Distinct Mechanisms of α5β1 Integrin Activation by Ha-Ras and R-Ras*

To investigate the possible roles of the Ras/Rho family members in the inside-out signals to activate integrins, we examined the ability of Ras/Rho small GTPases to stimulate avidity of α5β1 (VLA-5) to fibronectin in bone marrow-derived mast cells. We found that both Ha-RasVal-12 and R-RasVal-38 had strong stimulatory effects on adhesion and ligand binding activity of VLA-5 to fibronectin. However, only Ha-RasVal-12-, but not R-RasVal-38-induced adhesion was inhibited by wortmannin, which suggests that Ha-RasVal-12 is dependent on phosphatidylinositol (PI) 3-kinase on adhesion whereas R-RasVal-38 has another PI 3-kinase independent pathway to induce adhesion. The effector loop mutant Ha-RasVal-12E37G, but not Y40C retained the ability to stimulate adhesion of mast cells to fibronectin. Consistently, PI 3-kinase p110δ, predominantly expressed in mast cells, interacted with Ha-RasVal-12 E37G, but not Y40C, which was also correlated with the levels of Akt phosphorylation in mast cells. Furthermore, marked adhesion was induced by a membrane-targeted version of p110δ. These results indicate that Ha-RasVal-12 activated VLA-5 through PI 3-kinase p110δ. The mutational effects of the R-Ras effector loop region on adhesion were not correlated with PI 3-kinase activities, consistent with our contention that R-Ras has a distinct pathway to modulate avidity of VLA-5.

Adhesion mediated by integrins controls cell migration and localization. Adhesive interactions through integrins are modulated by adhesiveness (avidity) as well as expressions of integrins (1,2). Although both types of regulations are important, avidity modulation of integrins in particular plays a critical role in leukocyte migration and localization during inflammatory responses (3). Several external stimuli were reported to modulate avidity of integrins without changes of integrin expressions, such as antigen, chemokines, and cytokines (3)(4)(5). A rapid change of avidity of integrins occurs within minutes and is triggered by intracellular signaling pathways, which are referred to as inside-out signals (6).
Avidity modulation of integrins is regulated by increasing affinity to ligands, or by spatial redistribution of integrins on cell surface, which increases the number of integrins on the contact site (7)(8)(9)(10). Which types of avidity regulations are utilized largely depends on stimuli that induce adhesion. PMA 1 enhanced adhesion without detectable change in ligand-binding affinity of integrins (11)(12)(13). Recent studies have shown that an increase in lateral diffusion and clustering of integrins by PMA or cytochalasin D at low doses facilitates adhesion (10,14), suggesting that the adhesion is mediated by low affinity, but multivalent bindings of integrins. On the other hand, affinity modulation in integrins detected with soluble ligands or antibodies recognizing the high affinity state was reported for ␣ 4 ␤ 1 , ␣ 5 ␤ 1 , ␣ L ␤ 2 , and ␣ IIb ␤ 3 integrins in cells stimulated with activating antibodies, manganese ions, or cross-linking of the T cell receptor (12,13,(15)(16)(17)(18)(19). We previously demonstrated that PMA-stimulated mast cells adhered to fibronectin without accompanying affinity modulation of VLA-5, while Fc⑀RI crosslinked mast cells adhered to fibronectin by the high affinity state of VLA-5. Steel factor-induced adhesion was considered to be brought by both mechanisms (20). Changes in the affinity state of integrins influenced cell migratory speeds on substrates (21). However, the physiological significance of two modes of avidity modulation has not yet been demonstrated clearly.
