The small GTP-binding protein R-Ras can influence integrin activation by antagonizing a Ras/Raf-initiated integrin suppression pathway

Abstract

The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.

Figure 1

R-Ras(G38V) does not directly stimulate integrin α_IIbβ₃ activation in CHO cells. Both αβ-py and A5 cells were transiently transfected with 3 μg of an expression vector encoding R-Ras(G38V) and or an equivalent amount of an empty control vector. After 48 h, PAC1 binding was analyzed by flow cytometry as described in MATERIALS AND METHODS. Top, The mean activation indices ± SD of three independent experiments. Bottom, Immunoblot analysis of cell lysates illustrating the expression of myc-tagged R-Ras(G38V). Twenty micrograms of cell lysate from each transfection were separated on a 4–20% gradient gel and immunoblotted with the anti-Myc antibody 9E10.

Figure 2

Activated R-Ras rescues the suppression of integrin activation by H-Ras(G12V) and Raf-CAAX. (A) αβ-py cells were transiently transfected with 2 μg of an expression vector encoding Tac-α₅ alone and Tac-α₅ plus H-Ras(G12V). In a separate transfection Tac-α₅ plus H-Ras(G12V) was cotransfected with 3 μg of a plasmid encoding R-Ras(G38V). After 48 h, cells were harvested and stained for Tac expression (y-axis) and PAC1 binding (x-axis). Left, In the H-Ras(G12V)-transfected cells, there is a leftward shift of the dot plot in the upper quadrants representing a reduction in PAC1 binding. Middle, This shift is reversed by the cotransfection with activated R-Ras(G38V). Right, In the empty vector control transfection, there was no suppression of PAC1 binding in the Tac-α₅–expressing cells. (B) αβ-py cells were cotransfected with 4 μg of an expression vector encoding Raf-CAAX and H-Ras(G12V). In separate transfections Raf-CAAX or H-Ras(G12V) expression vectors were simultaneously cotransfected with 3 μg of a plasmid encoding R-Ras(G38V). After 48 h, integrin activation was determined by PAC1 binding. Depicted is the mean percent inhibition of integrin activation relative to that of the empty vector ± SE of three independent determinations.

Figure 3

Activated but not wild-type or dominant-negative R-Ras can reverse the suppressive effect of activated H-Ras(G12V). Top, αβ-py cells were cotransfected with an expression vector encoding H-Ras(G12V). They were simultaneously cotransfected with 2 and 4 μg of a plasmid encoding wild-type R-Ras (●) or R-Ras(S43N) (⋄) and 0.5, 1, 2, and 4 μg of a plasmid encoding R-Ras(G38V) (▪). After 48 h, integrin activation was determined by PAC1 binding. Depicted is the activation index in the presence of H-Ras(G12V) ± SE of three independent determinations. Bottom, The immunoblot analysis of cell lysates illustrates the expression of wild-type (Wt) R-Ras and R-Ras(G38V) in the presence of H-Ras(G12V). Twenty micrograms of cell lysate from each transfection were separated on a 4–20% gradient gel and immunoblotted with either an anti-HA antibody, 12CA5, or the anti-Myc antibody 9E10. The R-Ras(S43N) was also well expressed, as determined by immunoblot analysis of lysates from R-Ras(S43N)–transfected cells (our unpublished observations).

