Abstract

The G? subunits of heterotrimeric G proteins are constituted by a conserved GTPase 'Ras-like' domain (RasD) and by a unique ?-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All G? known functions, such as GTPase activity and receptor, effector, and G?? interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gs? we have previously identified a HD region, encompassing helices ?A, ?B, and ?C, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gs? proteins, in the present work we constructed two human Gs? mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGs??S133N, M135P, P138K, P143S) and M17 (hGs??S133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTP?S, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTP?S binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF4-, but a decreased activation with GTP?S, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gs? subunit.