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J. Biochem, 1995, Vol. 118, No. 3 650-657
© 1995 Japanese Biochemical Society

other

Purification and Characterization of the G203T Mutant{alpha}1-2 Subunit of GTP-Binding Protein Expressed in Baculovirus-Infected Sf9 Cells1

Shin-ichi Inoue, Shinichi Hoshino, Iwao Kukimoto, Michio Ui2 and Toshiaki Katada

Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113

2Present address: Ui Laboratory, Institute of Physical and Chemical Research, 2-1 Hirosawa, Wako, Saitama 351-01.

We expressed the Gly203{uparrow}Thr (G203T) mutant of G12{alpha} which was expected to show a dominant-negative phenotype in G12-mediated signal transduction, in baculovirus-infected Sf9 cells and purified the mutant {alpha} subunit for its characterization. The rate of dissociation of GDP from G203T G12{alpha} was 3-to4-fold faster than that from wild-type G12{alpha}but their kcat values for GTP hydrolysis were almost the same. The affinities of the two G12{alpha} proteins for the ß{gamma}subunits of G proteins to form {alpha}ß{gamma} trimers, which served as substrates for pertussis toxin-catalyzed ADP-ribosylation, were the same. In marked contrast, G203T G12{alpha} was unable to form a tight complex with a non-hydrolyzable analog (GTP[{gamma}S]) of GTP; bound GTP[{gamma}S] was readily released from the mutant G12{alpha} even in the presence of a high concentration of Mg2+ Its susceptibility to tryptic digestion also revealed that GTP[{gamma}S]-bound G203T G12{alpha} formed a conformation apparently different from that of the GTP[{gamma}S]-bound form of wild-type G12{alpha} Both the G203T and wild-type G12{alpha} proteins were capable of coupling with membrane-bound {alpha}2-adrenergic receptors, resulting in the formation of receptor-G protein complexes with high affinity for agonists. However, GTP[{gamma}S]-dependent uncoupling from the high-affinity receptors was markedly attenuated in the case of G203T G12{alpha}Thus, G203T-mutated G12{alpha} 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 G12{alpha}.

1This work was supported in part by research grants from the Scientific Research Funds of the Ministry of Education, Science and Culture of Japan.


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