G Protein betagamma dimer formation: Gbeta and Ggamma differentially determine efficiency of in vitro dimer formation
ABSTRACT 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.
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- "It has become increasingly clear in other systems, such as the cardiovascular and taste systems that following receptor activation, the Gβγ dimer dissociates from the Gα subunit and modulates a set of downstream components that are different from those activated by Gα (Dingus et al., 2005; Smrcka, 2008). For example, in mammalian taste receptor cells, upon tastant binding to the taste receptor, the Gβγ subunit dissociates from the α subunit gustducin (Gαgust) and activates phospholipase C β2 (PLC β2) (Huang et al., 1999) which in turn activates the DAG/IP3 pathway and transient receptor potential channel M5 (TRPM5) (Oike et al., 2006; Zhang et al., 2007). "
ABSTRACT: Heterotrimeric G-proteins mediate a variety of cellular functions, including signal transduction in sensory neurons of the olfactory system. Whereas the Gα subunits in these neurons are well characterized, the gene transcript expression profile of Gβγ subunits is largely missing. Here we report our comprehensive expression analysis to identify Gβ and Gγ subunit gene transcripts in the mouse main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Our reverse transcriptase PCR (RT-PCR) and realtime qPCR analyses of all known Gβ (β1,2,3,4,5) and Gγ (γ1,2,2t,3,4,5,7,8,10,11,12,13) subunits indicate presence of multiple Gβ and Gγ subunit gene transcripts in the MOE and the VNO at various expression levels. These results are supported by our RNA in situ hybridization (RISH) experiments, which reveal the expression patterns of two Gβ subunits and four Gγ subunits in the MOE as well as one Gβ and four Gγ subunits in the VNO. Using double-probe fluorescence RISH and line intensity scan analysis of the RISH signals of two dominant Gβγ subunits, we show that Gγ13 is expressed in mature olfactory sensory neurons (OSNs), while Gβ1 is present in both mature and immature OSNs. Interestingly, we also found Gβ1 to be the dominant Gβ subunit in the VNO and present throughout the sensory epithelium. In contrast, we found diverse expression of Gγ subunit gene transcripts with Gγ2, Gγ3, and Gγ13 in the Gαi2-expressing neuronal population, while Gγ8 is expressed in both layers. Further, we determined the expression of these Gβγ gene transcripts in three post-natal developmental stages (p0, 7, and 14) and found their cell-type specific expression remains largely unchanged, except the transient expression of Gγ2 in a single basal layer of cells in the MOE during P7 and P14. Taken together, our comprehensive expression analyses reveal cell-type specific gene expression of multiple Gβ and Gγ in sensory neurons of the olfactory system.Frontiers in Cellular Neuroscience 06/2013; 7:84. DOI:10.3389/fncel.2013.00084 · 4.18 Impact Factor
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- "Similar differences in the interaction between mammalian Gβ and Gγ proteins have also been observed. The human Gβ1-4 share 80–90% sequence identity; however, Gβ1 in general interacts with multiple Gγ isoforms, Gβ2 is more restricted in its interaction partners and Gβ3 displays significantly weaker interactions –. In most cases, however, the interaction data were based on in vitro assays and its relevance in the context of a specific cell type or a signal remains to be evaluated in both mammalian and plant systems. "
ABSTRACT: Heterotrimeric G-proteins comprised of Gα, Gβ and Gγ proteins are important signal transducers in all eukaryotes. The Gγ protein of the G-protein heterotrimer is crucial for its proper targeting at the plasma membrane and correct functioning. Gγ proteins are significantly smaller and more diverse than the Gα and Gβ proteins. In model plants Arabidopsis and rice that have a single Gα and Gβ protein, the presence of two canonical Gγ proteins provide some diversity to the possible heterotrimeric combinations. Our recent analysis of the latest version of the soybean genome has identified ten Gγ proteins which belong to three distinct families based on their C-termini. We amplified the full length cDNAs, analyzed their detailed expression profile by quantitative PCR, assessed their localization and performed yeast-based interaction analysis to evaluate interaction specificity with different Gβ proteins. Our results show that ten Gγ genes are retained in the soybean genome and have interesting expression profiles across different developmental stages. Six of the newly identified proteins belong to two plant-specific Gγ protein families. Yeast-based interaction analyses predict some degree of interaction specificity between different Gβ and Gγ proteins. This research thus identifies a highly diverse G-protein network from a plant species. Homologs of these novel proteins have been previously identified as QTLs for grain size and yield in rice.PLoS ONE 08/2011; 6(8):e23361. DOI:10.1371/journal.pone.0023361 · 3.23 Impact Factor
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ABSTRACT: The specificity of G protein betagamma signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of betagamma function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven beta1gamma complexes containing gamma1, gamma2, gamma5, gamma7, gamma10, gamma11, or gamma12 to interact with alphas and of these gamma subunits to compete for interaction with beta1 in live human embryonic kidney (HEK) 293 cells. betagamma complexes were imaged using bimolecular fluorescence complementation, in which fluorescence is produced by two nonfluorescent fragments (N and C) of cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) when brought together by proteins fused to each fragment. Plasma membrane targeting of alphas-CFP varied inversely with its expression level, and the abilities of YFP-N-beta1YFP-C-gamma complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-gamma subunits to compete for association with CFP-C-beta1. When the intensities of coexpressed CFP-C-beta1CFP-N-gamma (cyan) and CFP-C-beta1YFP-N-gamma2 (yellow) complexes were compared under conditions in which CFP-C-beta1 was limiting, the CFP-N-gamma subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-gamma2 for association with CFP-C-beta1. CFP-N-gamma12 and CFP-N-gamma1 were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for betagamma in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed beta and gamma subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.Molecular Pharmacology 08/2006; 70(1):194-205. DOI:10.1124/mol.106.022616 · 4.12 Impact Factor