activation by antagonizing the H-Rasinitiated integrin suppression pathway [12], through the activation of an as yet unidentified effector. We investigated various chimeras [16] between H-Ras and R-Ras (Figs. 1B and 2A) in order to map a region of R-Ras responsible for antagonizing suppression of integrin activation in the integrin modulation assay (Fig. 2B). The proteins contained activating mutations and were expressed to the same level in CHO cells [16]. All chimeras containing R-Ras residues Leu175 –Pro203 reversed suppression to a similar extent to R-Ras (Fig. 2B, chimeras C–E). Furthermore, none of these chimeras interfered significantly with ERK activation by H-Ras (data not shown). In contrast, those chimeras which lacked these sequences showed little or no capacity to rescue even if they contained all R-Ras sequence N-terminal of this region (Fig. 2A, chimeras A and B) or C-terminal of the

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Fig. 1. Integrin modulation assay and sequence alignment of the small GTPases R-Ras and H-Ras. (A) The effects of activated H-Ras and R-Ras on integrin activation in CHO cells expressing the chimeric integrin aIIb a6 b3 b1 . ab-py cells were transiently transfected with either vector, activated H-Ras, or activated H-Ras and activated R-Ras. The cells were harvested and stained for PAC1 binding. PAC1 is an activation-dependent monoclonal antibody against integrin aIIb b3 . The cells were finally analyzed by FACS to obtain an activation index (see Materials and methods). The vector-transfected CHO cells have a high activation index reflecting the constitutive activated stage of the chimeric integrins aIIb a6 b3 b1 , whereas activated H-Ras (noted ‘H-Ras’ in figure) suppresses this activation. Activated R-Ras (noted ‘R-Ras’ in figure) can completely reverse the integrin suppression induced by H-Ras. (B) Sequence alignment of R-Ras and H-Ras. Shared amino acids are indicated using the * symbol. Arrows indicate sites where exchange of H-Ras sequences for R-Ras sequences and vice versa was made to generate the chimeras investigated in this study.

region (Fig. 2A, chimera F). Thus, R-Ras residues 175– 203 (equivalent to H-Ras numbering 148–174) are critical for R-Ras’ capability to reverse the suppressive effect of activated H-Ras. These results extend previous findings [17] that the chimera H-Ras(165)R-Ras(193–218), an HRas chimera with amino acids 166–189 exchanged with the equivalent R-Ras amino acid 193–218, shows an RRas typic response in the PAC integrin modulation assay ([17], and Fig. 2B, asterisk). The combination of the data presented here and the previous data suggests the minimal sequence required for R-Ras reversal lies between amino acids 193 and 203 of R-Ras. Further characterization of the R-Ras proline-rich motif Notable features of the R-Ras segment required for integrin reversal are the overlapping proline-rich PXXP motifs encompassing residues 199-203 (Fig. 1A). This region has been implicated in integrin regulation by RRas via an association with the second SH3 domain of the adaptor protein Nck [28]. Furthermore, HRas(174)R-Ras(204–218) (chimera F), which does not contain this sequence, was unable to rescue H-Ras suppression (Fig. 2B). To determine whether a PPSP motif is sufficient for rescue of suppression, we generated an activated variant of H-Ras, named H-Ras(PPSP), in which a serine and a proline residue were inserted between residues 174 and

175 (Fig. 1B). H-Ras(PPSP) failed to antagonize H-Rasinduced suppression, demonstrating that the presence of this motif alone was not sufficient to convey R-Ras activity to H-Ras (Fig. 2B, chimera G). The H-Ras(PPSP), as H-Ras, was able to suppress integrin activation when expressed in ab-py cells (data not shown), demonstrating that this protein is functional with an H-Ras phenotype when expressed in CHO cells. We next asked whether the R-Ras PPSPP motif was necessary for R-Ras effects on H-Ras suppression. To examine this possibility, we generated the following mutants, R-Ras P199/200A, R-Ras P202/203A, and RRas 4A, in which all four of the prolines were substituted by alanine (Fig. 3A). We tested each of these for the ability to antagonize H-Ras suppression. Each one of these R-Ras mutants retained the R-Ras phenotype of suppression reversal (Fig. 3B), suggesting that this motif is not necessary for the regulation of integrin function by R-Ras in CHO cells. The proline-rich region of R-Ras has been shown to interact with the second SH3 domain of the adaptor protein Nck when both proteins were overexpressed in COS7 cells [28]. We therefore examined our alanine mutants of R-Ras on interactions with Nck using an affinity precipitation assay with a fusion protein comprised of the second SH3 domain of Nck joined to GST (GST-SH3B) as bait. In contrast to the positive control PAK1 [42–45] neither R-Ras nor any of the variants was

