Combinatorial Chemistry & High Throughput Screening, 2003, 6, 79-99 1 1386-2073/03 $41.00+.00 © 2003 Bentham Science Publishers Ltd. Established and Emerging Fluorescence-Based Assays for G-Protein Function: Ras-Superfamily GTPases Rafael J. Rojas 1 , Randall J. Kimple 1 , Kent L. Rossman 2 , David P. Siderovski 1,2,4 and John Sondek *1,2,3 1 Department of Pharmacology, 2 Lineberger Comprehensive Cancer Center, 3 Department of Biochemistry and Biophysics, 4 UNC Neuroscience Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Abstract: Ras and Rho GTPases are signaling proteins that regulate a variety of physiological events and are intimately linked to the progression of cancer. Recently, a variety of fluorescence-based assays have been refined to monitor activation of these GTPases. This review summarizes current fluorescence-based techniques for studying Ras superfamily GTPases with an emphasis on practical examples and high-throughput applications. These techniques are not only useful for biochemical characterization of Ras superfamily members, but will also facilitate the discovery of small molecule therapeutics designed to inhibit signal transduction mediated by GTPases. Keywords: Fluorescent nucleotide exchange assay, Ras, Rho, small GTPase. INTRODUCTION Ras-Superfamily GTPases: Molecular Switches of Signal Transduction The Ras-superfamily consists of over 150 members categorized into several subfamilies based upon sequence homology. The Ras, Rho, Ran, Rab, Arf, and Rem/Rad family proteins are monomeric GTP hydrolyzing proteins (GTPases or G-proteins) of ~21 kDa essential for a variety of biological phenomenon (reviewed in [1-4]). Similar to their large heterotrimeric G-protein α-subunit counterparts, small GTPases act as binary switches that cycle between active (GTP-bound) and inactive (GDP-bound) states. The GTP/GDP cycling of Ras superfamily GTPases, shown in Fig. (1), is highly regulated by classes of proteins specific to each subfamily. Guanine nucleotide exchange factors (GEFs) activate GTPases, while GTPase activating proteins (GAPs) cause inactivation. Small GTPases can additionally be regulated by guanine nucleotide dissociation inhibitors (GDIs) that prevent nucleotide exchange, sequester GTPases, and block associated downstream signaling. Members of the Ras and Rho subfamilies are the most extensively studied group of small GTPases and are essential components of the mitogenic signal transduction pathway. Extracellular stimulation of receptor tyrosine kinases, G- protein coupled receptors, or integrins can result in activation of Ras and the prototypical Rho subfamily members RhoA, Rac1, and Cdc42 [5, 6]. Once activated, these GTPases further propagate external signals by activating a multitude of downstream effector proteins, resulting in a diversity of cellular responses. Additionally, activation of Ras and Rho family GTPases is a critical step during tumor progression and acquisition of an invasive and *Address correspondence to this author at the Dept. of Pharmacology, University of North Carolina at Chapel Hill, CB #7365, 1106 M.E.J. Bldg, Chapel Hill, NC 27599-7365, USA; Phone: 919-966-7530; Fax: 919-966- 5640; e-mail: sondek@med.unc.edu metastatic phenotype [7-11]. These GTPases are highly oncogenic with over 30% of all human cancers and 90% of pancreatic cancers harboring activated Ras mutations [11]. Recent evidence also suggests a vital role for Rho family members during transformation and the acquisition of an invasive and metastatic phenotype by regulating the actin cytoskeleton [12-15]. Furthermore, GEFs specific to Rho GTPases (RhoGEFs) such as Dbl (diffuse B cell lymphoma), Tiam1 (T-cell invasion and metastasis factor), and LARG (leukemia associated RhoGEF) are routinely isolated during screens for transforming oncogenes and make up one of the largest classes of human proto-oncogenes with over 60 members (reviewed in [16, 17]). It is due to their inherent oncogenic signaling properties and role in cancer metastasis that Ras and Rho-mediated signaling events are emerging targets for anti-cancer drug discovery [18]. Furthermore, the complex pathways that regulate Ras superfamily GTPases are the current focus of intensive research. For many of these pursuits there is a critical need for sensitive, real-time measurements of GTPase activation and subsequent monitoring of signaling events. Recently, a variety of fluorescence-based assays have been refined to monitor these events using purified proteins in vitro as well as under more physiological conditions in vivo. This review will summarize current fluorescence-based techniques for studying Ras superfamily GTPases and will illustrate practical examples of applications demonstrating their utility. Such techniques are useful not only for the biochemical characterization of these signaling proteins, but will also be promising for future strategies in target-based drug design for the treatment of cancer. Anatomy of a Small GTPase The structural elucidation of a number of Ras superfamily members including Ras [19, 20], Rac [21], Rap [22], and Ran [23] has been instrumental in revealing a universal mechanism for nucleotide binding, GTP