Fluorescence approaches for monitoring interactions of Rho GTPases with nucleotides, regulators, and effectors
Introduction
Rho family GTPases act as tightly regulated molecular switches governing a variety of critical cellular functions [1], [2], [3], [4]. Their activity is controlled by two biochemical reactions, the GDP/GTP1 exchange and the GTP-hydrolysis, which can be catalyzed by two kinds of regulatory proteins [5]. While the guanine nucleotide exchange factors (GEFs) activate Rho GTPases by stimulating the slow exchange of bound GDP for the cellular abundant GTP, GTPase activating proteins (GAPs) accelerate the slow intrinsic rate of GTP-hydrolysis by several orders of magnitude, leading to the inactivation. The formation of the active GTP-bound state of the GTPase is accompanied by conformational changes mainly at two regions (called switch I and II) that provide a platform for a selective interaction with a multitude of downstream effectors and initiate downstream signaling [5], [6], [7], [8].
Our understanding of Rho GTPase regulation and signaling is becoming increasingly complex since more than 69 GEFs, 68 GAPs, and 90 effectors are considered as potential interacting partners of the 22 mammalian members of the Rho family [6], [9], [10], [11]. Only a sparse number of such intermolecular interactions have been primarily investigated in vitro with either solid phase methods like radioactive ligand overlay, pulldown assays or yeast two hybrid studies. To obtain a detailed picture of the molecular switch function of the Rho GTPases and their interaction with regulators and effectors we have established fluorescence-based methods for the time-resolved monitoring and quantification of Rho GTPase functions and interactions with their binding partners. Fluorescent guanine nucleotides are often ideally suited to fulfill these criteria, as it is known that they do not grossly disturb the biochemical properties of the GTPase and that the fluorescence reporter group is sensitive to changes in the local environment to produce a sufficiently large fluorescence change [12]. Furthermore, it is often sensitive to the interaction with partner proteins that happen to bind in its neighborhood.
This article describes the role of two different fluorescently labeled guanine nucleotides in the biochemical analysis of Rho GTPases. The fluorescent reporter groups such as methylanthraniloyl (mant) or tetramethylrhodamine (tamra) are located on either the 2′ or 3′oxygen of the ribose of GDP and GTP-analogues (Fig. 1) [12]. Fluorescent nucleotides can be used to determine the binding affinities of nucleotides and effector domains as well as to evaluate the GEF-catalyzed nucleotide exchange and the GAP-stimulated GTP-hydrolysis activities, respectively.
Section snippets
Fluorescent nucleotides
Two different fluorescence reporter groups, N-methylanthraniloyl (mant) and tetramethylrhodamine (tamra) attached to 2′(3′)-hydroxyl group of the ribose moiety of the guanine nucleotides (GDP, GTP, and Gpp(NH)p) (Fig. 1), were used in our research to monitor protein–ligand as well as protein–protein interactions, and to measure catalytic activities of regulatory proteins.
Nucleotide binding
Mant-nucleotides are exceedingly helpful as a large increase (∼40%) in fluorescence intensity upon binding to the nucleotide-free GTPase is observed. Obviously, the environment of the GTPase-bound mant-nucleotide is less polar compared to bulk aqueous solution and the bound mant-group experiences less quenching.
For quantifying nucleotide binding, fluorescence equilibrium titration is not amenable to most of the small GTPases because of their very high nucleotide affinity [18], [21]. The
Concluding remarks
Small GTPases of the Ras superfamily are molecular switches with central roles in virtually all signaling pathways of the cell. The fact that a large variety of regulators and effectors are identified provides an impression of the enormous dimensions of the signaling network relying on the small GTPases (e.g., 22 Rho GTPases with 69 GEFs, 68 GAPs, and 90 effectors). The elucidation of the molecular switch mechanism of the GTPases and particularly their specificities and affinities for
Acknowledgments
We thank A. Wittinghofer for continuous support. We appreciate the comments and discussions provided by C. Herrmann, R. Dvorsky, L. Gremer, L. C. Haeusler, L. Blumenstein, D. Fiegen, and A. Eberth. The Deutsche Forschungsgemeinschaft, Volkswagen-Stiftung, and European Community Marie Curie Fellowship support our research.
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