caracterización del dominio helicoidal de la subunidad Gas de la proteína G
G-proteins (Gajly} tranduce signals to the intracellular milieu by coupling to membrane receptors (GPCR) and to effector systems such as enzymes and ion channels. The Ga subunit Is fonned by a very well conserved domain that binds and hydrolizes GTP called the GTPase domain (DGTPase) and by a unique...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 1999 |
| País: | Chile |
| OAI Identifier: | oai:repositorio.anid.cl:10533/233003 |
| Acesso em linha: | https://hdl.handle.net/10533/233003 |
| Access Level: | acceso abierto |
| Palavra-chave: | Ciencias Naturales Otras Ciencias Naturales |
| Resumo: | G-proteins (Gajly} tranduce signals to the intracellular milieu by coupling to membrane receptors (GPCR) and to effector systems such as enzymes and ion channels. The Ga subunit Is fonned by a very well conserved domain that binds and hydrolizes GTP called the GTPase domain (DGTPase) and by a unique a-helical domain called helical domain (HD), whose function is not well understood. The binding of GTP to the DGTPase domain induces a conformational change in four different regions called "switch regions". Switch 1. 2 and 3 are present in the DGTPase domain and have been involved in the bindi09 of GDP and GTP and in the interaction with the Gjly heterodimer . The switch 4 region is localized in the HD. Through the use of chimeric Gsa proteins between human and Xenopus leavis we identified a HD region, encompassing helices aA. aB and aC that was responsible for the functional differences ~rv~ ~n both Gas proteins. Since switch 4 is located in this region and contains most of the nonconservative amino acid differences between the human and Xenopus laevis protein. we decided to mutate the human Gas subunit in the switch 4 region to analize In major detail its function. We changed different human Gas residues for the ones present in the Xenopus protein (8118• M120, V172, P123 y P"'). These mutants Gsa were expressed in vitro and in vivo systems and characterized by their abir.ty to activate the adenylyl cyclase. to bind GTPyS, to dissociate GDP and to hydrolyze GTP. The mutation of three °' more residues decreased the capacity of these mutants to activate the adenylyl cyclase activity and to exchange GDP by GTP. These results clearly indicate that the Gas HD and its switch 4 region play an important role. modulating the GDP/GTP exchanging rate through a mechanism that probably alters the distance between the GTPase and HD domains (nucleotide binding interphase). affecting the release of GDP and the entrance of GTP and as a consequence the activation of the Gas subunit. |
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