Specificities of Gßγ subunits for the SNARE complex before and after stimulation of α2a-adrenergic receptors.

Ligand binding to G protein­coupled receptors (GPCRs), such as the α2a-adrenergic receptor (α2aAR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gßγ dimers. α2aAR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble NSF attachment protein receptor (SNARE) complex, a Gßγ effector. To understand how the Gßγ-SNARE complex underlies the α2aAR-dependent inhibition of synaptic transmission, we examined the specificity of Gßγ subunits for the SNARE complex in adrenergic neurons, in which auto-α2aARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-α2aARs respond to epinephrine released from other neurons. We performed a quantitative, targeted multiple reaction monitoring proteomic analysis of Gß and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α2aARs, Gß1 and Gγ3 interacted with the SNARE complex. However, Gß1, Gß2, and Gγ3 were found in the complex when auto-α2aARs were activated by epinephrine. Further understanding of the specific usage of distinct Gßγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases.

Author Correction: The in vivo specificity of synaptic Gß and Gγ subunits to the α2a adrenergic receptor at CNS synapses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Disabling the Gßγ-SNARE interaction disrupts GPCR-mediated presynaptic inhibition, leading to physiological and behavioral phenotypes.

G protein-coupled receptors (GPCRs) that couple to Gi/o proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gßγ subunits from activated G proteins decreases the activity of voltage-gated Ca2+ channels (VGCCs), decreasing excitability. A less understood Gßγ-mediated mechanism downstream of Ca2+ entry is the binding of Gßγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABAB receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT1b receptors and adrenergic α2a receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by Gi/o-coupled GPCRs through the Gßγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.

The in vivo specificity of synaptic Gß and Gγ subunits to the α2a adrenergic receptor at CNS synapses.

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, ß, and γ subunits, with 16 Gα, 5 Gß and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gßγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gßγ specificity of presynaptic α2a-adrenergic receptors (α2aARs) in both adrenergic (auto-α2aARs) and non-adrenergic neurons (hetero-α2aARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gß and Gγ subunits, and co-immunoprecipitation of tagged α2aARs from mouse models including transgenic FLAG-α2aARs and knock-in HA-α2aARs, we investigated the in vivo specificity of Gß and Gγ subunits to auto-α2aARs and hetero-α2aARs activated with epinephrine to understand the role of Gßγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gß2, Gγ2, Gγ3, and Gγ4 with activated auto α2aARs, whereas we found Gß4 and Gγ12 preferentially interacted with activated hetero-α2aARs. Further understanding of in vivo Gßγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gß and Gγ, and the mechanisms underlying GPCR signaling through Gßγ subunits.

Muscle-specific stress fibers give rise to sarcomeres in cardiomyocytes.

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 µm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.

Quantitative Multiple-Reaction Monitoring Proteomic Analysis of Gß and Gγ Subunits in C57Bl6/J Brain Synaptosomes.

Gßγ dimers are one of the essential signaling units of activated G protein-coupled receptors (GPCRs). There are five Gß and 12 Gγ subunits in humans; numerous studies have demonstrated that different Gß and Gγ subunits selectively interact to form unique Gßγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gßγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple-reaction monitoring (MRM) studies of Gß and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified or compared their individual protein levels. In this study, we have developed a quantitative MRM method not only to quantify but also to compare the protein abundance of neuronal Gß and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gß and Gγ subunits and their subcellular localizations. For example, Gß1 was mostly localized at the membrane while Gß2 was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gß and Gγ subunits may affect the Gßγ dimerization and Gßγ-effector interactions. This study offers not only a new tool for quantifying and comparing Gß and Gγ subunits but also new insights into the in vivo distribution of Gß and Gγ subunits, and Gßγ dimer assembly in normal brain function.

Gßγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.

Gi/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gßγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gßγ subunits with the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gßγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gßγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gßγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gßγ for SNARE-binding sites in lipid environments. Mutant Gßγ subunits that were previously shown to be more efficacious at inhibiting Ca2+-triggered exocytotic release than wild-type Gßγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gßγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gß and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gßγ inhibited Ca2+/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gßγ-SNARE interaction and show that the target of Gßγ, downstream of VGCC, is the membrane-embedded SNARE complex.

Gßγ Binds to the Extreme C Terminus of SNAP25 to Mediate the Action of Gi/o-Coupled G Protein-Coupled Receptors.

