Mutagenesis in the switch IV of the helical domain of the human Gs alpha reduces its GDP/GTP exchange rate

The Ca subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, Il, III, and IV, have bee n identified as undergoing structural changes. Switch I, II, and III are lo cated in RasD and switch IV in I-ID. All Ca known functions, such as GTPase activity and receptor, effector, and G beta gamma interaction sites have b een 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 hum an and Xenopus Gs alpha we have previously identified a HD region, encompas sing helices alpha A, alpha B, and alpha C, that was responsible for the ob served functional differences in their capacity to activate adenylyl cyclas e (Antonelli et al. [1994]: FEES Lett 340:249-254). Since switch IV is loca ted within this region and contains most of the nonconservative amino acid differences between both Gs alpha proteins, in the present work we construc ted two human Gs alpha mutant proteins in which we have changed four and fi ve switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGs alpha alpha S133N, M135P, P138K, P143S) and M17 (hGsaaS133N, M135P , V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and ch aracterized by their ability to bind GTP gamma S, dissociate CDP, hydrolyze CTP, and activate adenylyl cyclase. A decreased rate of CDP release, GTP g amma S binding, and GTP hydrolysis was observed for both mutants, M17 havin g considerably slower kinetics than M15 for all functions tested. Reconstit uted adenylyl cyclase activity with both mutants showed normal activation i n the presence of AIF(4)(-), but a decreased activation with GTP gamma S, w hich is consistent with the lower GDP dissociating rate they displayed. The se data provide new evidence on the role that I-ID is playing in modulating the GDP/GTP exchange of the Gs alpha subunit. (C) 2000 Wiley-Liss, Inc.