Interaction Of Gβγ Research Articles

Structure of adenylyl cyclase 5 in complex with Gβγ offers insights into ADCY5-related dyskinesia.

The nine different membrane-anchored adenylyl cyclase isoforms (AC1-9) in mammals are stimulated by the heterotrimeric G protein, Gαs, but their response to Gβγ regulation is isoform specific. In the present study, we report cryo-electron microscope structures of ligand-free AC5 in complex with Gβγ and a dimeric form of AC5 that could be involved in its regulation. Gβγ binds to a coiled-coil domain that links the AC transmembrane region to its catalytic core as well as to a region (C1b) that is known to be a hub for isoform-specific regulation. We confirmed the Gβγ interaction with both purified proteins and cell-based assays. Gain-of-function mutations in AC5 associated with human familial dyskinesia are located at the interface of AC5 with Gβγ and show reduced conditional activation by Gβγ, emphasizing the importance of the observed interaction for motor function in humans. We propose a molecular mechanism wherein Gβγ either prevents dimerization of AC5 or allosterically modulates the coiled-coil domain, and hence the catalytic core. As our mechanistic understanding of how individual AC isoforms are uniquely regulated is limited, studies such as this may provide new avenues for isoform-specific drug development.

Gβγ-SNAP25 exocytotic brake removal enhances insulin action, promotes adipocyte browning, and protects against diet-induced obesity.

Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o GPCRs by Gβγ subunit inhibition of 2 mechanisms: decreased calcium entry and direct interaction of Gβγ with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) that decreased Gβγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact and removing GPCR-Gβγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. Snap25Δ3/Δ3 mice exhibited enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in Snap25Δ3/Δ3 mice challenged with a high-fat diet. Glucose homeostasis, whole-body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in Snap25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting that basal autonomic activity was required. Direct electrode stimulation of sympathetic neuron exocytosis from Snap25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combating metabolic disease.

Multiple potassium channel tetramerization domain (KCTD) family members interact with Gβγ, with effects on cAMP signaling

G protein-coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric G proteins (Gα and Gβγ subunits). Therefore, G protein modifiers are well positioned to shape GPCR pharmacology. A few members of the potassium channel tetramerization domain (KCTD) protein family have been found to adjust G protein signaling through interaction with Gβγ. However, comprehensive details on the KCTD interaction with Gβγ remain unresolved. Here, we report that nearly all the 25 KCTD proteins interact with Gβγ. In this study, we screened Gβγ interaction capacity across the entire KCTD family using two parallel approaches. In a live cell bioluminescence resonance energy transfer-based assay, we find that roughly half of KCTD proteins interact with Gβγ in an agonist-induced fashion, whereas all KCTD proteins except two were found to interact through coimmunoprecipitation. We observed that the interaction was dependent on an amino acid hot spot in the C terminus of KCTD2, KCTD5, and KCTD17. While KCTD2 and KCTD5 require both the Bric-à-brac, Tramtrack, Broad complex domain and C-terminal regions for Gβγ interaction, we uncovered that the KCTD17 C terminus is sufficient for Gβγ interaction. Finally, we demonstrated the functional consequence of the KCTD-Gβγ interaction by examining sensitization of the adenylyl cyclase-cAMP pathway in live cells. We found that Gβγ-mediated sensitization of adenylyl cyclase 5 was blunted by KCTD. We conclude that the KCTD family broadly engages Gβγ to shape GPCR signal transmission.

Disruption of the interaction between mutationally activated Gαq and Gβγ attenuates aberrant signaling

Heterotrimeric G protein stimulation via G protein-coupled receptors promotes downstream proliferative signaling. Mutations can occur in Gα proteins which prevent GTP hydrolysis; this allows the G proteins to signal independently of G protein-coupled receptors and can result in various cancers, such as uveal melanoma (UM). Most UM cases harbor Q209L, Q209P, or R183C mutations in Gαq/11 proteins, rendering the proteins constitutively active (CA). Although it is generally thought that active, GTP-bound Gα subunits are dissociated from and signal independently of Gβγ, accumulating evidence indicates that some CA Gα mutants, such as Gαq/11, retain binding to Gβγ, and this interaction is necessary for signaling. Here, we demonstrate that disrupting the interaction between Gβγ and Gαq is sufficient to inhibit aberrant signaling driven by CA Gαq. Introduction of the I25A point mutation in the N-terminal α helical domain of CA Gαq to inhibit Gβγ binding, overexpression of the G protein Gαo to sequester Gβγ, and siRNA depletion of Gβ subunits inhibited or abolished CA Gαq signaling to the MAPK and YAP pathways. Moreover, in HEK 293cells and in UM cell lines, we show that Gαq-Q209P and Gαq-R183C are more sensitive to the loss of Gβγ interaction than Gαq-Q209L. Our study challenges the idea that CA Gαq/11 signals independently of Gβγ and demonstrates differential sensitivity between the Gαq-Q209L, Gαq-Q209P, and Gαq-R183C mutants.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Cellular and Functional Defects in Aldosteronism-linked Cytosolic Domain Mutations in GIRK4 (KCNJ5)

