Synonyms

Low molecular mass GTPases; Small G proteins

Definition

Small GTPases are monomeric 20–40 kD GTP-binding proteins that interconvert between an active (GTP-bound) and an inactive (GDP-bound) state. As molecular switches they are involved in the regulation of complex cellular processes.

Basic Characteristics

Regulation

Activation of small GTPases occurs by GDP/GTP exchange catalyzed by guanine nucleotide exchange factors (GEFs) (Fig. 1). They stimulate the dissociation of GDP in response to an upstream signal which results in binding of GTP. In the GTP-bound form the GTPases are active, and bind to and activate a number of effector molecules. The small G proteins are able to hydrolyze the bound nucleotide to GDP. This inactivation step is accelerated by GTPase activating proteins (GAPs). In the GDP-bound form the GTPases are inactive. Some GTPases bind to guanine nucleotide dissociation inhibitors (GDIs) that stabilize the inactive form and cover the lipid modification of the GTPase forming a cytosolic complex. Novel roles of GDI in GTPase regulation like the delivery of GTPases to specific sites within the cell are discussed. Moreover, local synthesis and degradation of GTPases seem to play additional roles in the spatial regulation of small GTPases. In mammalian cells, each family of the regulating proteins GAPs, GEFs, and GDIs comprise numerous members that are more or less specific for individual GTPases, cell types, GTPase functions, and signaling pathways.

Small GTPases, Fig. 1
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The GTPase cycle: GTPases are inactive in the GDP-bound form. In a complex with guanine nucleotide dissociation inhibitors (GDIs) the inactive form is stabilized (note: not all small GTPases are regulated by typical GDIs, e.g., not Ras, Ran, and Arf subfamily proteins). Guanine nucleotide exchange factors (GEFs) cause the release of GDP and binding of GTP, and thereby the activation of the proteins. The active state of the GTPases is turned off by GTP hydrolysis catalyzed by GTPase activating proteins (GAPs)

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General Structural Properties

All small GTPases are folded in a similar way. They possess four consensus amino acid sequences in common, which are involved in nucleotide binding and hydrolysis: GXXXXGK, DXXG, NKXD, and EXSAX. Two highly flexible regions (Switch I and Switch II regions) determine the nucleotide-dependent activation state of the GTPases and the protein–protein interactions with effectors and regulatory proteins.

Posttranslational Modification

All small GTPases (except Ran) are posttranslationally modified. Most important is the isoprenylation of the C-terminus. The type of modification is determined by the COOH-terminal amino acid sequence. GTPases with a C-terminal CAAX-box (A = aliphatic amino acid, X = any amino acid) are farnesylated at the cysteine residue followed by the proteolytic degradation of the last three amino acids and subsequent methylation of the carboxy-terminus. In the case of CAAL or CAC, the cysteines are modified by geranylgeranylation. In some cases an additional cysteine is palmitoylated or N-terminal myristoylation occurs. All these posttranslational modifications allow the interaction of GTPases with the phospholipid bilayer. Lipid modification of the GTPases is required for membrane localization and GDI binding.

Families

The superfamily of small GTPases consists of more than 100 members from yeast to human with more than 80 members expressed in mammalian cells. Based on structural and functional similarities the GTPases are subdivided into five major classes.

Ras GTPases

The mammalian family of Ras GTPases consists of more than 15 members, which share high homology to each other and include Ha-Ras, Ki-Ras, N-Ras, R-Ras, Rap, Ral, Rheb, Rin, and Rit proteins (Hancock 2003). Ras proteins have achieved attention with the discovery that they contain point mutations in 15% of all human tumors (more than 90% in pancreatic tumors), leading to the exchange of conserved amino acids, for example, at positions 12 and 61. Amino acid exchanges at these positions block the GTP hydrolyzing capacity of the GTPases, resulting in constitutive activation. Ras GTPases are involved in proliferation and/or differentiation. They couple receptor tyrosine kinases with a cascade of cytosolic kinases termed Raf/ERK kinase pathway (also known as MAP kinase cascade). Activation of this pathway leads to phosphorylation and activation of transcription factors like Elk-1, and stimulate gene expression. Activated Ras has been shown to transform culture cells and to produce tumors in nude mice. Besides the Raf kinase, also RalGDS, which is an activator of the Ral subfamily proteins, and the PI3 kinase involved in inositol signaling are important effectors of Ras signaling. Ral GTPases (<50% identical with Ras) control cell proliferation, Ras-mediated cell transformation, vesicle traffic, phospholipase D, and cytoskeleton organization. Rap GTPases have been identified in a screen for cDNAs that are able to revert the transforming phenotype of Ki-Ras (Kirsten Ras) and, therefore, were also termed K-rev proteins.

Rho GTPases

Members (>20) of the Rho family of GTPases, including RhoA, B and C, Cdc42 and Rac1, 2 and 3, share more than 50% sequence identity. These GTPases are important regulators of the actin cytoskeleton (Etienne-Manneville and Hall 2002). RhoA regulates the formation of actin stress fibers, whereas Cdc42 is known to induce filopodia. Rac is involved in the formation of lamellipodia and membrane ruffles. Rho GTPases are involved in migration, phagocytosis, endo- and exocytosis, and cell–cell and cell–matrix contact. Rac regulates NADPH oxidase. Furthermore, Rho GTPases are involved in transcriptional activation, cell transformation, and apoptosis. Considering their diverse functions, Rho GTPases are regulated by a large number of GEFs and GAPs (>60 members of each family have been identified), suggesting spatiotemporal, function-specific regulation.

