The Small GTP-Binding Proteins Cdc42, Rac, and Rho Control Actin Organization

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A striking feature of a moving cell is its polarity: it has a front and a back. When a cell makes a turn, a new leading edge forms in the new direction of movement. If these extensions formed in all directions at once, the cell would be unable to pick a new direction of movement. To sustain movement in a particular direction, a cell requires signals to coordinate events at the front of the cell with events at the back and, indeed, signals to tell the cell where its front is. Our understanding of how this coordination occurs emerged from studies with growth factors.

Growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), bind to specific cell-surface receptors (see Chapter 16) and stimulate cells to move and then to divide. For example, in a wound, blood platelets become activated by being exposed to collagen in the extracellular matrix at the wound edge, which helps the blood to clot. Activated platelets also secrete PDGF to attract fibroblasts and epithelial cells to enter the wound and repair it. It is possible to watch part of this process in vitro. If you grow cells in a culture dish and, after starving them of growth factors, you add some fresh growth factor, within a minute or two the cells respond by forming membrane ruffles. Membrane ruffles are very similar to the lamellipodia of migrating cells: they are a result of activation of the machinery that controls exocytosis of endosomes coupled with actin assembly.

Scientists knew that growth factors bind to very specific receptors on the cell surface and induce a signal transduction pathway on the inner surface of the plasma membrane (see Chapter 15), but how that process linked up to the actin machinery was mysterious. Research then revealed that the signal transduction pathway activates Rac, a member of the small GTPase superfamily of Ras-related proteins (see Chapter 15). Rac is one member of a family of proteins that regulate microfilament organization; two others are Cdc42 and Rho. Unfortunately, due to the history of their discovery, the family of proteins of which Cdc42, Rac, and Rho are members has also been collectively named “Rho proteins.” To understand how these proteins work, we first have to recall the way small GTP-binding proteins function.

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Like all small GTPases of the Ras superfamily, Cdc42, Rac, and Rho act as molecular switches, inactive in the GDP-bound state and active in the GTP-bound state (Figure 17-41). In their GDP-bound state, they exist free in the cytoplasm in an inactive form bound to a protein known as guanine nucleotide dissociation inhibitor (GDI). Growth factors can bind and activate their receptors to turn on specific membrane-bound regulatory proteins, called guanine nucleotide exchange factors (GEFs), which activate Rho proteins at the membrane by releasing them from GDI and catalyzing the exchange of GDP for GTP. The GTP-bound active Rho protein associates with the plasma membrane, where it binds effector proteins to transmit the biological response. The small GTPase remains active until the GTP is hydrolyzed to GDP, a process that is stimulated by specific GTPase-activating proteins (GAPs).

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FIGURE 17-41 Regulation of the Rho family of small GTPases. The small GTPases of the Rho family are molecular switches regulated by accessory proteins. Rho proteins exist in the Rho-GDP bound form complexed with a protein known as GDI (guanine nucleotide dissociation inhibitor), which keeps them in an inactive state in the cytosol. Membrane-bound signaling pathways bring Rho proteins to the membrane and, through the action of a GEF (guanine nucleotide exchange factor), exchange the GDP for GTP, thus activating them. Membrane-bound activated Rho-GTP can then bind effector proteins that cause changes in the actin cytoskeleton. The Rho protein remains in the active Rho-GTP state until acted on by a GAP (GTPase-activating protein), which allows it to interact with the GDI and be returned to the cytoplasm. See S. Etienne-Manneville and A. Hall, 2002, Nature 420:629.

An important approach to unraveling the functions of the Rho proteins has been to introduce into cells mutant proteins that are locked either in the active—Rho-GTP—state or in the inactive—Rho-GDP—state. A mutant small GTPase that is locked in the active state, called a dominant-active protein, binds the effector molecules constitutively, and one can then assess the biological outcomes. Alternatively, one can introduce a different mutant that is dominant negative, which binds and inhibits the relevant GEF protein. Thus introduction of a dominant-negative protein, even in the presence of the endogenous wild-type protein, interferes with the signal transduction pathway, so one can now assess what processes are blocked.

Cdc42, Rac, and Rho were implicated in the regulation of microfilament organization because introduction of dominant-active mutant proteins had dramatic effects on the actin cytoskeleton, even in the absence of growth factors. It was discovered that dominant-active Cdc42 results in the appearance of filopodia, dominant-active Rac results in the appearance of membrane ruffles, and dominant-active Rho results in the formation of stress fibers, which then contract (Figure 17-42). How can one tell if dominant-active Rac and growth factor stimulation, both of which stimulate membrane ruffle formation, operate in the same signal transduction pathway? If growth factor stimulation leads to Rac activation, then introduction of a dominant-negative Rac protein into a cell should block the ability of a growth factor to induce membrane ruffling. This is precisely what was found. Using this and many other biochemical strategies, scientists have identified the signaling pathways involving Cdc42, Rac, and Rho (Figure 17-43).

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EXPERIMENTAL FIGURE 17-42 Dominant-active Rac, Rho, and Cdc42 induce different actin-containing structures. To look at the effects of constitutively active Rac, Rho, and Cdc42, growth-factor-starved fibroblasts were microinjected with plasmids to express dominant-active versions of the three proteins. The cells were then treated with fluorescent phalloidin, which stains filamentous actin. Dominant-active Rac induces the formation of peripheral membrane ruffles, whereas dominant-active Rho induces abundant contractile stress fibers, and dominant-active Cdc42 induces filopodia.
[Republished with permission of AAAS, from Hall, A., “Rho GTPases and the actin cytoskeleton,” Science, 1998, 279 (5350):509-14; permission conveyed through Copyright Clearance Center, Inc.]
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FIGURE 17-43 Summary of signal-induced changes in the actin cytoskeleton. Specific signals, such as growth factors and lysophosphatidic acid (LPA), are detected by cell-surface receptors. Detection leads to the activation of the small GTP-binding proteins, which then interact with effectors to bring about cytoskeletal changes as indicated.

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Some of the pathways that these proteins regulate contain proteins we are familiar with. Activation of Cdc42 stimulates actin assembly by Arp2/3 through activation of WASp, a nucleation promoting factor (NPF) (see Figure 17-16), resulting in the formation of filopodia. Activation of Rac also induces Arp2/3, mediated by the WAVE complex, leading to the assembly of branched actin filaments in the leading edge. Activation of Rho has at least two effects. First, it can activate a formin for unbranched actin filament assembly. Second, through activation of Rho kinase, it can phosphorylate the myosin light chain to activate nonmuscle myosin II and can also inhibit light-chain dephosphorylation by phosphorylating myosin light-chain phosphatase to inhibit its activity. Both actions of Rho kinase lead to a higher level of myosin light-chain phosphorylation and therefore higher myosin activity and contraction. The three Rho proteins, Cdc42, Rac, and Rho, are also linked by activation and inhibition pathways, as shown in Figure 17-43.