Search
Close
Search
Advanced Search
Search Menu
AI Discovery Assistant

The crystal structures of the GTP- and GDP-bound α subunits of heterotrimeric GTP-bind-ing proteins were recently determined, and a conserved Thr residue in the G2 (linker 2) region of the a subunits, which corresponds to Thrl82 in G12α, was deduced to interact with the γ-phosphate of GTP and Mg2+. To investigate biochemically the significance of the Thr residue, we produced a mutant G12α, in which Thr 182 was substituted for Ala (T182A), in Escherichia coli. The rate of guanosine 5‘-(γ-thio)tri-phosphate (GTPγS) binding to T182A was higher than that to the wild-type G12αa, especially with a high concentration (10 mM) of Mg2+. The rate of dissociation of bound GDP from T182A was also much faster than that from the wild-type with the high Mg2+ concentration. Moreover, T182A had much lower GTPase activity than the wild-type, like the gip mutant (R179C) of Gl2α found in human endocrine tumors. The ability of T182A to interact with βγ subunits and membrane-bound receptors was the same as that of the wild-type a subunit. T182A could take on a GTP-bound active conformation, as judged from its sensitivity to tryptic digestion. These results indicated that Thr182 plays an important role not only in the Mg2+-sensitive GDP-GTP exchange reaction but also in the GTPase activity of G12α. The T182A mutant of G12α, characterized by the faster GDP release and the slower GTP hydrolysis, would be a novel mutant that retains the ability to interact with receptors and βγ subunits.

G protein, GDP-GTP exchange, GTPase, Mg2+-binding site
This content is only available as a PDF.
© Oxford University Press
Topic:
You do not currently have access to this article.
Download all slides