Jane Dingus

Role of the Chaperonin CCT/TRiC Complex in G Protein βγ-Dimer Assembly

Gβγ dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, 35S-labeled Gγ subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, 35S-labeled Gβ subunits traveled at a high apparent molecular mass (∼700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gβ isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gβ subunits to efficiently form dimers with Gγ. When translated Gγ was added to translated Gβ, a new band of low apparent molecular mass (∼50 kDa) was observed, which was labeled by either 35S-labeled Gβ or Gγ, indicating that it is a dimer. Formation of the Gβγ dimer was ATP-dependent and inhibited by either adenosine 5′-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg2+. This inhibition led to increased association of Gβ with CCT/TRiC. Although Gγ did not bind CCT/TRiC, addition of Gγ to previously synthesized Gβ caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gβ subunits and that CCT/TRiC mediates Gβγ dimer formation by an ATP-dependent reaction.

Synthesis and use of biotinylated beta gamma complexes prepared from bovine brain G proteins.

Publisher Summary This chapter describes a method developed for directly studying the association and interaction of the α subunits with the βγ complex. Modified βγ is immobilized on agarose beads, which allows a straightforward binding assay to be performed with α subunits. Biotinylated βγ is prepared by treating intact bovine brain G protein with NHS-biotin, activating with AlF 4 - , and separating the subunits on ω -aminooctyl-agarose column. The b βγ is immobilized on streptavidin–agarose. This allows the association of the various α subtypes with βγ to be studied, and it permits the investigation of factors affecting the interaction of the subunits with one another. Because intact G protein is biotinylated, the binding site(s) on βγ for α is protected from modification. Although the α subunit is heavily biotinylated with this procedure, βγ is minimally modified and appears fully functional. The b βγ is further purified by anion-exchange chromatography. The b βγ is further purified by anion-exchange chromatography. This highly purified, biotinylated βγ appears to maintain all the functional properties of unmodified βγ .

Proteomic Analysis of Bovine Brain G Protein γ Subunit Processing Heterogeneity

We characterized the variable processing of the G protein γ subunit isoforms associated with bovine brain G proteins, a primary mediator of cellular communication. Gγ 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 Gγ isoforms were detected. Significant variation in processing was found at both the amino termini and particularly the carboxyl termini of the proteins. All Gγ 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.

Identification of a region in G protein γ subunits conserved across species but hypervariable among subunit isoforms

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: α, β, and γ. There are 12 known mammalian γ subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain γ10 protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N‐terminus. Comparison of γ isoform subunit sequences indicated that they vary substantially more at the N‐terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 γ subunit sequences from four different species shows that the N‐terminus of a given γ subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N‐terminus of γ is a functionally important part of the protein exhibiting substantial isoform‐specific variation.

Characterization of the Major Bovine Brain Go α Isoforms MAPPING THE STRUCTURAL DIFFERENCES BETWEEN THE α SUBUNIT ISOFORMS IDENTIFIES A VARIABLE REGION OF THE PROTEIN INVOLVED IN RECEPTOR INTERACTIONS

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas αoA and αoB arise from a single Goα gene as alternatively spliced mRNAs, αoA and αoC are thought to differ by covalent modification. To test the hypothesis that αoA and αoC have different N-terminal lipid modifications, proteolytic fragments of αo isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three αoisoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between αoAand αoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 m urea. Based upon the αosubunit fragments produced under activating and nonactivating conditions, differences between αoA and αoCwere localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two αo proteins. Finally, the structural difference between αoA and αoCis associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.

The Relationship of Goα Subunit Deamidation to the Tissue Distribution, Nucleotide Binding Properties, and βγ Dimer Interactions of Goα Subunit Isoforms

Abstract : The distribution and properties in brain of the α subunits of the major bovine brain Go isoforms, GoA, GoB and GoC, were characterized. The αoA and αoB isoforms arise from alternative splicing of RNAs from a single αo gene, whereas αoC is a deamidated form of αoA. All three Go isoforms purify from brain with different populations of βγ dimers. This variable subunit composition of Go heterotrimers is likely a consequence of their functional differences. This study examined the biochemical properties of the αo isoforms to see if these properties explain the variable βγ composition of their heterotrimers. The brain distribution of αoB differed substantially from that of αoA and αoC, as did its guanine nucleotide binding properties. The unique subunit composition of GoB can be explained by its expression in different brain regions. The αoA and αoC showed slight differences in guanine nucleotide binding properties but no preference for particular βγ dimers when reassociated with a heterogeneous βγ pool. The αoC protein occurred in a constant ratio to αoA throughtout the brain, but was a much larger percent of total brain αo than previously thought, ~35%. These results suggest that αoA is a precursor of αoC and that the association of Goα subunits with different βγ dimers reflects the function of an adaptive, G‐protein signaling mechanism in brain.

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. The assembly of the Gβγ 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.

The N54-αs mutant has decreased affinity for βγ 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 βγ, 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 βγ 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 βγ; implying that N54-αS has reduced affinity for βγ 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 βγ activation of phospholipase Cβ2. Further, in vitro synthesized N54-αs bound biotinylated-βγ with lower apparent affinity than did WT-αs. The Cys54 mutation also decreased βγ binding but less effectively than N54-αs. Substitution of the conserved Ser in αo with Cys or Asn increased βγ 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.