The Ras/Rho family of small GTPases regulates the actin cytoskeleton and contributes to the formation of membrane ruffling and focal adhesion (22,23). Cytoskeletal reorganization subsequent to attachment to substrate leads to marked cell shape changes and strengthens adhesive interactions. Several members of the Ras/Rho family have been reported to influence integrin-mediated adhesion. Ha-Ras was shown to suppress the active form of ␣ IIb ␤ 3 chimeras through the MAP kinase pathway (24). A constitutively active R-Ras was found to enhance cellular adhesion to fibronectin by enhancing ␤ 1integrin ligand-binding affinity (25). We have recently shown that Rap1 has a unique property that causes an increase of ligand binding affinity of the ␤ 2 integrin LFA-1, and that Rap1 was critically involved in T-cell receptor-mediated LFA-1/ ICAM-1 adhesion (26). However, there are few comprehensive studies that examine whether or not the Ras/Rho family of small GTPases can modulate avidity of ␤ 1 integrins directly.
To gain a clearer understanding of avidity regulations of ␤ 1 integrins by the Ras/Rho family of small GTPases, we employed bone marrow-derived mast cells as a model system to analyze their ability to modulate avidity of VLA-5, because mast cells have been shown to adhere to fibronectin through VLA-5 upon physiologically relevant stimulation such as steel factor (27), or antigen cross-linking of Fc⑀RI (20) and are considered to be suitable for activation signal-dependent adhesion.
With mast cells, one can also examine the affinity state of VLA-5 as we demonstrated that with antigen cross-linking of Fc⑀RI (20).
Here we report that the active mutants of Ha-Ras and R-Ras among the Ras/Rho family member lead to strong adhesion to fibronectin with the high affinity state of VLA-5. Furthermore, our study reveals distinct mechanisms of Ha-Ras and R-Ras in regulation of avidity of VLA-5 through analyses of effector mutants and constitutively active downstream signaling molecules: PI 3-kinase p110␦ is critical to avidity modulation by Ha-Ras, while R-Ras has other mechanisms to regulate avidity of VLA-5.
Preparation of Fibronectin and the 80-kDa Fragment, and Integrin Affinity Measurement-Fibronectin and its 80-kDa tryptic fragment that contains the RGD binding motif for VLA-5 were produced as described (35,36). The 80-kDa fibronectin fragment was radioiodinated with a modified method using chloramine T (37). The typical specific activity of the labeled 80-kDa fragment used in our experiments was about 3.5 ϫ 10 8 dpm/nmol. Its binding to cells was measured as described (20). Briefly, mast cells were washed once with binding buffer containing RPMI 1640 (Sigma), 0.1% BSA (Life Technologies, Inc.), and 10 mM HEPES, pH 7.4 (Sigma), and suspended with the same buffer at 1 ϫ 10 7 cells/ml. In a typical binding assay, performed in a 1.5-ml microcentrifuge tube, 100 l of cells (1 ϫ 10 6 cells per tube) were mixed with 100 l of the radiolabeled 80-kDa fragment. For inhibition with antibodies or wortmannin, mast cells were preincubated with antibodies (20 g/ml), or wortmannin for 15 min at 25°C before assays. After incubation for 30 min at 37°C, samples were oil-separated by centrifugation at 8000 rpm for 1 min. The tip of tubes was amputated from the body with a blade and applied to a ␥-counter to measure radioactivity of the bound (the tip) and the unbound (the body). The nonspecific binding was determined at each data point in the presence of a 50-fold excess of the unlabeled 80-kDa fragment. The specific binding was calculated by subtracting the nonspecific binding from the total binding.
Flow Cytometric Analysis-Cells (1 ϫ 10 6 ) were incubated on ice for 30 min with 50 l of staining buffer (phosphate-buffered saline, 0.1% BSA, 0.05% sodium azide) containing 1 g of monoclonal Rat anti-VLA-5 antibody (MFR-5H10) or isotype-matched rat antibody. After washing with staining buffer three times, cells were stained with fluorescein isothiocyanate-labeled anti-Rat IgG as above. The stained cells were analyzed by FACScan (Becton Dickinson, San Jose, CA).
