Purification and characterization of the G203T mutant alpha i-2 subunit of GTP-binding protein expressed in baculovirus-infected Sf9 cells.
We expressed the Gly203-->Thr (G203T) mutant of Gi2alpha, which was expected to show a dominant-negative phenotype in Gi2-mediated signal transduction, in baculovirus-inefected Sf9 cells and purified the mutant alpha subunit for its characterization. The rate of dissociation of GDP from G203T Gi2alpha was 3- to 4-fold faster than that from wild type Gi2alpha, but their kappacat values for GTP hydrolysis were almost the same. The affinities of the two Gi2alpha proteins for the beta gamma subunits of G proteins to form alpha beta gamma timers, which served as substrates for pertussis toxin-catalyzed ADP-ribosylation, were the same. In marked contrast, G203T Gi2alpha was unable to form a tight complex with a non- hydrolyzable analog (GTP[gammaS) of GTP; bound GTP[gammaS] was readily released from the mutant Gi2alpha even in the presence of a high concentration of Mg2+. Its susceptibility to tryptic digestion also revealed that GTP[gammaS]-bound G203T Gi2alpha formed a conformation apparently different from that of the GTP[gammaS]-bound form of wild-type Gi2alpha. Both the G203T and wild-type Gi2alpha proteins were capable of coupling with membrane-bound alpha2-adrenergic receptors, resulting in the formation of receptor-G protein complexes with high affinity for agonists. However, GTP[gammaS]-dependent uncoupling from high-affinity receptors was markedly attenuated in the case of G203T Gi2alpha. Thus, G203T-mutated Gi2alpha had a unique property in terms of coupling to membrane receptors, in addition to the previously expected defect in the active conformation of the GTP-bound form of Gi2alpha.
Inoue
S
S
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
Hoshino
S
S
Kukimoto
I
I
Ui
M
M
Katada
T
T
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D018411:Spodoptera / Q000821:virology; D014357:Trypsin / Q000494:pharmacology
D018076:DNA, Complementary; D046911:Macromolecular Substances; D018341:Receptors, Adrenergic, alpha-2; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D014357:Trypsin; D019204:GTP-Binding Proteins