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Fig. 2. R-Ras C-terminal amino acids 175–203 are required for rescue ability. (A) Schematic representation of H-Ras and R-Ras chimeras. An open box indicates H-Ras sequences, whereas a filled box indicates the R-Ras portion of each chimera. The PPSP mutation in H-Ras(PPSP) introduces amino acids Pro and Ser to create a motif (PPSP) present in R-Ras. Numbers over boxes indicate R-Ras amino acid numbers whereas numbers below boxes indicate H-Ras amino acid numbers. Chimera name coding is used in (B). (*) Chimeras H-Ras(165)R-Ras(193–218) and H-Ras(180)RRas(208–218) have previously been published as HR166 and HR181, respectively [17]. (B) ab-py cells were transiently transfected in duplicate with either control expression vector, vector expressing H-Ras 12V, or a combination of H-Ras 12V with the indicated chimeras to address the effect of the indicated chimeras on H-Ras-induced suppression. Depicted is the mean percent rescue of integrin suppression obtained with activated H-Ras 12V  SEM of at least four independent experiments related to the rescue observed for that of R-Ras 38V in the same experiment. (*) Previously published data on the chimera H-Ras(165)R-Ras(193–218) [17].

precipitated efficiently by GST-SH3B (Fig. 4). These data suggest that interactions between the second SH3 domain of Nck and R-Ras are likely to be weaker than those with PAK1. Activated TC21 can rescue H-Ras-initiated integrin suppression The small GTPase TC21 is homologous to R-Ras and contains a similar C-terminus (Fig. 3A). However,

in contrast to R-Ras, TC21 is a strong activator of ERK MAP kinase [46]. In order to investigate the biological phenotype of TC21 for affinity modulation of the chimeric integrin, ab-py cells were transiently co-transfected with H-Ras 12V and either activated R-Ras (R-Ras 38V) or activated TC21 (TC21 23V) and after 48 h integrin activation was assessed by measuring binding. H-Ras-induced integrin suppression was reversed by co-transfection with activated TC21 to the same degree as observed with co-transfection of

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Fig. 3. Mutation of R-Ras proline-rich site has no impact on the ability of R-Ras to rescue H-Ras-mediated integrin suppression. (A) Sequence of the R-Ras C-terminus containing the proline-rich site and the various investigated alanine mutations (bold). The C-terminus of TC21 is also shown. R-Ras numbering is shown in uppercase, TC21 numbering is shown in lowercase. (B) ab-py cells were transiently transfected in duplicate with either control expression vector, vector expressing HRas 12V, or a combination of H-Ras 12V with either R-Ras 38V, or the various alanine mutants, or TC21 23V, respectively, to address the effect of the indicated chimeras on H-Ras-induced suppression. Depicted is the mean percent rescue of integrin suppression obtained with activated H-Ras 12V  SEM of three independent experiments.

activated R-Ras (Fig. 3B). Wildtype TC21 did not reverse H-Ras-induced integrin suppression (data not shown), as was also observed for wildtype R-Ras [12]. Activated TC21 did not alter integrin affinity when transfected alone into abpy-cells but did activate ERK (data not shown). Thus, TC21 showed integrin modulating activities similar to those of R-Ras in ab-py cells. R-Ras microlocalization in the plasma membrane differs from that of H-Ras One possible way that H-Ras and R-Ras could produce their contrasting biological responses is by residing in different membrane subdomains and thereby, having differing access to downstream effectors. H-Ras redistributes from lipid rafts into non-raft microdomains upon GTP-loading [23–25] and this redistribution is