Adhesion Assays-Assays of adhesion to fibronectin were performed as described (27). Briefly, mast cells labeled with 2Ј,7Ј-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in 96-well plates precoated with fibronectin (1 g/well) or 1% bovine serum albumin (BSA) were incubated in triplicate at 37°C for 30 min, or in the presence of PMA as indicated. After washing the plate four times, bound fluorescence was measured with a fluorescence concentration analyzer (IDEXX Laboratories, Westbrook, ME). The level of adhesion was calculated by dividing bound fluorescence by input fluorescence. For the assay with antibodies or wortmannin, labeled mast cells were preincubated at room temperature for 15 min with 20 g/ml antibodies or wortmannin as indicated before assays.
Western Blot-Mast cells were prepared for cell lysates as described (28). Equal amounts of protein were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE, the separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane. After blocking with 5% BSA, the membrane was incubated with antibodies as indicated and detected with the appropriate secondary antibody conjugated with horseradish peroxidase. The bands were visualized using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Stripping and reprobing were performed according to the manufacturer's instructions.

The Effects of the Ras/Rho Family of Small GTPases on
Adhesion to Fibronectin through VLA-5-To examine the ability of the Ras/Rho small GTPases to modulate avidity of VLA-5 to fibronectin, we established bone marrow-derived mast cells by culturing bone marrow cells with interleukin-3 for 4 weeks, and then introduced active forms of Ha-Ras (Ha-Ras Val-12 ), Rap1 (T7-tagged Rap1 Val-12 ), RalA (RalA Leu-72 ), R-Ras (Myc-tagged R-Ras Val-38 ), Rac (Myc-tagged Rac Val-12 ), Rho (Myctagged Rho Val-14 ), or neomycin only by retrovirus. Following drug selection, we examined adhesion of these transfectants to fibronectin. Control mast cells (neo) and uninfected mast cells did not adhere to fibronectin significantly when compared with BSA ( Fig. 1). However, cells expressing Ha-Ras Val-12 and R-Ras Val-38 adhered strongly to fibronectin without stimulation. In both cases, adhesion to fibronectin was blocked by an anti-VLA-5 antibody, 5H10, indicating that adhesion was mediated by VLA-5 (Fig. 1). Adhesion to fibronectin was also induced slightly in Rap1 Val-12 or Rac Val-12 expressing cells, which was inhibited with anti-VLA-5 antibody, but RalA Leu-72 or Rho Val-14 transfectants did not change the level of adhesion significantly. Expressions of VLA-5 of transfectants were similar to control cells (Neo) ( Fig. 2A), or uninfected cells (not shown). We demonstrated expression of introduced active forms of the Ras/Rho small GTPases in mast cells by Western blot analysis (Fig. 2B). These results indicated that adhesiveness of VLA-5 was increased in cells expressing Ha-Ras Val-12 and R-Ras Val-38 . Transfectants expressing Rap1 Val-12 , RalA Leu-72 , Rac Val-12 , or Rho Val-14 adhered to fibronectin when stimulated with steel factor or PMA, indicating that these active forms of small GTPases did not exert inhibitory effects on activation-dependent adhesion of mast cells (data not shown).
Ha-Ras Val-12 and R-Ras Val-38 Increase Ligand Binding Activity of VLA-5-R-Ras Val-38 was previously shown to augment ligand binding activity to fibronectin (25). To examine whether ligand binding activities of VLA-5 are augmented in cells expressing Ha-Ras Val-12 and R-Ras Val-38 , we measured ligandbinding affinity using a soluble 80-kDa fibronectin fragment (FN80) containing the RGD motif that was recognized by VLA-5. We previously demonstrated that ligand bindings of unstimulated mast cells was low, but increased by Fc⑀RI crosslinking, but not PMA stimulation (20). In mast cells expressing either Ha-Ras Val-12 or R-Ras Val-38 , ligand bindings were augmented compared with those of control cells (Fig. 3). The level of ligand bindings of cells expressing Ha-Ras Val-12 was higher than that in R-Ras Val-38 expressing cells. The increased ligand bindings were inhibited by anti-VLA-5 antibody, indicating that ligand binding activity of VLA-5 was increased in these cells.
