Significance of Thr182 in the nucleotide-exchange and GTP-hydrolysis reactions of the alpha subunit of GTP-binding protein Gi2.
The crystal structures of the GTP- and GDP-bound alpha subunits of heterotrimeric GTP-binding proteins were recently determined, and a conserved Thr residue in the G2 (linker 2) region of the alpha subunits, which corresponds to Thr182 in Gi2 alpha, was deduced to interact with the gamma-phosphate of GTP and Mg2+. To investigate biochemically the significance of the Thr residue, we produced a mutant Gi2 alpha, in which Thr182 was substituted for Ala (T182A), in Escherichia coli. The rate of guanosine 5'-(gamma-thio)tri-phosphate (GTP gamma S) binding to T182A was higher than that to the wild-type Gi2 alpha, 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 Gi2 alpha found in human endocrine tumors. The ability of T182A to interact with beta gamma subunits and membrane-bound receptors was the same as that of the wild-type alpha 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 Mg(2+)-sensitive GDP-GTP exchange reaction but also in the GTPase activity of Gi2 alpha. The T182A mutant of Gi2 alpha, 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 beta gamma subunits.
Nishina
H
H
Department of Life Science, Tokyo Institute of Technology, Midori-ku, Yokohama.
Nimota
K
K
Kukimoto
I
I
Maehama
T
T
Takahashi
K
K
Hoshino
S
S
Kanaho
Y
Y
Katada
T
T
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000737:chemistry; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013912:Threonine / Q000737:chemistry*
D009711:Nucleotides; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D013912:Threonine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C498017:Gnai2 protein, rat; D008274:Magnesium; D000409:Alanine