Gat-GTP

Cholera toxin catalyzes the ADP-ribosylation of an arginine residue (Argl74 in Gat, Arg201 in Gas) in various a-subunits. The Argl74 residue of G t contacts the phosphate group of the bound GTP and is thus directly involved in GTP binding and possibly also in GTP hydrolysis. Modification of Argl74 by ADP-ribosylation interferes with this... 

Fig. 5.21 GTP and GDP structures of Transducin. The Gat subunit of transducin possesses - in contrast to Ras protein and to other small regulatory GTPases - an a-helical domain that hides and closes the G-nucleotide binding pocket. The conformational changes that accompany the transition from the inactive GajtGDP form (a) into the active GatGTP from (b) are restricted to three structural sections that are known as switches I, II and III. Switch I includes the link ofthe a-helical domain with pi, switch II affects in particular helix al, and switch III, the/ 3-a3 loop. Switch III includes a sequence that is characteristic for the a-subunits ofthe heterotrimeric G proteins. The conformational changes of switches II and III affect structural sections that are assumed to be binding sites forthe effector molecule adenylyl cyclase (AC) and the y-subunit of cGMP-dependent phosphodiesterase (PDEy), based on mutation experiments and biochemical investigations. MOLSKRIPT representation according to Kraulis (1991).

These data obtained by two-hybrid and pull-down experiments consistently indicate that the GAT domains from all GGAs interact with GTP-bound Arfs, and that the GAT domain interacts with all classes of Arfs with comparable efficiencies. 

Cellular levels of activated, GTP-bound Arfl can be monitored with a recently described pull-down assay using a GST-GGA fusion protein as a probe (Lorraine et al., 2001). This fusion protein is composed of the VHS and GAT domains of human GGA3 (Golgi-associated, 7-adaptin homologous, Arf-interacting protein 3), an ubiquitously expressed coat protein localized in... 

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