To explore whether PI 3-kinase is involved in affinity modulation of VLA-5 by Ha-Ras Val-12 and R-Ras Val-38 , as is the case in mast cells stimulated with Fc⑀RI cross-linking (20), ligand binding assays were performed in the presence of wortmannin. Bindings to FN80 in both Ha-Ras Val-12 and R-Ras Val-38 expressing cells were completely inhibited with low doses of wortmannin (Fig. 3) or LY294002 (data not shown), suggesting that PI 3-kinase is involved in affinity modulation by Ha-Ras Val-12 and R-Ras  .
Differential Effects of Wortmannin on Adhesion to Fibronectin of Cells Expressing Ha-Ras Val-12 and R-Ras Val-38 -Wortmannin also inhibited adhesion of Ha-Ras Val-12 expressing cells to fibronectin at a concentration similar to those abolished bindings to FN80 (Fig. 4). On the other hand, adhesion of R-Ras Val-38 expressing cells was resistant to treatment of wortmannin even at 100 nM, the dose of which completely blocked the bindings to FN80 (Fig. 3). LY294002 also failed to inhibit adhesion to fibronectin (data not shown). Anti-VLA-5 inhibited adhesion of R-Ras Val-38 expressing cells to fibronectin in the presence of wortmannin (Fig. 4). These results indicate that the high affinity state of VLA-5 by PI 3-kinase likely accounted for adhesion induced by Ha-Ras Val-12 , while R-Ras Val-38 -induced adhesions were mostly independent from PI 3-kinase activities, and did not require the high affinity state of VLA-5 for adhesion.
The T35S and Ha-Ras Val-12 D38E, and RalGDS and PI 3-kinase p110␣ interacted only with Ha-Ras Val-12 E37G and Y40C, respectively. To confirm PI 3-kinase dependence of Ha-Ras for adhesion, mast cells expressing Ha-Ras Val-12 , T35S (Ser 35 ), E37G (Gly 37 ), D38E (Glu 38 ), or Y40C (Cys 40 ) were established (Fig. 5B). There were no significant changes in surface levels of VLA-5 in these transfectants (data not shown). Contrary to our expectation, cells expressing the Gly 37 , but not Cys 40 mutant showed the levels of adhesion equivalent to, or more than, cells expressing Ha-Ras Val-12 , whereas those of adhesion of mast cells expressing the Ser 35 , Glu 38 , or Cys 40 mutants were reduced considerably (Fig. 5A). We also examined the phosphorylation of Akt in effector mutant expressing cells as its phosphorylation is dependent on activities of PI 3-kinase (39). The level of Akt phosphorylation was augmented only in mast cells expressing the Gly 37 mutant, the level of which was more than that of the Val 12 mutant (Fig. 5C). The Ser 35 slightly increased Akt phosphorylation compared with control (Fig. 5C). The phosphorylation of Akt was completely abolished with treatment of wortmannin, confirming the requirement of PI 3-kinase activity for Akt phosphorylation (data not shown). Thus the levels of Akt phosphorylation were in good correlation with those of adhesion to fibronectin (Fig. 5C), which is consistent with the notion that PI 3-kinase is critically involved downstream of Ha-Ras for adhesion, and suggest that the interaction of Ha-Ras and PI 3-kinase occurs in the Gly 37 , but not Cys 40 mutants in mast cells.
