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Rho-family GTPases (p21s) are molecular switches related to the protooncogene Ras: these function in complementary pathways to orchestrate the actin cytoskeleton, regulate cell polarity, microtubule dynamics, membrane transport pathways and modulate a variety of transcriptional events. Rho GTPases are biochemically and structually simple proteins: they cycle between two conformational states, with the GTP-bound form regarded as active, while the GDP-bound state is essentially inactive. Conversion between these states is brought about by hydrolysis of bound GTP. The GTPases in their active form can recognize a variety of target proteins and thereby generate a response until GTP hydrolysis returns the switch to an ‘off’ state. The Rho family of GTPases is now intensively studied by cell biologists, as its members have turned out to be key regulators of many aspects of cell behaviour. In this introductory chapter I discuss how Rho proteins become activated by GEFs in response to extracellular signals, and in particular how integrin signaling might impinge on Rhos. At this stage our knowledge of many of these pathways is rudimentary, thus much still needs to be done to uncover specific pathways that regulate of Rho proteins in vivo . This volume covers what is known of Rho proteins in vertebrate systems with some reference to lower model organisms. The molecular and structural data is covered in some depth; in this ‘post genomic’ era we are also able to provide a panoramic view of the various protein families that have emerged as key players in Rho GTPases signaling.

The Rho GTPases form a relatively old family of signalling proteins, with representation across all branches of the eukaryote lineage. As a consequence of this, there is a large amount of sequence information relating to Rho GTPases in the rapidly increasing number of genome and cDNA databases. This kind of data is never a substitute for ‘wet’ experimental work in terms of understanding protein function; however, it can tell us something about the history of the proteins we work with. By understanding the history of these signalling proteins, we can more accurately draw together the growing body of experimental work on Rho GTPase function in various genetic model organisms. In this short review we look at the origins of the human Rho GTPase family and attempt to trace the emergence of specific isoforms. We also examine two other factors that contribute to the overall diversity of human Rho GTPase proteins: genetic polymorphism and alternative splicing.

The structural GDP/GTP cycle of Arf proteins is implemented by switch regions that change their conformations in response to the nature of their interactions. They include the classical switch regions in the nucleotide-binding site, and a unique switch region located on the opposite side of the protein. These switch regions communicate with each other and couple activation of Arfs by GTP to their recruitment to membranes. Variations in this cycle determine the specificity and differences in Arf1 and Arf6 signaling. The role of guanine nucleotide exchange factors (GEFs) as key players in the implementation of the nucleotide/membrane coupling is also described. A particular emphasis is given to the inhibition of GEF-activated nucleotide exchange by the drug Brefeldin A and by a GEF point mutation, as tools to trap intermediate states of the reaction. Taken together, structural and biochemical studies yield a comprehensive model for one of the most remarkable nucleotide cycle found in G proteins.

Members of the Dock180 superfamily of proteins have been recently identified as novel guanine nucleotide exchange factors (GEF) for Rho family of small GTPases. While conventional GEFs use a conserved DH-PH cassette for catalyzing GTP/GDP nucleotide exchange on Rho family GTPases, the Dock180 family members mediate nucleotide exchange via a novel evolutionarily conserved Docker/DHR2/CZH2 domain. The activation of distinct Rho-family GTPases by particular members of the Dock180 family have been linked to a multitude of biological processes. Based on sequence similarity, Dock180 family proteins can be broadly divided into four subfamilies, DOCK-A, DOCK-B, DOCK-C and DOCK-D, based on overall domain structure and substrate specificity. The function of proteins in DOCK-A and DOCK-B subfamilies is subject to regulation by their interacting partners such as ELMO and CrkII. In particular, the Dock180/ELMO complex functions as a bi-partite GEF for the GTPase Rac. This review discusses the features of the Dock180 family members, their regulation by binding partners and their relevance to biology.

The cycling of Rho proteins between GTP bound ‘on’ and GDP- bound ‘off’ states is essential for their transient signalling to downstream effector proteins, which include kinases, cytoskeletal regulatory proteins and enzymes. Rho GTPase-activating proteins (RhoGAPs) can terminate Rho GTPases by stimulating their intrinsic rate of GTP hydrolysis. This rapidly converts Rhos to their GDP-bound state, and these membrane-bound proteins are then thought to be sequestered by guanine nucleotide dissociation inhibitors, allowing subsequent transport between membranes. RhoGAPs are multi-domain proteins with various protein and lipid interactive domains capable of precise targeting and regulation in signalling complexes. Some have more than one catalytic activity and potentially signal to other pathways in addition to terminating Rho signals. In this respect Rho GTPases may provide spatial and temporal information to GAPs that in turn serve as protein adaptors in a variety of intracellular compartments. Certainly much has yet to be learnt about this diverse family of proteins.

