GTPases

A critical component of the G-protein effector cascade is the hydrolysis of GTP by the activated a-subunit (GTPase). This provides not only a component of the amplification process of the G-protein cascade (63) but also serves to provide further measures of dmg efficacy. Additionally, the scheme of Figure 10 indicates that the coupling process also depends on the stoichiometry of receptors and G-proteins. A reduction in receptor number should diminish the efficacy of coupling and thus reduce dmg efficacy. This is seen in Figure 11, which indicates that the abiUty of the muscarinic dmg carbachol [51 -83-2] to inhibit cAMP formation and to stimulate inositol triphosphate, IP, formation yields different dose—response curves, and that after receptor removal by irreversible alkylation, carbachol becomes a partial agonist (68). 

The ability of receptors to couple to G-proteins and initiate GTPase activity may also be independent of ligand. Thus, specific mutations in a- and P-adrenergic receptors have led to receptors that mediate agonist-independent activation of adenylyl cyclase (69,70). These mutations presumably mimic the conformational state of the ligand-activated receptor when they are activated conventionally by ligands. 

GTPases hydrolyze GTP through nucleophilic attack by a water molecule... 

All the residues involved in important functions in the catalytic mechanism are strictly conserved in all homologous GTPases with one notable exception. Ras does not have the arginine in the switch 1 region that stabilizes the transition state. The assumption that the lack of this catalytically important residue was one reason for the slow rate of GTP hydrolysis by Ras was confirmed when the group of Alfred Wittinghofer, Max-Planck Institute,... 

In summary, structural studies of Ras and Gq with GTP-yS and a transition state analog have illuminated the catalytic mechanism of their GTPase activity, as well as the mechanism by which GTP hydrolysis is stimulated by GAP and RGS. In addition, these structural studies have shown how tumor-causing mutations affect the function of Ras and Gq. 

The GTPase domain ofGa binds to Gp in the heterotrimeric Gapy complex... 

Gq-GDP binds to the Gpy dimer through its GTPase domain in a region of the p propeller opposite to where G is bound (Figure 13.15). There are, therefore, no contacts between Gq and Gy in the heterotrimeric G py complex. The... 

Rittinger, K. et al. Crystal structure of a small G protein in complex with GTPase-activating protein rhoGAP. Nature 388 693-697, 1997. 

Rittinger, K., et al. Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389 758-762, 1997. 

Scheffzek, K., et al. The Ras-RasGAP complex structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277 333-338, 1997. 

Sondek, J., et al. GTPase mechanism of G proteins from the 1.7 A crystal structure of transducin a.GDP.AlF4 . Nature 372 276-279, 1994. 

FIGURE 15.21 Hormone (H) binding to its receptor (R) creates a hormone receptor complex (H R) that catalyzes GDP-GTP exchange on the o -subunit of the heterotrimer G protein (G ), replacing GDP with GTP. The G -subunit with GTP bound dissociates from the /37-subunits and binds to adenylyl cyclase (AC). AC becomes active upon association with G GTP and catalyzes the formation of cAMP from ATP. With time, the intrinsic GTPase activity of the G -subunit hydrolyzes the bound GTP, forming GDP this leads to dissociation of G GDP from AC, reassociation of G with the /Sy subunits, and cessation of AC activity. AC and the hormone receptor H are integral plasma membrane proteins G and G are membrane-anchored proteins. 

G-proteins, trim eric membrane-bound proteins that have intrinsic GTPase activity and act as intermediaries between 7TM receptors and a host of cellular effectors see Section 2.2. 

Adaptor Proteins. Figure 1 Adaptor protein domains. A scheme of the domain structures of some well-characterized adaptor proteins is shown. Descriptions of domain characteristics are in main text except C2, binds to phospholipids GTPase activating protein (GAP) domain, inactivates small GTPases such as Ras Hect domain, enzymatic domain of ubiquitin ligases and GUK domain, guanylate kinase domain. For clarity, not all domains contained within these proteins are shown. 

---