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

Ser54 of Gsα binds guanine nucleotide and Mg2+ 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 αi/o, this results from persistent binding of α to ßγ, whereas for small GTP binding proteins and αs this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of ßγ interactions in mediating the properties of the Ser54-like mutants of Gα subunits. Unexpectedly, WT-αs or N54-αs coexpressed with α1B-adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT-αs was more effective than the mutant. One explanation for this result would be that αs, like Ser47 αi/o, blocks receptor activation by sequestering ßγ; implying that N54-αS has reduced affinity for ßγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT-αs was more effective than the mutant in inhibiting ßγ activation of phospholipase Cß2. Further, in vitro synthesized N54-αs bound biotinylated-ßγ with lower apparent affinity than did WT-αs The Cys54 mutation also decreased ßγ binding but less effectively than N54-αs Substitution of the conserved Ser in αo with Cys or Asn increased ßγ binding, with the Cys mutant being more effective. This suggests that Ser54 of αs is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins.

Synthesis and assembly of G protein ßγ 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 Gßγ 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. The assembly of the Gßγ dimer is a complex process involving multiple accessory proteins and organelles. The mechanisms involved are becoming increasingly appreciated, but are still incompletely understood. In vitro and in vivo (cellular) studies provide different perspectives of these processes, and a comparison of them can provide insight into both our current level of understanding and directions to be taken in future investigations.

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). Different FLAG-Gbeta isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gbeta subunits to efficiently form dimers with Ggamma. When translated Ggamma was added to translated Gbeta, a new band of low apparent molecular mass (approximately 50 kDa) was observed, which was labeled by either (35)S-labeled Gbeta or Ggamma, indicating that it is a dimer. Formation of the Gbetagamma dimer was ATP-dependent and inhibited by either adenosine 5'-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg(2+). This inhibition led to increased association of Gbeta with CCT/TRiC. Although Ggamma did not bind CCT/TRiC, addition of Ggamma to previously synthesized Gbeta caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gbeta subunits and that CCT/TRiC mediates Gbetagamma dimer formation by an ATP-dependent reaction.

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. Significant variation in processing was found at both the amino termini and particularly the carboxyl termini of the proteins. All Ggamma isoforms contain a carboxyl-terminal CAAX motif for prenylation, carboxyl-terminal proteolysis, and carboxymethylation. Characterization of these proteins indicates significant variability in the normal processing of all of these steps in the prenylation reaction, including a new variation of prenyl processing resulting from cysteinylation of the carboxyl terminus. These results have multiple implications for intracellular signaling mechanisms by G proteins, for the role of prenyl processing variation in cell signaling, and for the site of action and consequences of drugs that target the prenylation modification.

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. The translation products were combined and dimers isolated by immunoprecipitation. Gbeta1 and Gbeta4 formed dimers with all Ggamma subunit isoforms, generally with Gbeta/Ggamma stoichiometries between 0.2:1 and 0.5:1. Gbeta5, Gbeta5L, and Gbeta3s did not form significant amounts of dimer with any of the gamma subunit isoforms. Gbeta2 and Gbeta3 formed dimers with selected Ggamma isoforms to levels intermediate between that of Gbeta1/Gbeta4 and Gbeta3s/Gbeta5/Gbeta5L. We also expressed selected Gbetagamma in HEK293 cells and measured PLCbeta2 activity. Gbetagamma dimer-dependent increases in IP3 production were seen with most Gbeta1, Gbeta2, and Gbeta5 combinations, indicating functional dimer expression in intact cells. These results define the complete set of G protein betagamma dimers that are formed using a single biochemical assay method and suggest that there are Gbeta isoform-specific factors in rabbit reticulocyte lysates that determine the efficacy of Gbetagamma dimer formation.