In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/CZH2 domains.
Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.
Côté
Jean-François
JF
Institut de Recherches, Cliniques de Montréal, Canada.
Vuori
Kristiina
K
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase / Q000302:isolation & purification / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D020764:cdc42 GTP-Binding Protein / Q000302:isolation & purification; D020744:rac GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
C099818:DOCK1 protein, human; C402110:DOCK2 protein, human; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D005982:Glutathione Transferase; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins