A new approach to producing functional G alpha subunits yields the activated and deactivated structures of G alpha(12/13) proteins.

Abstract

The oncogenic G(12/13) subfamily of heterotrimeric G proteins transduces extracellular signals that regulate the actin cytoskeleton, cell cycle progression, and gene transcription. Previously, structural analyses of fully functional G alpha(12/13) subunits have been hindered by insufficient amounts of homogeneous, functional protein. Herein, we report that substitution of the N-terminal helix of G alpha(i1) for the corresponding region of G alpha12 or G alpha13 generated soluble chimeric subunits (G alpha(i/12) and G alpha(i/13)) that could be purified in sufficient amounts for crystallographic studies. Each chimera bound guanine nucleotides, G betagamma subunits, and effector proteins and exhibited GAP responses to p115RhoGEF and leukemia-associated RhoGEF. Like their wild-type counterparts, G alpha(i/13), but not G alpha(i/12), stimulated the activity of p115RhoGEF. Crystal structures of the G alpha(i/12) x GDP x AlF4(-) and G alpha(i/13) x GDP complexes were determined using diffraction data extending to 2.9 and 2.0 A, respectively. These structures reveal not only the native structural features of G alpha12 and G alpha13 subunits, which are expected to be important for their interactions with GPCRs and effectors such as G alpha-regulated RhoGEFs, but also novel conformational changes that are likely coupled to GTP hydrolysis in the G alpha(12/13) class of heterotrimeric G proteins.

• Authors: Barry Kreutz, Douglas M Yau, Mark R Nance, Shihori Tanabe, John J G Tesmer, Tohru Kozasa
• Journal: Biochemistry (Biochemistry) Vol. 45 Issue 1 Pg. 167-74 (Jan 10 2006) ISSN: 0006-2960 [Print] United States
• PMID: 16388592 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• G-protein Beta gamma
• GTP-Binding Protein beta Subunits
• GTP-Binding Protein gamma Subunits
• GTPase-Activating Proteins
• Guanine Nucleotide Exchange Factors
• Guanine Nucleotides
• Proto-Oncogene Proteins
• Receptors, G-Protein-Coupled
• Rho Guanine Nucleotide Exchange Factors
• GTP-Binding Protein alpha Subunits, G12-G13

Topics

• Animals
• Binding Sites
• Cells, Cultured
• Chimera (genetics, metabolism)
• Crystallography, X-Ray
• GTP-Binding Protein alpha Subunits, G12-G13 (chemistry, genetics, isolation & purification, metabolism)
• GTP-Binding Protein beta Subunits (chemistry, genetics, metabolism)
• GTP-Binding Protein gamma Subunits (chemistry, genetics, metabolism)
• GTPase-Activating Proteins (chemistry, genetics, metabolism)
• Guanine Nucleotide Exchange Factors (genetics, metabolism)
• Guanine Nucleotides (chemistry, genetics, metabolism)
• Leukemia (metabolism)
• Protein Conformation
• Proto-Oncogene Proteins (chemistry, genetics, metabolism)
• Receptors, G-Protein-Coupled (genetics, metabolism)
• Rho Guanine Nucleotide Exchange Factors