Shutes, Adam; (2002) Studies on the small G protein Rac1: Interaction with IQGAP and the role of Mg2+ during nucleotide exchange. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This Thesis is centred around the small G-protein Rac1, and investigates: Rac's interaction with the effector protein IQGAP; the role of Mg2+ during Rac's nucleotide exchange; and Rac1's proposed self-stimulatory activity. Rac1's C-terminal tail has been suggested to posses GTPase self-stimulatory activity. An investigation of this property is described. The effects on Rac's intrinsic GTPase rate were observed using full-length and C-terminal truncated forms of the protein, as well as Raco GMPPNP forms and peptides of Rac's C-terminal tail. In contrast to the previously published data, no significant increase in rate was observed in the presence or absence of the C-terminal tail. The role of Mg2+ in nucleotide exchange is examined using both novel (MBC: 7- Monoethylamino 8-bromocoumarin) and well characterised (Mant: N-methylanthraniloy1) fluorophores, covalently bound to Rac-complexed nucleotides. Fluorescent intensity measurements suggest a two step model of release of Mg2+ and nucleotide from Raco nucleotide complexes during exchange. This model is supported by anisotropy data and use of Mg2+ as a substitute for Mg2+, which allows examination of each of the two steps separately. Further work examines the Mg2+ binding to Raco nucleotide complexes, and combined with the release data allows calculation of dissociation constants for Mg2+ from the Rac complexes. IQGAP has been suggested to associate with Rac1 and inhibit its intrinsic GTPase activity. These observations are confirmed in this report by measurements made on single turnover GTP hydrolysis of Rac in the presence or absence of IQGAP. The kinetics of this association are measured using fluorescent intensity and fluorescent anisotropy techniques. The use of a panel of Rac mutants provides a method to asses the importance of different Rac regions in the interaction with IQGAP. IQGAP exhibits a relatively high affinity with wild type Rac. The mutant studies suggest that the main contribution to association of Rac with IQGAP occurs via the Effector Region, with a small contribution from the Insert Loop.

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