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

Abstract

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 ␣ 5 ␤ 1 (VLA-5) to fibronectin in bone marrow-derived mast cells. We found that both HaRas Val-12 activated VLA-5 through PI 3-kinase p110␦. 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 (3-5). 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 (7-10). 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 (11-13). 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. Sever...

The effector loop and prenylation site of R-Ras are involved in the regulation of integrin function

“…Alternatively, the dierences between HRas and R-Ras could be the result of the activation of distinct isoforms of PI 3-kinase by these two proteins. In support of this hypothesis, recent studies have demonstrated that dierent isoforms of PI 3-kinase couple to H-Ras with dierent binding speci®cities (Kinashi et al, 2000). Thus, eector loop mutations in R-Ras disrupt coupling to PI 3-kinase in a manner distinct from the eects of similar mutations in H-Ras.…”

“…Thus far, H-Ras has been shown to suppress integrin activation in Chinese Hamster ovary (CHO) cells via its eector serine/threonine kinase Raf-1 (Hughes et al, 1997) and activated R-Ras can antagonize the Ras/Raf suppressor pathway (Sethi et al, 1999). In both myeloid cells and bone marrow derived mast cells activated R-Ras can stimulate both integrin a5b1 activation and a5b1 dependent-adhesion to ®bronectin (Kinashi et al, 2000;Zhang et al, 1996).…”

“…R-Ras and H-Ras have distinct roles in regulating integrin function (Kinashi et al, 2000;Zou et al, 1999;Sethi et al, 1999;Zhang et al, 1996). A characteristic feature of integrins is their ability to dynamically regulate their anity for soluble ligands in response to intracellular signals, a process de®ned as`activation'.…”

R‐Ras promotes tumor growth of cervical epithelial cells

“…22,42 In those reports, the expression of a constitutively active form of R-Ras resulted in increased cell adhesion to fibronectin and collagen. This was interpreted as an increase in binding affinity of the integrins involved.…”

Interactions between Ras1, dMyc, and dPI3K signaling in the developing Drosophila wing

“…For example, Ras V12S35 specifically binds and activates mammalian Raf, but not RalGDS or PI3K Kinashi et al 2000;Pacold et al 2000). Ras V12G37 interacts specifically with RalGDS (resulting in activation of the Ral GTPase; RodriguezViciana et al 1997), PI3K␥ (Pacold et al 2000), and PI3K␦ (Kinashi et al 2000), but not with PI3K␣ or Raf. A third variant, Ras V12C40 , specifically interacts with PI3K␣ but not with PI3K␥, PI3K␦, Raf, or RalGDS.…”