Fig. 4. Immunoprecipitation of Nck does not bring down R-Ras. (A) To assess the interactions of R-Ras and the double alanine mutants with the GST-Nck fusion protein, cell lysates of transiently transfected CHO cells were incubated with GST-tagged Nck SH3B (second SH3 domain of Nck) coupled to glutathione beads. Protein associated with the beads was Western blotted to assay for the recovery of PAK1 or RRas proteins. (B) Twenty micrograms of cell lysate was blotted for PAK1 and R-Ras expression by immunoblotting with a mix of the anti-HA antibody, 12CA5, and the anti-myc antibody, 9E10. The experiment was done twice with similar results. All R-Ras constructs carried the 38V activating mutation.

important in its capacity to activate ERK. We therefore investigated the membrane localization of R-Ras in more detail. Initially, fusion proteins were made between GFP and either wildtype or an activated version of R-Ras, R-Ras 87L. The R-Ras proteins were fused onto the C-terminus of GFP. Expression of the constructs in BHK and CHO cells showed plasma membrane staining and strong Golgi and endosomal labelling (data not shown). In addition, electron microscopy of plasma membrane sheets showed both wildtype R-Ras and activated R-Ras localized throughout in the plasma membrane (data not shown).

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Fig. 5. Subcellular localization of R-Ras constructs and H-Ras/R-Ras chimeras, H-Ras(165)R-Ras(193–218) and H-Ras(180)R-Ras(208– 218). Sucrose fractionation of BHK cells expressing GFP-tagged versions of all the indicated constructs. Lower fraction numbers indicate proteins present in caveolin-containing lipid rafts/caveolae, whereas higher fraction numbers indicate localization to disordered plasma membrane [25]. The experiment was done three times with similar results. H-Ras(165)R-Ras(193–218) and H-Ras(180)R-Ras(208–218) are H-Ras chimeras with either the complete hypervariable region (amino acids 166–189) or the membrane attachment region (amino acids 181– 189), respectively, exchanged with the equivalent R-Ras sequences. Both chimeras contain activating mutations. H-Ras(180)R-Ras(208– 218) shows an H-Ras-typic rescue of integrin suppression and H-Ras(165)R-Ras(193–218) shows an R-Ras-typic rescue of integrin suppression when measuring integrin affinity by PAC1 binding in ab-py cells [17].

The microlocalization of the GFP-R-Ras constructs in the plasma membrane was subsequently investigated in sucrose gradient experiments (Fig. 5). In this assay, cell membranes were prepared in sodium carbonate and floated through a 10–45% gradient to separate lipid rafts/caveolae from non-raft microdomains [25]. It appeared that both GFP-R-Ras and GFP-R-Ras 87L were primarily located in fractions 2–5 (lipid rafts/caveolae, as indicated by caveolin marker) and that GFP-R-Ras 87L seemed to be more enriched in lipid rafts/caveolae (Fig. 5). This localization pattern contrasts with that of H-Ras, which upon activation migrates out of lipid rafts/caveola ([25,26]; and Fig. 5). This observation is not unexpected since there appears to be no homology between H-Ras and R-Ras C-terminal membranedirecting sequences, and the C-terminus of H-Ras is required for microlocalization of H-Ras [25]. Subcellular localization of H-Ras-like and R-Ras-like chimeras is identical Since activated versions of H-Ras and R-Ras seem to occupy different regions in the plasma membrane, we wanted to compare the localization pattern of chimeras which show either H-Ras-like or R-Ras-like properties in integrin activation. We GFP-tagged and investigated two H-Ras chimeras which have different segments of their C-terminus exchanged with the equivalent R-Ras sequences (Fig. 2A). The chimera H-Ras(165)R-