GIRK4 (KCNJ5) is one of four subunits of the G-protein gated inwardly rectifying potassium channels (GIRK1-4). GIRK4 subunits are abundant in heart and adrenal cortex, and form homo- or heterotetramers with GIRK1. GIRK channels mediate inhibitory transmission via Gi/o-protein coupled receptors, with Gβγ subunits as their main gating factor. KCNJ5 mutations were causally linked to primary aldosteronism (PA). The leading concept for PA-related mutations in KCNJ5 is that mutations in the pore region cause loss of K+ selectivity, which increases Na+ conductance and membrane depolarization followed by Ca2+ influx and aldosterone secretion. This mechanism later was extended to mutations in cytosolic N- and C-terminal domains far from the selectivity filter: GIRK4R52H, GIRK4E246K, and GIRK4G247R. Previously, using Xenopus laevis oocytes, we have shown that the activity of heterotetrameric GIRK1/4R52H and GIRK1/4E246K channels was reduced. We also showed that the mutations did not affect the selectivity or inward rectification. Here, we show that the activity of GIRK1/4G247R was not different from GIRK1/4WT. We corroborated these results in human adrenal cortex cell line (HAC15). Confocal imaging revealed that abundance in plasma membrane of mutated homo- or heterotetrameric channels was significantly reduced. Moreover, mutated homotetrameric channels were non-functional; however, VU0529331, an opener of GIRK4 homotetramers, rescued the activity of GIRK4G247R. Using Fluorescence Resonance Energy Transfer (FRET), we demonstrate that interaction of Gβγ with GIRK1/4R52H and GIRK1/4E246K is significantly impaired in a wide range of acceptor/donor molar ratios. Interaction with GIRK1/4G247R was only slightly impaired. Our results suggest an impairment of both Gβγ interaction and expression for GIRK4R52H and GIRK4E246K, but mainly a reduction in expression for homotetrameric (and not heterotetrameric) GIRK4G247R. Furthermore, these results rule out a previously proposed mechanism for R52H and E246K mutants, and indicate potential ways of clinical treatment.

The Arabidopsis heterotrimeric G-protein β subunit, AGB1, is required for guard cell calcium sensing and calcium-induced calcium release.

Cytosolic calcium concentration ([Ca2+ ]cyt ) and heterotrimeric G-proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Cao ) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Cao responses: induction of stomatal closure; inhibition of stomatal opening; [Ca2+ ]cyt oscillation; and inositol 1,4,5-trisphosphate (InsP3) production. Stomata in wild-type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Cao . By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double-mutants, as well as those of the agg1agg2Gγ double-mutant, were insensitive to Cao . These behaviors contrast with ABA-regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G-protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus-specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6-detected [Ca2+ ]cyt oscillations in response to Cao , initiating only a single [Ca2+ ]cyt spike. Experimentally imposed [Ca2+ ]cyt oscillations restored stomatal closure in agb1. Yeast two-hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Cao induced InsP3 production in Col but not in agb1. In sum, G-protein signaling via AGB1/AGG1/AGG2 is essential for Cao -regulation of stomatal apertures, and stomatal movements in response to Cao apparently require Ca2+ -induced Ca2+ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.

Gβγ SNARE Interactions and Their Behavioral Effects.

Presynaptic terminals possess interlocking molecular mechanisms that control exocytosis. An example of such complexity is the modulation of release by presynaptic G Protein Coupled Receptors (GPCRs). GPCR ubiquity at synapses-GPCRs are present at every studied presynaptic terminal-underlies their critical importance in synaptic function. GPCRs mediate presynaptic modulation by mechanisms including via classical Gα effectors, but membrane-delimited actions of Gβγ can also alter probability of release by altering presynaptic ionic conductances. This directly or indirectly modifies action potential-evoked presynaptic Ca2+ entry. In addition, Gβγ can interact directly with SNARE complexes responsible for synaptic vesicle fusion to reduce peak cleft neurotransmitter concentrations during evoked release. The interaction of Gβγ with SNARE is displaced via competitive interaction with C2AB-domain containing calcium sensors such as synaptotagmin I in a Ca2+-sensitive manner, restoring exocytosis. Synaptic modulation of this form allows selective inhibition of postsynaptic receptor-mediated responses, and this, in combination with Ca2+ sensitivity of Gβγ effects on SNARE complexes allows for specific behavioral outcomes. One such outcome mediated by 5-HT receptors in the spinal cord seen in all vertebrates shows remarkable synergy between presynaptic effects of Gβγ and postsynaptic 5-HT-mediated changes in activation of Ca2+-dependent K+ channels. While acting through entirely separate cellular compartments and signal transduction pathways, these effects converge on the same effect on locomotion and other critical functions of the central nervous system.

Modulation of Synaptic transmission: Quantitative analysis of Gβγ specificity to adrenergic α2a receptor and SNARE.

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.

