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

Echeverria, V; Hinrichs, MV; Torrejón M.; Ropero, S; Martinez, J.; Toro, MJ; Olate, J


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 been identified as undergoing structural changes. Switch I, II, and III are located 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 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 alpha we have previously identified a HD region, encompassing helices alpha A, alpha B, and alpha C, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEES Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gs alpha proteins, in the present work we constructed two human Gs alpha mutant proteins in which we have changed four and five 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 characterized by their ability to bind GTP gamma S, dissociate CDP, hydrolyze CTP, and activate adenylyl cyclase. A decreased rate of CDP release, GTP gamma 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 AIF(4)(-), but a decreased activation with GTP gamma S, which is consistent with the lower GDP dissociating rate they displayed. These 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.

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Título según WOS: Mutagenesis in the switch IV of the helical domain of the human Gs alpha reduces its GDP/GTP exchange rate
Volumen: 76
Número: 3
Editorial: Wiley
Fecha de publicación: 2000
Página de inicio: 368
Página final: 375
Idioma: English
Notas: ISI