Rho belongs to the Rho family guanosine triphosphatases (GTPases) including Rho, Rac, Cdc42, TC10, and so on. Rho is categorized into RhoA, B, and C. The Rho family GTPases exhibit guanine nucleotide-binding activity and function as molecular switches by cycling between an inactive guanosine diphosphate (GDP)-bound form and an active GTP-bound form. Rho participates in the regulation of actin cytoskeletons, cell adhesions, cytokinesis, smooth muscle contraction, cell morphology, cell motility, neurite retraction, and polarity formation through their specific effectors. The characterization of these effectors has begun to clarify how Rho regulates some phenotypes. This article focuses on the roles of RhoA/C and their effectors.

Cell shape changes are critical to cell differentiation, movement and motility. Reorganization of the actin cytoskeleton is crucial to these changes, which are elicited in response to extracellular stimuli. This reorganization is achieved by the action of kinases, phosphatases and effectors of Rho GTPases on actincontaining filaments. In this review, we shall discuss how the different proteins such as WASP, WAVE, PAK and IQGAP, which are downstream of Cdc42 and Rac affect the actin cytoskeleton. Guanine nucleotide exchange factors that activate different Rho GTPases also contribute towards the regulation of the cytoskeleton. Many of the Rho GTPase targets are kinases or are regulated by phosphorylation. Thus phosphatases are essential in the control of cell adhesion and spreading as well. The integration and modulation of the different signalling pathways downstream of the Rho proteins are key to the final cellular responses. How the different downstream proteins are shuttled and shuffled is also of interest because the same molecules may participate in pathways that are regulated by Cdc42, Rac and the antagonistic RhoA.

Cell-cell adhesion is a fundamental determinant of tissue organization. Adhesive interactions between cells help model body plan and histoarchitecture during development, while disorders of cell adhesion contribute to common diseases, including cancer and inflammation. Given their wide-ranging ramifications, it is not surprising that these interactions are subject to strict cellular regulation. In particular, classical cadherins, major mediators of cell-cell adhesion in many tissues, are key targets of Rho GTPase signalling. In this chapter we review recent developments in understanding the interrelationship between cadherin function and Rho family members. It is increasingly apparent that cadherin function is tightly regulated by membrane-local GTPase signals localized to cell-cell contacts. These may be activated both by cadherins themselves or by cadherin-dependent juxtacrine signalling receptors. These GTPases exert profound, but often pleiotropic effects on cadherin function, through their ability to regulate both cadherin-actin cooperativity and cadherin trafficking.

Membrane trafficking includes a highly dynamic and intricate set of intracellular pathways responsible for the transport of molecules in and out of the cell, and between the different intracellular compartments. A lot of attention has been paid in the past decades to the role played by distinct classes of small GTPases on the regulation of membrane trafficking, with special emphasis on the Rab and Arf families. More recently, Rho GTPases have been implicated in several important aspects of membrane trafficking. The initial indications that Rho proteins might be involved in membrane trafficking came from the observation of the localization of some of these proteins at specific intracellular compartments. These observations are corroborated by the findings of specific effects of these proteins on different membrane transport pathways. The role played by Rho family members in different aspects of membrane trafficking will be considered in this chapter.

p21-activated kinase (PAK) was the first identified serine/threonine protein kinase to bind and be activated by Rho GTPases. Since it was discovered some 10 years ago, PAK has been intensively studied and represents the best understood of the Rho-associated kinases. PAK family kinases are encoded by six mammalian genes with three conventional PAKs (PAK1–3) and three non-conventional PAKs (PAK4–6). Cdc42/Rac activates conventional PAKs through modulation of a kinase inhibitory (KI) domain by the overlapping Cdc42/Rac interaction/binding (CRIB) domain. Nonetheless PAKs can also be activated in GTPase independent manners, including by protease cleavage, translocation to membranes and binding to lipids. As a key signaling component, PAK is evolutionarily conserved from yeast to man. In mammalian cells PAKs play roles in many cellular signaling pathways such as the regulation of focal adhesion and actin dynamics, changes in cell morphology, cell motility, and the regulation of gene expression. Recently a number of studies show PAK is implicated disease states. As a result PAK family kinases are becoming good candidates for drug development.