Associations of PI 3-Kinase p110␦ with Ha-Ras Effector Mutants-To confirm the possibility that the Gly 37 , but not Cys 40 mutant associates with PI 3-kinase in mast cells, we examined the isotypes of the p110 catalytic subunit that were expressed in mast cells. Mast cells expressed predominantly p110␦, while p110␣ and ␤ were barely detected (Fig. 6A). The interactions of Ha-Ras effector mutants and Myc-tagged p110␦ were examined by co-transfection into COS cells and immunoprecipitation with either anti-Myc antibody (Fig. 6B) or anti-Ha-Ras antibody (Fig. 6C) for associations with Ha-Ras mutants or p110␦, respectively. In both cases, p110␦ was co-immunoprecipitated with the Gly 37 as efficiently as Ha-Ras Val-12 . This result is consistent with strong Akt phosphorylation in the Gly 37 expressing mast cells (Fig. 5C).
p110␦-CAAX Induces Adhesion to Fibronectin-To directly demonstrate that p110␦ itself is sufficient to induce adhesion to fibronectin, we introduced an activated membrane-targeted version of p110␦, p110␦-CAAX, into mast cells. We also tested membrane-targeted versions of two known effector molecules that bind to Ha-Ras, Raf-CAAX and Rlf-CAAX (Fig. 7A). Mast cells expressing p110␦-CAAX strongly adhere to fibronectin without stimulation. In contrast, cells expressing Raf-CAAX or Rlf-CAAX failed to adhere to fibronectin while they responded well to PMA to adhere to fibronectin. As shown in Fig. 7B, mast cells expressing p110␦-CAAX showed marked cell attachment and spreading on fibronectin compared with control cells (neo). Cell attachment and spreading of p110␦-CAAX expressing mast cells were comparable to those of Ha-Ras Val-12 , while R-Ras Val-38 expressing cells tended to spread more on fibronectin (Fig. 7B).
We further examined the effect of activation of the Raf-MAP kinase pathway on adhesion by introducing a chimera of the Raf kinase domain and the hormone-binding domain of the estrogen receptor (rafER) (33). A conditional activation of the Raf kinase activity by estradiol increased the kinase activity of ERK2 more than that by steel factor (Fig. 8B). However, mast cells did not adhere to fibronectin by estradiol, while they responded to steel factor for adhesion to fibronectin (Fig. 8A). Stimulation with estradiol did not affect adhesion to fibronectin by steel factor and PMA (data not shown).
Mutational Analysis of the Effector Loop of R-Ras-The differential sensitivity to wortmannin on adhesion induced by Ha-Ras Val-12 and R-Ras Val-38 suggests PI 3-kinase independent activation mechanisms of VLA-5 in R-Ras Val-38 -expressing cells. Since R-Ras has the identical amino acid sequences of the effector loop region with Ha-Ras (40), we compared effects of mutations in the Ha-Ras and R-Ras effector loop on adhesion to fibronectin. We introduced R-Ras Val-38 effector mutants that carry the same replacement mutations as Ha-Ras at the corresponding sites in the background of the Val 38 mutation. Established mast cells expressed comparable amounts of R-Ras mutants (Fig. 9B). When they were subjected to adhesion assays, only the Glu 64 mutant of R-Ras Val-38 lost the ability to stimulate adhesion to fibronectin (Fig. 9A), while the Ser 61 , Gly 63 , or Cys 64 expressing cells still adhered to fibronectin. On the other hand, the levels of Akt phosphorylation were not in correlation with adhesion, and the mutations in the effector loop reduced Akt phosphorylation to the comparable degrees in all effector mutants (Fig. 9C). This result was in contrast to that of Ha-Ras Val-12 , in which the levels of adhesion paralleled with PI 3-kinase activities, and supports PI 3-kinase independent mechanisms of R-Ras Val-38 in stimulating adhesion to fibronectin.