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Ras(193–218) carries the HVR of R-Ras and is able to rescue integrin suppression by active H-Ras as R-Ras 38V does (R-Ras-typic response; Fig. 2B, asterisk and [17]). The chimera H-Ras(180)R-Ras(208–218) has the membrane attachment signal exchanged and is able to suppress integrin activation (H-Ras-typic response, [17] and cannot rescue integrin suppression by H-Ras (Fig. 2B)). Both activated chimeras were found to localize to lipid rafts and thus resembled the localization pattern of R-Ras (Fig. 5). Thus, these two chimeras with contrasting biological phenotypes did not differ with respect to localization in these microdomains. This suggests that the differences between H-Ras and R-Ras effects on integrins are not ascribable solely to differences in microcompartmentalization at the plasma membrane. Raf-1 activation is abrogated when H-Ras is confined to lipid rafts by deletion of sequences in the hypervariable domain [26]. We therefore asked whether HRas(180)R-Ras(208–218), which is confined to lipid rafts and shows strong H-Ras biological phenotypes in suppression of integrin activation, phosphorylation of ERK, and focus formation in NIH3T3 clone 7 cells [17], was capable of activating Raf-1. We found that wholecell lysates of NIH3T3 UNC cells stably expressing HRas(180)R-Ras(208–218) [17] possessed very low levels of Raf-1 kinase activity (data not shown), supporting the idea that Raf-1 becomes activated by H-Ras only in non-raft microdomains [25,26].

Discussion In this study, we have performed a structure–function analysis of H-Ras and R-Ras using the contrasting phenotypes of the small GTPases and defined critical sequences in activation of integrins. In addition, we have characterized the plasma membrane localization of RRas and a subset of H-Ras/R-Ras chimeras and found that the microdomain localization of these proteins specifies their capacity to activate the downstream effector Raf-1, but does not correlate with their effects on integrins. We also found that the homologous small GTPase TC21 exhibits features of both H-Ras and RRas in that it strongly activates ERK yet also promotes integrin activation. Taken together, these data indicate that the hypervariable region of R-Ras is essential for its capacity to promote integrin activation and these effects are not mediated by altering the membrane microdomain localization of the GTPase or of its capacity to activate the ERK MAP kinase pathway. Analysis of the panel of H-Ras and R-Ras chimeras here has mapped the critical sequences to the C-terminal region of amino acids 175–203 of R-Ras required for increasing integrin activation. Combined with previous work [17], this suggests a redefined segment of amino

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acids 193–203 in the hypervariable region of R-Ras to be required for R-Ras reversal. However, an internal chimera, which has amino acids 166–180 of H-Ras replaced with R-Ras sequences (equivalent to R-Ras amino acids 193–209), suppresses integrin activation [17]. Thus, RRas amino acids 193–203 are necessary but not sufficient for increased integrin activation; hence, sequences in either the N- and/or the C-terminus of R-Ras may also be involved in the response. We investigated an N-terminal deletion mutant of R-Ras 38V, which has 26 amino acids unique to R-Ras deleted; however, this protein showed similar properties to activated R-Ras (data not shown), suggesting that the extreme N-terminus of R-Ras is not involved in integrin regulation in ab-py cells. A notable feature contained in R-Ras’ amino acid 193–203 region is the proline-rich site, which has been reported to bind the second SH3 domain of the adaptor protein Nck and to be required for integrin activation in cell adhesion [28]. None of the alanine variants we created, including mutation of all four prolines in the site, had any impact on the R-Ras modulation of integrin function, suggesting that the PPSPP motif in R-Ras is not involved in antagonizing integrin suppression by HRas. In analogy with this finding, Furuhjelm and Peranen [9] recently reported that the R-Ras C-terminal proline-rich site is not required for targeting activated R-Ras to focal adhesions in HeLa cells. We examined the alanine mutants for binding to the second SH3 domain of Nck but did not find detectable levels of R-Ras following pull down of Nck. Thus, in contrast to the finding by Wang et al. [28], our data suggest that the interaction between R-Ras and Nck fell below the level of detection of this particular assay. The H-Ras region equivalent to R-Ras amino acids 175–203 is required for suppression of integrin activation [16], which suggests that the highly divergent C-termini of the two small GTPases are involved in directing specificity in regard to integrin regulation. The HVR of H-Ras has been implicated in directing microlocalization of H-Ras in the plasma membrane; the specific localization is important for the activation of H-Ras effectors [25,26]. To address the possibility that the equivalent sequences in R-Ras also play a role in localization, we investigated the subcellular localization of R-Ras in the cell by using GFP-tagged constructs of either wildtype or activated R-Ras. R-Ras was localized to the plasma membrane, the Golgi and to endosomes. Microlocalization studies of both GFP-R-Ras and GFP-R-Ras 87L showed that these proteins were primarily in fractions containing lipid rafts/caveolae, and that GFP-R-Ras 87L seemed to be more enriched in lipid rafts/caveolae fractions than wildtype R-Ras. Thus, in contrast to H-Ras, which upon activation migrates out of lipid rafts/caveolae, R-Ras does not and in fact is slightly more enriched in these fractions when activated. This observation is consistent with the recent finding by others that activated

