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Mutagenesis in the switch IV of the helical domain of the human Gsα reduces its GDP/GTP exchange rate
Author(s) -
Echeverría Valentina,
Hinrichs María Victoria,
Torrejón Marcela,
Ropero Santiago,
Martinez José,
Toro María Jose,
Olate Juan
Publication year - 2000
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(20000301)76:3<368::aid-jcb4>3.0.co;2-b
Subject(s) - adenylyl cyclase , gtp' , gtpase , g protein , heterotrimeric g protein , gs alpha subunit , xenopus , mutant , gtp binding protein regulators , gtpase activating protein , mutagenesis , biology , guanosine diphosphate , biochemistry , guanosine triphosphate , chemistry , microbiology and biotechnology , receptor , enzyme , gene
The Gα subunits of heterotrimeric G proteins are constituted by a conserved GTPase “Ras‐like” domain (RasD) and by a unique α‐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 Gα known functions, such as GTPase activity and receptor, effector, and Gβγ 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 Gsα we have previously identified a HD region, encompassing helices αA, αB, and αC, 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 Gsα proteins, in the present work we constructed two human Gsα mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsααS133N, M135P, P138K, P143S) and M17 (hGsααS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli , purified, and characterized by their ability to bind GTPγS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPγS 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 AlF 4 − , but a decreased activation with GTPγS, 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 Gsα subunit. J. Cell. Biochem. 76:368–375, 2000. © 2000 Wiley‐Liss, Inc.