Figure 4

R-Ras reversal of H-Ras– and Raf-1–initiated suppression is not caused by inhibition of Ras/Raf activation of ERK MAP kinase. (A) αβ-py cells were cotransfected with an expression vector encoding either Raf-BXB or a control cDNA. In a separate transfection Raf-BXB cDNA was cotransfected with a plasmid encoding activated R-Ras(G38V). After 48 h, integrin activation was determined by PAC1 binding. Depicted is the mean percent inhibition relative to that of the empty vector control ± SE of three independent determinations. (B) αβ-py cells were cotransfected with an expression vector encoding HA-tagged ERK2. The cells were also cotransfected with a control expression vector or vectors containing inserts encoding either R-Ras(G38V), Raf-BXB, or H-Ras(G12V). In separate transfections expression vectors encoding either Raf-BXB or H-Ras(G12V) were simultaneously cotransfected with a plasmid encoding R-Ras(G38V). The transfected ERK2 kinase was immunoprecipitated with the anti-HA antibody 12CA5. ERK-2 activity was measured by phosphorylation of myelin basic protein (MBP) using an immunocomplex kinase assay. Top, The relative ERK activation is shown. Bottom, Immunoblots with the anti-HA (12CA5) antibody illustrate the comparable expression of HA-tagged ERK2 in all transfections. The H-Ras(G12V) construct bore an HA-tag and was detected as the lower band in lanes transfected with that construct. Note the similar expression of recombinant activated H-Ras(G12V) in both the control and R-Ras(G38V)–cotransfected cells.

Figure 5

PI 3-kinase activation does not mediate H-Ras– or R-Ras–dependent regulation of integrin affinity. (A) αβ-py cells were transiently transfected with either a control expression vector or vectors containing inserts encoding either R-Ras(G38V) and H-Ras(G12V) or H-Ras(G12V). Each transfection was performed in duplicate; 24 h after transfection, the PI 3-kinase inhibitor LY294002 was added at a final concentration of 20 μM to one of the duplicates. After 48 h, integrin activation was determined by PAC1 binding. Depicted are the activation indices ± SE of three independent determinations. (B) αβ-py cells were transiently transfected with an expression vector encoding HA-tagged Akt and either a control expression vector or vectors containing inserts encoding either R-Ras(G38V) and H-Ras(G12V) or H-Ras(G12V). Each transfection was performed in duplicate, and 24 h after transfection, the PI 3-kinase inhibitor LY294002 was added at a final concentration of 20 μM to one of the duplicates. Forty-eight hours after transfection, the cells were lysed, and the transfected Akt was immunoprecipitated with the anti-HA antibody 12CA5. Akt activity was then assayed using an immunocomplex kinase assay with histone 2B as a substrate. Top, The relative Akt activation is depicted; note the inhibition of Akt activity by LY294002. Bottom, Immunoblots with the anti-HA (12CA5) antibody illustrate comparable expression of HA-tagged Akt in all transfections.

Figure 6

Ral activation does not mediate H-Ras– or R-Ras–dependent regulation of integrin affinity. (A) αβ-py cells were cotransfected with expression vectors encoding H-Ras(G12V),R-Ras(G38V), or Rlf-CAAX in the combinations depicted on the y-axis. After 48 h, integrin activation was determined by PAC1 binding. Depicted are the activation indices ± SE of three independent determinations. Immunoblot analysis of cell lysates demonstrated that HA-tagged Rlf-CAAX was well expressed in all conditions (our unpublished observations). (B) αβ-py cells were cotransfected with expression vectors encoding H-Ras(G12V), R-Ras(G38V), or dominant-negative RalA(T28N) in the combinations depicted on the y-axis. After 48 h, integrin activation was determined by PAC1 binding. Depicted are the activation indices ± SE of three independent determinations. (C) CHO cells were transfected with HA-RalA, HA-RalA(G23V), HA-RalA(T28N), and HA-Rlf-CAAX as indicated and grown in media containing 0.5% FCS before cell lysis. Ral-GTP was precipitated from the clarified cell lysates with glutathione-sepharose–bound GST-RalBD. Precipitated HA-Ral (bottom) and HA-Ral present in the cell lysate (top) were then identified by Western analysis using the anti-HA monoclonal 12CA5.

Figure 7

A hypothetical scheme of how the GTP-binding cycles of Ras and R-Ras could influence integrin affinity modulation. In this model, ligation of integrins and other cell surface receptors leads to the formation of the GRB2–SOS complex necessary for Ras activation. Ras can then activate its downstream effectors, including Raf-1, stimulating the MAP kinase–dependent negative feedback loop that can suppress activation of integrin. The activation of R-Ras, by as yet undefined stimuli, could then antagonize the Ras/Raf suppressor pathway downstream of MAP kinase.