Gi/o-coupled G protein-coupled receptors can exert an inhibitory effect on vesicle release through several G protein-driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. The synaptosomal-associated protein of 25 kDa (SNAP25) is a key downstream effector of Gßγ subunits. It has previously been shown that proteolytic cleavage of SNAP25 by botulinum toxin A reduces the ability of Gßγ to compete with the calcium sensor synaptotagmin 1 (Syt1) for binding to SNAP25 in a calcium-dependent manner. These truncated SNAP25 proteins sustain a low level of exocytosis but are unable to support serotonin-mediated inhibition of exocytosis in lamprey spinal neurons. Here, we generate a SNAP25 extreme C-terminal mutant that is deficient in its ability to bind Gßγ while retaining normal calcium-dependent Syt1 binding to soluble N-ethylmaleimide attachment protein receptor (SNARE) and vesicle release. The SNAP25Δ3 mutant, in which residue G204 is replaced by a stop codon, features a partial reduction in Gß1γ2 binding in vitro as well as a partial reduction in the ability of the lamprey 5-hydroxytryptamine1b-type serotonin receptor to reduce excitatory postsynaptic current amplitudes, an effect previously shown to be mediated through the interaction of Gßγ with SNAP25. Syt1 calcium-dependent binding to SNAP25Δ3 was reduced by a small extent compared with the wild type. We conclude that the extreme C terminus of SNAP25 is a critical region for the Gßγ-SNARE interaction.

Differential localization of G protein ßγ subunits.

G protein ßγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gß and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gß and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gß and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gß and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.

Gßγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.

Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein ßγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gßγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gßγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gßγ binding. Peptides that bound Gßγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gßγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gßγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gß1γ1. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gßγ, but it still interacted with synaptotagmin-1 in a Ca²âº -dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.

Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the mu- and delta-opioid receptors regulate opioid receptor signaling.

To define receptor subdomains important for protein interaction and identify components of novel signal transduction complexes for the mu- and delta-opioid receptors (mu-OR, delta-OR), we generated glutathione S-transferase fusion proteins of the carboxyl-termini of the mu-opioid receptor (mu-CT), the delta- (delta-CT), and the third intracellular loop of the delta-opioid receptor (delta-i3L) to search for interactive proteins. Results from pull down experiments have demonstrated for the first time that Gbetagamma complexes, derived from the heterotrimeric Galphatbeta1gamma1, purified Gbeta1gamma1, or Gbeta endogenously present in cell lysates and rat striatal extracts, interact with all mu- and delta-opioid receptor subdomains. On the other hand, the C-terminal peptides of the delta- and the mu-ORs exhibit differential profiles for Galpha subunit binding. Indeed, while mu-CT was unable to bind any form of Galpha, both the delta-CT and the delta-i3L displayed interactive regions for heterotrimeric Galphatbeta1gamma1, inactive Galpha(GDP) and active Galpha(GTPgammaS). Regulators of G protein signaling (RGS) proteins are another class of proteins that can modulate G protein signaling events. We demonstrate for the first time that RGS4 directly interacts with the mu-CT, the delta-CT as well as delta-i3L in a dose dependent manner. Moreover, RGS4 modulates mu-OR signaling and can form stable heterotrimeric complexes with the activated Galpha. Collectively, our data demonstrate that the C-termini of the mu- and delta-ORs provide direct physical scaffolding in which G protein subunits and RGS4 protein can be bound.

Novel tumor necrosis factor alpha-regulated genes in rheumatoid arthritis.

OBJECTIVE: To determine novel genes regulated by tumor necrosis factor alpha (TNFalpha) signaling in primary rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Oligonucleotide microarrays were used to measure gene expression levels in 6 independent replicate samples of RASFs. RASFs were transfected for 18 hours with AdIkappaB-dominant negative (AdIkappaB-DN) (n = 3) or with control AdTet expressing the reverse tetracycline trans-activator (n = 3). The cells were stimulated for 3 hours with TNFalpha, and total RNA was prepared. Several novel parametric and nonparametric methods were used to rank genes in terms of the magnitude and significance of intergroup differences. Microarray expression differences were confirmed by real-time quantitative reverse transcription-polymerase chain reaction. Small interfering RNA (siRNA) was used to specifically down-modulate microarray-identified genes to demonstrate their role in the promotion of apoptosis, proliferation, or matrix metalloproteinase (MMP) expression. RESULTS: Blocking of NF-kappaB by AdIkappaB-DN was associated with a down-modulation of antiapoptosis genes, including BIRC-3, and several novel genes, including GG2-1, a TNFalpha-inducible FLIP-like gene. Other families of genes that were significantly down-regulated by AdIkappaB-DN included cytokines/chemokines (interleukin-1beta [IL-1beta], IL-8, IL-15, and RANTES), adhesion molecule (vascular cell adhesion molecule 1, intercellular adhesion molecule 1), and unique genes that have not previously been reported to be regulated by TNFalpha in RA. Inhibition of the GG2-1 gene using the siRNA technique resulted in significantly enhanced apoptosis, decreased proliferation, and decreased production of MMP-1 in TNFalpha-stimulated RASFs. CONCLUSION: These studies provide a comprehensive analysis of genes that are differentially regulated by TNFalpha signaling and NF-kappaB nuclear translocation in RASFs and demonstrate methods for confirming the expression and functional significance of such genes.