A subfamily of Rho proteins, the Rnd proteins are always GTP-bound and seem to be regulated by expression and localization rather than by nucleotide exchange and hydrolysis. Many Rho GTPase effectors have been identified, including protein and lipid kinases, phospholipase D, and numerous adaptor proteins. One of the best characterized effector of RhoA is Rho kinase, which phosphorylates and inactivates myosin phosphatase; thereby RhoA causes activation of actomyosin complexes. Rho proteins are preferred targets of bacterial protein toxins (Bacterial Toxins).

Rab GTPases

The largest family of small GTPases with more than 40 members identified is the family of Rab GTPases (Zerial and McBride 2001). Rab proteins are important regulators of specific steps of intracellular vesicle trafficking, including budding, targeting, docking, and fusion with acceptor membranes. Each Rab protein has an organelle-specific subcellular localization and seems to be functionally specialized. Rab1A and Rab1B are two of the most extensively studied members of the Rab family. Both proteins are found in membranes of the ER, Golgi apparatus, and intermediate vesicles between these compartments. They appear to function in the anterograde trafficking of proteins from the ER to the Golgi compartment. Rab4 and Rab5 are present on early endosomes and are involved in the endocytic process, whereas Rab6 is localized at the Golgi apparatus regulating processes of the secretory pathway. One of the best studied members of the Rab protein family is Rab3a. This GTPase is a key regulator of Ca2+-induced exocytosis, particularly in nerve terminals. Several effectors of Rab proteins like Rabphilin, Rabaptin, and Rim have been identified and characterized as essential for vesicle trafficking. Recently, the Rab effector Rabkinesin6 has been identified that links Rab proteins to the microtubule cytoskeleton. Rabkinesin6 may be the motor driving vesicles along microtubules from the Golgi apparatus to the periphery.

Arf/Sar1 GTPases

The name Arf (ADP-ribosylation factor) stems from its discovery as a cytosolic factor with the ability to enhance the ADP-ribosylation of the α-subunit of the G protein GS by cholera toxin. Arf is known to regulate phospholipid metabolism. Studies with dominant active or dominant negative mutants of Arf proteins in mammalian cells suggest the involvement of these GTPases in the trafficking of coated vesicles, and it is now known that Arf1 regulates the formation of COPI-coated vesicles for retrograde transport between Golgi apparatus and endoplasmic reticulum (Souza-Schorey and Chavrier 2006). Sar1, which is 37% identical to Arf1, is needed for the assembly of COPII proteins for vesicle transport in the opposite direction. Taken together, Arf and Sar proteins play crucial roles in the recruitment of COP components to vesicles thereby regulating vesicle budding. In contrast to the other small GTPases, Arf/Sar1 proteins are not regulated by GDI proteins, whereas different GEF and GAP proteins have been identified. Myristoylation of Arf proteins at the N-terminus is required for its membrane localization.

Ran GTPases

In mammalian cells there is only one Ran gene, which was discovered as a Ras-like gene (Ran: Ras-related nuclear protein) (Dasso 2002). In contrast, in yeast more than one related Ran genes have been identified. The predominant nuclear localization of the GTPase was the first hint that Ran is involved in nucleocytoplasmic transport processes. Interestingly, the only Ran GEF present in mammalian cells, RCC1 (regulator of chromatin condensation), is localized exclusively in the nucleus, whereas the single Ran GAP (Ran GAP1) is in the cytoplasm. This specialized localization of the regulators is the prerequisite for the asymmetric distribution of the GDP- and GTP-bound form of Ran and for its role as a nucleocytoplasmic transporter. In contrast to other GTPases, the activity of Ran is dependent on the gradient of the GTP-bound GTPase from cytoplasm to nucleoplasm that allows the transport of cargo proteins. Ran is involved in nuclear import as well as in export of proteins through the nuclear pore complex. Both processes require the formation of protein complexes, including Ran, the cargo protein, and Ran-binding proteins like importins or exportins. In addition to its transporter function, Ran has been shown to participate in microtubule organization during the M phase of the cell cycle.

Cascades and Cross-Talk

Small GTPases are not isolated molecular switches. Signaling cascades within one subfamily and cross-talk between members of different subfamilies are known. For example, Cdc42/Rac/Rho is sequentially activated after extracellular stimuli in quiescent Swiss 3T3 cells. Moreover, reciprocal modulation between Rho GTPases has been described. Ras and Rho proteins act in a cooperative manner in Ras-induced transformation. A further example of cross-talk between GTPase families is the cooperative function of Rho and Rab proteins during cell migration, with Rho proteins controlling the actin cytoskeleton, and Rab proteins regulating vesicular traffic for the recruitment of membrane material, and the recycling of proteins like integrins. Arfaptin connects signaling via Arf and Rac in regulating fundamental processes like endocytosis and secretion.

Drugs

Small GTPases, among other activities, regulate cell growth, neurite outgrowth, and signaling of immune cells involved in inflammation. Pharmacological modulation of the activity of small GTPases is thus a useful aim in cancer and anti-inflammatory therapies. Despite many years of scientific work, specific inhibition of Ras is not yet possible; however, the prenyl binding protein PDEdelta sequesters oncogenic Ras within the cytosol to suppress its membrane localization and signaling. Moreover, inhibitors for an activating mutation of the Ras effector Raf are in clinical use. Farnesyltransferase inhibitors block the posttranslational modification of several GTPases, to block correct cellular localization and therefore the transforming activity of Ras or Rho GTPases. Moreover, inhibitors of Rho effectors like Rho kinase inhibitors have been generated.

Cross-References