Purification and Characterization of the G203T Mutant α_i-2 Subunit of GTP-Binding Protein Expressed in Baculovirus-Infected Sf9 Cells

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We expressed the Gly203→Thr (G203T) mutant of G_i2α, which was expected to show a dominant-negative phenotype in G_i2-mediated signal transduction, in baculovirus-infected Sf9 cells and purified the mutant α subunit for its characterization. The rate of dissociation of GDP from G203T G_i2α was 3- to 4-fold faster than that from wild-type G_i2α, but their k_cat values for GTP hydrolysis were almost the same. The affinities of the two G_i2α proteins for the βγ subunits of G proteins to form αβγ trimers, which served as substrates for pertussis toxin-catalyzed ADP-ribosylation, were the same. In marked contrast, G203T G_i2α was unable to form a tight complex with a non-hydrolyzable analog (GTP[γS]) of GTP; bound GTP[γS] was readily released from the mutant G_i2α even in the presence of a high concentration of Mg²⁺. Its susceptibility to tryptic digestion also revealed that GTP [γS]-bound G203T G_i2α formed a conformation apparently different from that of the GTP[γS]-bound form of wild-type G_i2α. Both the G203T and wild-type G_i2α proteins were capable of coupling with membrane-bound α₂-adrenergic receptors, resulting in the formation of receptor-G protein complexes with high affinity for agonists. However, GTP [γS] -dependent uncoupling from the high-affinity receptors was markedly attenuated in the case of G203T G_i2α. Thus, G203T-mutated G_i2α 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 G_i2α.