Rho-GTPases in Embryonic Stem Cells

Abstract

The Rho family of small GTP-binding proteins is comprised of 22 members, including the most well characterized members RhoA, Rac1 and Cdc42 (Jaffe and Hall 2005). The Rho family proteins share a high degree of homology with the Ras proto-oncogene, and indeed were first identified as a result of this similarity (Ras homologue). Activity of these proteins is dependent upon their nucleotide binding state; inactive when associated with GDP but active following exchange of GDP for GTP, which induces conformational changes that promote association/activation of downstream effector proteins. The GDP/GTP cycle is regulated by GAPs that accelerate GTP hydrolysis by providing a critical catalytic amino acid leading to a return to the inactive state (Bernards and Settleman 2005), and GEFs that promote guanine nucleotide exchange and consequent Rho activation (Rossman et al. 2005). The number of GAPs and GEFs far exceeds the number of Rho proteins, and the roles of individual GAPs and GEFs in specific cell types and biological processes is currently an intensively studied field. Although united by homology and function as regulators of the actin cytoskeleton, each of RhoA, Rac1 and Cdc42 has a distinct role in the organization of actin structures (Figure 1). RhoA is principally involved with the production of actin-myosin bundles and the generation of actomyosin contractile force. Rac1 contributes to the formation of actin meshworks that result in the emergence of large protrusive structures that lead to spreading or, if occurring in a polarized manner, will contribute to motility. Cdc42 promotes the formation of actin-rich filopodia. Together, coordinated programs of RhoA, Rac1 and Cdc42 activation/inactivation play prominent roles in processes such as endocytosis/exocytosis, adhesion and motility, which may subsequently impact upon proliferation and death/survival. Recent advances in the development of activation-state sensitive fluorescent probes have allowed temporal and spatial analysis of Rho protein activation, which has added significantly to our appreciation of Rho regulation and function (Hodgson et al. 2010). Much of the early research on Rho protein function relied upon over-expression of dominant-negative mutants that reduced affinity for GTP and constitutively-active mutants that reduced GTP hydrolysis; however, more refined analysis has become possible with the rise of RNAi and knockout methodologies (Heasman and Ridley 2008). The study of Rho family proteins has historically focused on their roles as molecular switches acting downstream of cell surface receptors to regulate the actin cytoskeleton (Jaffe and Hall 2005). Significant effort has gone into classifying signaling from Rho proteins into