Newcombe, Anthony R.; (2000) The biochemical role of the small G protein Rac1 in cell signalling pathways - Interaction with RhoGDI and the phagocyte NADPH oxidase component, p67phox. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Rac is a small G protein with a number of signalling roles. Along with other members of the Rho family of small GTPases, it is involved in the control of the actin cytoskeleton (Hall, 1992) and an apparently separate role in the activation of the NADPH oxidase, an enzymatic mechanism in phagocytes which forms superoxide in response to bacterial infection. In an inactive state, Rho family proteins exist in a complex with a second cytosolic protein, Rho guanine nucleotide dissociation inhibitor, RhoGDI. Activation causes dissociation of the Rac GDI complex and movement of Rac to the membrane. Spectroscopic studies have been used to investigate the interaction of Racl with other molecules, such as p67phox, a component of the NADPH oxidase complex. Complexes of racl with 2'(3')O-(N-methylanthraniloyl) (mant) fluorescent nucleotide analogues (eg. mantGDP) have been used to try to develop methods to study the interaction of Racl and p67phox. Although a previous report indicates a fluorescent change when Racl (complexed to a fluorescent nucleotide analogue) is incubated with p67phox, these experiments could not be repeated. A number of other approaches have been taken to develop a system to monitor the interaction of Rac and p67phox. Fluorescent approaches have also been developed to study the interaction of Rac and RhoGDI. GDI has previously been labelled with the fluorophore N-[2-1-(maleimidyl)ethyl]-7- (diethylamino)coumarin-3-carboxamide (MDCC) on a single cysteine in our laboratory and shows a large fluorescence decrease on Rac binding. Rac requires a lipid modification at the C-terminus to interact with RhoGDI, which presents a number of experimental difficulties. A system has been developed using C-terminally truncated (E.coli expressed) Rac and a farnesylated C-terminal peptide that mimics full length Racl that has been lipid modified in vivo (Newcombe et al., 1999). We are currently using this system to study the interaction of Racl with GDI and a number Racl point mutants have been made in the major regions of divergence between Ras superfamily proteins, based on the crystal structure of Racl (Hirshberg et al., 1997). In addition, a Racl/H-Ras chimaeric protein has been made and expressed in this laboratory. Results indicate that a region of the Racl effector loop is important for the Racl GDI interaction, with mutations in the insertion loop of Racl having little or no affect on the affinity of the Racl GDI interaction (Newcombe, Hunter & Webb, unpublished results). In addition, the interaction of Racl with a number of novel fluorescent nucleotide analogues including 3'-O-[N-[2-(7-diethylaminocoumarin-3-carboxamido)ethyl]carbamoyl] GTP (cou-edaGTP) and coumarin343-edaGTP (but-edaGTP) have been tested, and Racl complexes have shown that the rate of nucleotide hydrolysis and exchange by Racl shows them to be good analogues of GTP. It is hoped that these analogues will be useful to study the interaction of Racl with other proteins, such as GDI and p67phox.

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