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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Binding Sites
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Cells, Cultured
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Chimera / genetics
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Chimera / metabolism
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Crystallography, X-Ray
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GTP-Binding Protein alpha Subunits, G12-G13 / chemistry*
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GTP-Binding Protein alpha Subunits, G12-G13 / genetics
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GTP-Binding Protein alpha Subunits, G12-G13 / isolation & purification
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GTP-Binding Protein alpha Subunits, G12-G13 / metabolism*
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GTP-Binding Protein beta Subunits / chemistry
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GTP-Binding Protein beta Subunits / genetics
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GTP-Binding Protein beta Subunits / metabolism
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GTP-Binding Protein gamma Subunits / chemistry
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GTP-Binding Protein gamma Subunits / genetics
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GTP-Binding Protein gamma Subunits / metabolism
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GTPase-Activating Proteins / chemistry
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GTPase-Activating Proteins / genetics
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GTPase-Activating Proteins / metabolism
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Guanine Nucleotide Exchange Factors / genetics
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Guanine Nucleotide Exchange Factors / metabolism
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Guanine Nucleotides / chemistry
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Guanine Nucleotides / genetics
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Guanine Nucleotides / metabolism
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Leukemia / metabolism
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Protein Conformation
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Proto-Oncogene Proteins / chemistry
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Receptors, G-Protein-Coupled / genetics
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Receptors, G-Protein-Coupled / metabolism
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Rho Guanine Nucleotide Exchange Factors
Substances
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G-protein Beta gamma
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GTP-Binding Protein beta Subunits
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GTP-Binding Protein gamma Subunits
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GTPase-Activating Proteins
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Guanine Nucleotide Exchange Factors
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Guanine Nucleotides
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Proto-Oncogene Proteins
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Receptors, G-Protein-Coupled
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Rho Guanine Nucleotide Exchange Factors
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GTP-Binding Protein alpha Subunits, G12-G13