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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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 01/2013; 7:84. · 4.18 Impact Factor
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ABSTRACT: Synaptic transmission is a finely regulated mechanism of neuronal communication. The release of neurotransmitter at the synapse is not only the reflection of membrane depolarization events, but rather, is the summation of interactions between ion channels, G protein coupled receptors, second messengers, and the exocytotic machinery itself which exposes the components within a synaptic vesicle to the synaptic cleft. The focus of this review is to explore the role of G protein signaling as it relates to neurotransmission, as well as to discuss the recently determined inhibitory mechanism of Gβγ dimers acting directly on the exocytotic machinery proteins to inhibit neurotransmitter release.Progress in Neurobiology 01/2012; 96(3):304-21. · 10.30 Impact Factor
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ABSTRACT: A splice variant of Gβ3, termed Gβ3s, has been associated with the C825T polymorphism in the Gβ3 gene and linked with many human disorders. However, the biochemical properties and functionality of Gβ3s remain controversial. Here, using multidisciplinary approaches including co-immunoprecipitation analysis and bioluminescence resonance energy transfer (BRET) measurements, we showed that unlike Gβ3, Gβ3s failed to form complexes with either Gγ or Gα subunits. Moreover, using a mutant Gγ2 deficient in lipid modification to purify Gβ3s from Sf9 cells without the use of detergents, we further showed that the failure of Gβ3s to form dimers with Gγ was not due to the instability of the dimers in detergents, but rather, reflected the intrinsic properties of Gβ3s. Additional studies indicated that Gβ3s is unstable, and unable to localize properly to the plasma membrane and to activate diverse Gβγ effectors including PLCβ2/3, PI3Kγ, ERKs and the Rho guanine exchange factor (RhoGEF) PLEKHG2. Thus, these data suggest that the pathological effects of Gβ3 C825T polymorphism may result from the downregulation of Gβ3 function. However, we found that the chemokine SDF1α transmits signals primarily through Gβ1 and Gβ2, but not Gβ3, to regulate chemotaxis of several human lymphocytic cell lines, indicating the effects of Gβ3 C825T polymorphism are likely to be tissue and/or stimuli specific and its association with various disorders in different tissues should be interpreted with great caution.Cellular Signalling 08/2012; 24(12):2349-59. · 4.47 Impact Factor