The N54- Mutant Has Decreased Affinity for and Suggests a Mechanism for Coupling Heterotrimeric G Protein Nucleotide Exchange with Subunit Dissociation.

Ser54 of G binds guanine nucleotide and Mg as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For , this results from persistent binding of to , whereas for small GTP binding proteins and this results from persistent binding to guanine nucleotide exchange factor or receptor.

Synthesis and assembly of G protein βγ dimers: comparison of in vitro and in vivo studies.

The heterotrimeric GTP-binding proteins (G proteins) are the canonical cellular machinery used with the approximately 700 G protein-coupled receptors (GPCRs) in the human genome to transduce extracellular signals across the plasma membrane. The synthesis of the constituent G protein subunits, and their assembly into Gβγ dimers and G protein heterotrimers, determines the signaling repertoire for G-protein/GPCR signaling in cells. These synthesis/assembly -processes are intimately related to two other overlapping events in the intricate pathway leading to formation of G protein signaling complexes, posttranslational modification and intracellular trafficking of G proteins.

Role of the chaperonin CCT/TRiC complex in G protein betagamma-dimer assembly.

Gbetagamma dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, (35)S-labeled Ggamma subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, (35)S-labeled Gbeta subunits traveled at a high apparent molecular mass (approximately 700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC).

Proteomic analysis of bovine brain G protein gamma subunit processing heterogeneity.

We characterized the variable processing of the G protein gamma subunit isoforms associated with bovine brain G proteins, a primary mediator of cellular communication. Ggamma subunits were isolated from purified brain G proteins and characterized by Edman sequencing, by MALDI MS, by chemical and/or enzymatic fragmentation assayed by MALDI MS, and by MS/MS fragmentation and sequencing. Multiple forms of six different Ggamma isoforms were detected.

G Protein betagamma dimer formation: Gbeta and Ggamma differentially determine efficiency of in vitro dimer formation.

The Gbeta and Ggamma subunit of the heterotrimeric G proteins form a functional dimer that is stable once assembled in vivo or in vitro. The requirements, mechanism, and specificity of dimer formation are still incompletely understood, but represent important biochemical processes involved in the specificity of cellular signaling through G proteins. Here, seven Gbeta and 12 FLAG-epitope-tagged Ggamma subunits were separately synthesized in vitro using a rabbit reticulocyte lysate expression system.