Ligand binding to G protein–coupled 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 Gβγ 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 Gβγ effector. To understand how the Gβγ-SNARE complex underlies the α2aAR-dependent inhibition of synaptic transmission, we examined the specificity of Gβγ 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 Gβ and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α2aARs, Gβ1 and Gγ3 interacted with the SNARE complex. However, Gβ1, Gβ2, and Gγ3 were found in the complex when auto-α2aARs were activated by epinephrine. Further understanding of the specific usage of distinct Gβγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases.

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Modulation of neurotransmitter exocytosis by activated Gi/o coupled G‐protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is Gβγ interaction with soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE). There are 5 Gβ and 12 Gγ subunits, but specific Gβγs activated by a given GPCR and the specificity to effectors in vivo are not known. Presynaptic α2a‐adrenergic receptors (α2aARs) in both adrenergic (auto α2aARs) and non‐adrenergic neurons (hetero α2aARs) inhibit neurotransmitter release and affect various physiological functions such as anesthetic sparing and working memory enhancement. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits to detect neuronal Gβ and Gγ subunits, several mouse models including transgenic Flag‐α2aARs, knock‐in HA‐α2aARs, and other biochemical techniques such as co‐immunoprecipitation, we investigate the specificity of Gβ and Gγ subunits to α2aARs in both adrenergic(auto α2aARs) and non‐adrenergic neurons (hetero α2aARs), and SNARE in presence of epinephrine. Gβ2, Gγ2, Gγ3, and Gγ4 preferentially interact with activated auto α2aARs while Gβ4 and Gγ12 preferentially interact with activated hetero α2aARs. We also detect a subset of Gβ and Gγ subunits interacting with SNARE upon α2aARs activation. Further understanding of Gβγ specificity on its downstream signaling, especially Gβγ‐SNARE interaction, offers new insights into the normal functioning of the brain and lead to an identification of potential pathophysiological states in which the Gβγ‐SNARE interaction may be dysregulated. These studies yield additional insights into Gi/o‐coupled GPCR‐mediated regulation of exocytosis.Support or Funding InformationThis work was supported by the National Institutes of Health (EY10291, MH101679, DK109204, and T32 GM07628).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of neurotransmitter exocytosis by activated Gi/o‐type G‐protein coupled receptors (GPCRs) is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. One of the known modulation mechanisms is Gβγ and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) complex interaction. There are 5 Gβ and 12 Gγ subunits, but specific Gβγs activated by a given GPCR in vivo are not known. Presynaptic α 2a ‐adrenergic receptors (α 2a ‐ARs) in both adrenergic (auto α 2a ‐ARs) and non‐adrenergic neurons (hetero α 2a ‐ARs) inhibit neurotransmitter release and affect various physiological function such as anesthetic sparing and working memory enhancement. Here, we investigate whether auto α 2a ‐ARs in sympathetic neurons use the same Gbg subunits as hetero α 2a ‐ARs in other neuronal types to inhibit exocytosis by interacting with SNARE. Using several mice models including transgenic Flag‐α 2a ‐ARs, knock‐in HA‐α 2a ‐ARs, co‐immunoprecipitation, mass spectrometry analysis, we have determined the Gb and Gg subunits that interact with α 2a ‐ARs and SNARE complexes. So far, we find Gβ2 preferentially interacting with activated auto α 2a ‐ARs. We also see a basal Gβγ‐SNARE interaction and the 2 fold enhancement of this interaction upon the auto α 2a ‐ARs activation. Further understanding Gβγ specificity and Gβγ‐SNARE interaction may offer new insights into the normal functioning of the brain, as well as better understanding of disease progression. Support or Funding Information This work was supported by the National Institutes of Health (EY10291, MH101679, DK109204, and T32)

Modulation of neurotransmitter exocytosis by activated Gi/o‐type G‐protein coupled receptors (GPCRs), such as the α2a adrenergic receptor (α2a‐ARs), is a universal regulatory mechanism used both to avoid overstimulation and to influence circuitry. Gβγ and soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) interaction is known as one of mechanisms that inhibit neurotransmitter release. There are 5 Gβ and 12 Gγ subunits, but protein level of each Gβs and Gγs in brain is not known. It is also not known whether specific Gβγs are activated by a given GPCR in vivo. However, presynaptic α2a‐ARs in both adrenergic (auto α2a‐ARs) and non‐adrenergic neurons (hetero α2a‐ARs) work in a similar manner to inhibit neurotransmitter release and have various physiological functions such as anesthetic sparing and working memory enhancement. In this project, we examine the protein level of each Gβ and Gγ in whole brain synaptosomes and in fractions of synaptosomes. In whole brain synaptosome, we have identified Gβ1 and Gγ2 as dominant subunits present. Although all Gβs and Gγs are highly present in cytosolic fractions, Gγs show interesting changes in protein expression patterns by fractions. We also investigate whether autoreceptors in sympathetic neurons use the same Gβγ subunits as heteroreceptors in other neuronal types. Using several mice models including transgenic Flag‐α2a‐ARs, knock‐in HA‐α2a‐ARs, and wild‐type mice, co‐immunoprecipitation, mass spectrometry analysis, we test our hypothesize that specific Gβγ subunits interact with activated auto‐ and hetero α2a‐ARs and inhibit exocytosis by interacting with SNARE. We have identified Gβ1, Gβ2, Gβ4, Gβ5, Gγ2, Gγ3, Gγ4, Gγ7, Gγ12, and Gγ13 which interact with α2a‐ARs and SNARE. Out of these G proteins, we found Gβ2 and Gγ13 preferentially interacting with activated auto α2a‐ARs. However, we found no difference in Gβγ specificity to SNARE following α2a‐ARs activation. Further understanding Gβγ specificity may offer new insights into the normal functioning of the brain, as well as better understanding of disease progression. The Gβγ‐SNARE interaction may be a new therapeutic target to modulate exocytosis in neural disorders in combination with drugs targeted to Gi/o‐type GPCRs.Support or Funding InformationThis work was supported by the National Institutes of Health (EY10291, MH101679, and T32).

Evaluating sites of protein-protein interactions can be an arduous task involving extensive mutagenesis work and attempts to express and purify individual proteins in sufficient quantities. Peptide mapping is a useful alternative to traditional methods as it allows rapid detection of regions and/or individual residues important for binding, and it can be readily applied to numerous proteins at once. Here we describe the use of the ResPep SL SPOT method to evaluate protein-protein binding interactions such as that between G-protein βγ subunits and SNARE proteins, identifying both regions of interest and subsequently individual residues which can then be manipulated in further biochemical assays to confirm their validity.

It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium‐dependent vitamin C transporter‐2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter‐2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2‐mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft. © 2015 Wiley Periodicals, Inc.

G protein βγ 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 Gβ 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 Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ 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 Gβ 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.

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.

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 Gβγ 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 Gβγ-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 Gβγ-SNARE interaction.

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 βγ 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. Gβγ 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 Gβγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gβγ binding. Peptides that bound Gβγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gβγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gβγ 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 Gβ1γ1. 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 Gβγ, but it still interacted with synaptotagmin-1 in a Ca2+-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.