DISCUSSION
In this study, we examined the ability of a series of the Ras/Rho family of small GTPases to activate VLA-5 to adhere to fibronectin in bone marrow-derived mast cells. We found that the active forms of Ha-Ras (Ha-Ras Val-12 ) and R-Ras (R-Ras Val-38 ) were most potent in stimulating adhesion to fibronectin. Both Ha-Ras Val-12 and R-Ras Val-38 induced the high affinity state of VLA-5. However, PI 3-kinase inhibitors abrogated adhesion by Ha-Ras Val-12 , but not R-Ras Val-38 . Among effector loop mutants of Ha-Ras Val-12 , only the Gly 37 mutant retained the ability to stimulate adhesion and also the ability to associate with p110␦, which is consistent with strong phosphorylation of Akt in the Gly 37 mutant expressing cells. The membrane-targeted version of p110␦ was sufficient to stimulate adhesion to fibronectin. These results indicate that Ha-Ras Val-12 depends on PI 3-kinase ␦ to activate VLA-5 in mast cells.
We showed that wortmannin had the marginal effect on adhesion induced by R-Ras Val-38 , whereas it abolished the increase in ligand binding activity of VLA-5. This result suggests that R-Ras depends on PI 3-kinase on induction of the high  affinity state of VLA-5, which only made a minor contribution to R-Ras Val-38 -induced adhesion. The effects on adhesion of mutations in the effector loop region of R-Ras Val-38 were distinct from those of Ha-Ras Val-12 and the decrease of phosphorylation of Akt did not result in loss of adhesion, which further support the PI 3-kinase independent mechanism of R-Ras Val-38 to activate VLA-5. Although it is currently unclear about adhesion mechanisms of R-Ras, it is conceivable that R-Ras Val-38 has other mechanisms that induce adhesion mediated through the low affinity state of VLA-5, possibly by modulating lateral diffusion/clustering on VLA-5 on cell surface.
We have recently shown that PI 3-kinase is an affinity modulator of VLA-5, which is critically involved in adhesion induced by Fc⑀RI (20). Our results that PI 3-kinase was involved in Ha-Ras Val-12 and R-Ras Val-38 induced the high affinity state of VLA-5 are in line with previous reports (38,41) on activation of PI 3-kinase by these small GTPases, and further implicate physiological roles in Ha-Ras and R-Ras in Fc⑀RI-induced adhesion. In fact, activation of Ha-Ras, but not R-Ras was most seen in mast cells when stimulated with cross-linking of Fc⑀RI. The kinetics of Ha-Ras activation paralleled with that of adhesiveness of VLA-5 increased by Fc⑀RI. 2 Thus, Ha-Ras could contribute to the high affinity state of VLA-5 by Fc⑀RI. However, we could not demonstrate that Ha-Ras was responsible for the high affinity state of VLA-5 by Fc⑀RI, because a domi-nant negative Ha-Ras was not expressed in mast cells, possibly due to the inhibition of their growth.
Ha-Ras was reported to suppress the activation of integrins through the Raf/MAP kinase pathway. In this study, chimeras of the extracellular regions of ␣ IIb ␤ 3 and intracellular regions of ␣ 5 ␤ 1 or ␣ 6 ␤ 1 integrins were used in adherent Chinese hamster ovary cells (24). In the present study, we showed that Raf-CAAX did not affect adhesion induced by PMA (Fig. 7A). We also showed that a conditional activation of the Raf/MAP kinase pathway did not increase adhesion (Fig. 8), and had no effect on steel factor-and Fc⑀RI-induced adhesion (data not shown). In fact, it has not been demonstrated directly whether Ha-Ras plays an inhibitory role in integrin activation by insideout signals. The reason of the discrepancy is not known at present, but it is likely due to the differences in experimental systems including integrin adhesive property and structure. The other investigators reported that Ha-Ras was involved in adhesion induced by interleukin-3 and the activated form of Ha-Ras induced adhesion to fibronectin in an interleukin-3-dependent cell line Ba/f3, which was blocked by an inhibitor of phospholipase C, U-73122, but not inhibitors for PI 3-kinase (42). At present, we cannot explain this discrepancy. It could be due to the difference in cell context, or the other effects of 2 T. Kinashi, unpublished data. U-73122, which caused marked morphological changes leading to cytolysis at high doses (43,44).
