This chapter describes methods for the expression of Dbl and the Dbl homology (DH) domain in baculovirus expression systems and discusses the in vitro assays to demonstrate that Dbl and its DH domain contain guanine nucleotide exchange factors (GEFs) activity for the Cdc42Hs and Rho GTP-binding proteins. GEFs activate Ras family members of GTP-binding proteins by accelerating their rate of GDP release and, therefore, facilitating their GTP binding in cells. Unlike the case for Ras–GEFs for which the in vivo activities can be examined by the extent of conversion of Ras-bound GDP to GTP by the immunoprecipitation of Ras and thin-layer chromatography separation of the bound nucleotides, the GEF activities for Rho-type GTPases are difficult to determine in this manner because of the fast hydrolysis rates of the members of this family and the presence of abundant Rho–GDP-dissociation inhibitor (GDI), which renders most of the Rho-type proteins cytosolic and blocks the GEF action. Thus, the in vitro reconstituted assay is indispensable for detecting and quantitating the activities of potential Rho family GEF molecules.
ASJC Scopus subject areas
- Molecular Biology