R-Ras and phospho-caveolin co-localize in focal adhesions [9]. Some amount of both GFP-R-Ras and GFP-R-Ras 87L was present in the fractions representing non-raft microdomains. This bimodal distribution of R-Ras both within and outside lipid rafts is probably due to the presence of the geranyl-geranyl group, which is predicted to be repelled from lipid rafts [47]. Interestingly, the small GTPase K-Ras, which possesses a very different hypervariable domain from both H-Ras and RRas, resides in non-raft microdomains irrespective of being in an activated or inactive form [22,24,25]; the underlying mechanism of either retaining a GTPase in its respective microdomain following activation, e.g., for RRas and K-Ras, or dynamically translocating it between different domains following GTP-loading, as seen for HRas, is not currently understood but may direct the biological responses to Ras GTPases. The differential microcompartmentalization of R-Ras and H-Ras was not mirrored by phenotypically divergent chimeras. The chimeras H-Ras(180)R-Ras(208–218) and H-Ras(165)R-Ras(193–218) have the H-Ras C-terminal membrane attachment signal and the complete HVR, respectively, exchanged with the equivalent R-Ras sequences and behave differently in the integrin modulation assay: H-Ras(180)R-Ras(208–218) suppresses integrin activity (like H-Ras) whereas H-Ras(165)R-Ras(193– 218) rescues H-Ras-induced integrin suppression (like R-Ras) [17]. Both chimeras localized to lipid rafts, as did R-Ras, indicating that the R-Ras C-terminus directs binding to lipid rafts. Therefore, additional regulatory events, such as binding of specific downstream effectors, must be involved in defining specificity of the chimera. In conclusion, by analyzing a panel of chimeras between H-Ras and R-Ras [16], we have found that sequences in the hypervariable C-terminus of R-Ras (amino acids 175–203) are involved in rescue of H-Rasinduced integrin suppression. In addition, we have found that activated R-Ras localized predominantly to lipid rafts and not to non-raft microdomains as activated H-Ras; sequences in the R-Ras extreme C-terminus are involved in this lipid-raft confinement. The divergent membrane localization of the two small GTPases cannot, however, explain the basis for functionally distinct H-Ras/R-Ras chimeras, suggesting that additional levels of regulation are involved. The findings of this study therefore support the hypothesis that sequences in the C-terminal hypervariable region of not just founder Ras proteins but also Ras-related proteins are involved in directing the differences in action of these small GTPases and their specific use of effectors.

Acknowledgments The Ph.D. fellowship of M. Hansen was funded by Grant 9600821 from the Danish Medical Research council to B.M. Willumsen.

M. Hansen et al. / Biochemical and Biophysical Research Communications 311 (2003) 829–838 The work was supported by Grant 9710013 from the Danish Cancer Society and Grant 9600821 from the Danish Medical Research council to B.M. Willumsen, and Grants HL31950, HL57900, and HL48728 from the NIH to M.H. Ginsberg, and grants from the NHMRC, Australia, to I. Prior and J.F. Hancock.

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