The activated Ha-Ras interacts with and activates the PI 3-kinase catalytic subunit, p110␣ (38). The association of Ha-Ras and p110␣ was further characterized with the effector loop mutations of Ha-Ras (31). Notably the Cys 40 mutant was shown to interact with p110␣. p110␦ belongs to class IA of PI-3 kinase (45,46). It is specifically expressed in leukocytes (30). Ha-Ras was also shown to interact with p110␦. p110␦ has biochemical properties similar to p110␣, including the sensitivity of PI 3-kinase inhibitors (30). However, p110␦ was coimmunoprecipitated with the Gly 37 mutant, but poorly with the Cys 40 mutant, as we showed in this study. The amino acid sequence of p110␦ in the Ras-binding domain is considerably diverged from that of p110␣ and p110␤ (30,47). The sequence divergence in this region among p110 subunits likely contributes to the difference in the specific interaction sites of the effector loop region of Ha-Ras. The Gly 37 mutant was previously shown to interact with RalGDS and Rlf, GTP exchange factors for Ral (31,34). However, the experiments with active forms of Ral, Rlf, or RalGDS (data not shown) rule out their critical roles in activation of integrins. Instead, the fact that Akt phosphorylation was augmented in the Gly 37 , but not Cys 40 mutant expressing mast cells indicates that the association and activation of p110␦ with the Gly 37 mutant occur in mast cells. Taken together, our results demonstrate that p110␦ is a critical effector molecule of Ha-Ras in activating integrins in mast cells.
R-Ras Val-38 was reported to increase ligand-binding affinity to ␣ 5 ␤ 1 in an interleukin-3-dependent myeloid cell line, 32D cells (25). We showed here that both R-Ras Val-38 and Ha-Ras-Val-12 induced the high affinity state of VLA-5 in mast cells. The ligand binding activity was higher in Ha-Ras Val-12 transfectants than in R-Ras Val-38 transfectants. Our preliminary experiments showed that the dissociation constant of VLA-5 in Ha-Ras Val-12 transfectants was between 20 and 50 nM, which was higher than that reported in R-Ras Val-38 expressing 32D cells (250 nM) and equivalent to that induced by Fc⑀RI cross-linking (20). Importantly, our study revealed that the high affinity state of VLA-5 in R-Ras Val-38 expressing cells was dispensable for adhesion to fibronectin, since the treatment of wortmannin abolished the high affinity state with a small inhibitory effect on adhesion. This is in contrast with Ha-Ras Val-12 expressing cells, in which wortmannin abolished both ligand binding activity and adhesion to fibronectin at the similar doses. The PI 3-kinase independent adhesion by R-Ras was also supported by the analysis using the effector loop mutants. All of the effector loop mutations at the homologous sites of Ha-Ras resulted in decrease of Akt phosphorylation at the similar degree, but did not parallel levels of adhesion. Recently it has been reported that the R-Ras Gly 63 mutants among other mutants interacted more with the Ras-binding domain of PI 3-kinase p110␣, and that adhesion by the Gly 63 mutant was partially inhibited by a dominant negative Rac or Ral (48). We failed to detect distinct sites of the R-Ras effector loop region to interact with p110␦ by co-immunoprecipitation. 3 The difference could be due to low homologies in the Ras-binding domain between p110␣ and p110␦ as discussed above. In addition, Rac Val-12 or RalA Leu-72 by itself failed to induce adhesion in our study, ruling out their critical roles downstream of R-Ras in activating integrins in our case, although they might promote adhesion by modulating cytoskeletal organization such as cell spreading.
Adhesion through integrins is mediated through multiple steps initiated by integrin activation by inside-out signals, leading to cytoskeletal reorganization and firm attachment by outside-in signals upon adhesion. Here in this study, we examined the ability of a series of Ras/Rho family of small GTPase to activate integrins in search for the possible inside-out signals. Our study clearly demonstrated that distinct members of small GTPases had the ability to regulate adhesiveness of integrins, which gives important clues to dissect regulatory processes of adhesion through integrins.