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.

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.

Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex.

G(i/o)-protein-coupled receptors (GPCRs) ubiquitously inhibit neurotransmission, principally via Gßγ, which acts via a number of possible effectors. GPCR effector specificity has traditionally been attributed to Gα, based on Gα's preferential effector targeting in vitro compared with Gßγ's promiscuous targeting of various effectors. In synapses, however, Gßγ clearly targets unique effectors in a receptor-dependent way to modulate synaptic transmission. It remains unknown whether Gßγ specificity in vivo is due to specific Gßγ isoform-receptor associations or to spatial separation of distinct Gßγ pathways through macromolecular interactions. We thus sought to determine how Gßγ signaling pathways within axons remain distinct from one another. In rat hippocampal CA1 axons, GABA(B) receptors (GABA(B)Rs) inhibit presynaptic Ca(2+) entry, and we have now demonstrated that 5-HT(1B) receptors (5-HT(1B)Rs) liberate Gßγ to interact with SNARE complex C terminals with no effect on Ca(2+) entry. Both GABA(B)Rs and 5-HT(1B)Rs inhibit Ca(2+)-evoked neurotransmitter release, but 5-HT(1B)Rs have no effect on Sr(2+)-evoked release. Sr(2+), unlike Ca(2+), does not cause synaptotagmin to compete with Gßγ binding to SNARE complexes. 5-HT(1B)Rs also fail to inhibit release following cleavage of the C terminus of the SNARE complex protein SNAP-25 with botulinum A toxin. Thus, GABA(B)Rs and 5-HT(1B)Rs both localize to presynaptic terminals, but target distinct effectors. We demonstrate that disruption of SNARE complexes and vesicle priming with botulinum C toxin eliminates this selectivity, allowing 5-HT(1B)R inhibition of Ca(2+) entry. We conclude that receptor-effector specificity requires a microarchitecture provided by the SNARE complex during vesicle priming.

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.

Docking of protein models.

An adequate description of entire genomes has to include information on the three-dimensional (3D) structure of proteins. Most of these protein structures will be determined by high-throughput modeling procedures. Thus, a structure-based analysis of the network of protein-protein interactions in genomes requires docking methodologies that are capable of dealing with significant structural inaccuracies in the modeled structures of proteins. We present a systematic study of the applicability of our low-resolution docking method to protein models of different accuracies. A representative nonredundant set of 475 cocrystallized protein-protein complexes was used to build an array of models of each protein in the set. A sophisticated procedure was created to generate the models with RMS deviations of 1, 2, 3,., 10 A from the crystal structure. The docking was performed for all the models, and the predictions were compared with the configurations of the original cocrystallized complexes. Statistical analysis showed that the low-resolution docking can determine the gross structural features of protein-protein interactions for a significant percent of complexes of highly inaccurate protein models. Such predictions may serve as starting points for a more detailed structural analysis, as well as complement experimental and computational data on protein-protein interactions obtained by other techniques.

GPCR mediated regulation of synaptic transmission.

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 Gßγ dimers acting directly on the exocytotic machinery proteins to inhibit neurotransmitter release.

Label-free detection of G protein-SNARE interactions and screening for small molecule modulators.

G(i/o)-coupled presynaptic GPCRs are major targets in neuropsychiatric diseases. For example, presynaptic auto- or heteroreceptors include the D(2) dopamine receptor, H(3) histamine receptor, 5HT(1) serotonin receptors, M(4) acetylcholine receptors, GABA(B) receptors, Class II and III metabotropic glutamate receptors, opioid receptors, as well as many other receptors. These GPCRs exert their influence by decreasing exocytosis of synaptic vesicles. One mechanism by which they act is through direct interaction of the Gßγ subunit with members of the SNARE complex downstream of voltage-dependent calcium channels, and specifically with the C-terminus of SNAP25 and the H3 domain of syntaxin1A(1-3). Small molecule inhibitors of the Gßγ-SNARE interaction would allow the study of the relative importance of this mechanism in more detail. We have utilized novel, label-free technology to detect this protein-protein interaction and screen for several small molecule compounds that perturb the interaction, demonstrating the viability of this approach. Interestingly, the screen also produced enhancers of the Gßγ-SNARE interaction.

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.

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.