Interaction between the dopamine transporter and the Gβγ subunits of the heterotrimeric G‐protein modifies transporter function and response to psychostimulants

Monoaminergic neurotransmission (dopamine, serotonin, and norepinephrine) is altered in complex psychiatric conditions such as depression, attention‐deficit hyperactivity disorder (ADHD) and drug use disorders. The dopamine transporter (DAT) clears extracellular dopamine (DA) through a sodium‐coupled transport mechanism. The fine‐tuning of DA homeostasis is maintained by multiple mechanisms involving protein‐protein interactions. We recently reported that G‐protein beta‐gamma (Gβγ) subunits bind directly to DAT. The binding of Gβγ results in a net change in the direction of the DA flux, increasing extracellular DA levels. The activation of Gβγ by a binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous systems and primary dopaminergic neurons in culture. The activation of a Gq‐subtype GPCR like the muscarinic receptor 5 (hM5R), also stimulates efflux that can be blocked with gallein, a small molecule that block the Gbg “hot spot” for binding to different effectors. A permeable TAT‐peptide containing the Gβγ interacting domain of DAT (human DAT 582 to 586) blocked the ability of mSIRK and carbachol to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. To further understand the mechanism of Gβγ regulation on DAT function, we examined whether the functional effects were linked to conformational changes in the DAT. An extracellularly‐oriented cysteine residue (C306) was used to probe conformational changes induced by the Gβγ activator peptide, mSIRK, and ligands such as cocaine, ibogaine and other modulators of DAT function. The accessibility of C306 to an impermeant thiol‐reactive reagent was significantly reduced in cells treated with mSIRK. It has previously been shown that cocaine increases the accessibility of C306 by stabilizing an outward facing conformation of the DAT. However, pretreatment of cells with mSIRK reduced the cocaine‐induced increase in C306 reactivity, suggesting that Gβγ activation limits the available binding sites for cocaine, perhaps by stabilizing an inward conformation. Finally, we wanted to test if the observed conformational changes were result of the Gβγ binding to DAT or if the binding of Gβγ is conformation‐dependent. For that we used the proximity‐ligation assay, new approach to study protein‐protein interactions, to study how the interaction DAT‐Gβγ is modified in the presence of different conformation‐specific DAT‐ligands. Collectively, our data show that Gβγ interacts directly with DAT and facilitate a shift in DAT conformation towards an inwardly‐oriented efflux mode. This novel mechanism may have important implications in the regulation of brain DA homeostasis.Support or Funding InformationNIMH IRP:ZIA MH002946‐04This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gβγ subunit activation promotes dopamine efflux through the dopamine transporter.

The dopamine transporter (DAT) is an important regulator of brain dopamine (DA) homeostasis, controlling the intensity and duration of DA signaling. DAT is the target for psychostimulants-like cocaine and amphetamine-and plays an important role in neuropsychiatric disorders, including attention-deficit hyperactivity disorder and drug addiction. Thus, a thorough understanding of the mechanisms that regulate DAT function is necessary for the development of clinical interventions to treat DA-related brain disorders. Previous studies have revealed a plethora of protein-protein interactions influencing DAT cellular localization and activity, suggesting that the fine-tuning of DA homeostasis involves multiple mechanisms. We recently reported that G-protein beta-gamma (Gβγ) subunits bind directly to DAT and decrease DA clearance. Here we show that Gβγ induces the release of DA through DAT. Specifically, a Gβγ-binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous cells and primary dopaminergic neurons in culture. Addition of the Gβγ inhibitor gallein or DAT inhibitors prevents this effect. Residues 582 to 596 in the DAT carboxy terminus were identified as the primary binding site of Gβγ. A TAT peptide containing the Gβγ-interacting domain of DAT blocked the ability of mSIRK to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. Finally, activation of a G-protein-coupled receptor, the muscarinic M5R, results in DAT-mediated DA efflux through a Gβγ-dependent mechanism. Collectively, our data show that Gβγ interacts with DAT to promote DA efflux. This novel mechanism may have important implications in the regulation of brain DA homeostasis.

Comparative electrostatic analysis of adenylyl cyclase for isoform dependent regulation properties

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second-messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein Gi α at a pseudo-symmetric position to the stimulatory Gs α binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform-specific modulatory effects on AC. Proteins 2016; 84:1844-1858. © 2016 Wiley Periodicals, Inc.

The molecular requirements for the G protein betagamma‐SNARE interaction

Inhibitory G‐protein coupled receptors (GPCRs) can attenuate the secretion of hormones and neurotransmitters in an Ca2+‐dependent fashion. It has been shown that one mechanism for this inhibition involves the direct interaction of G‐protein βγ dimers (Gβγ) with the soluble N‐ethylmaleimide sensitive attachment protein receptor (SNARE) proteins syntaxin1A (Stx1A) and SNAP‐25. We have previously shown that Gβγ competes with the calcium sensor synaptotagmin 1 (Syt1) for binding sites upon SNARE proteins in a Ca2+ dependent manner. The goal of this study is to determine the individual residues required for the Gβγ‐SNARE interaction. To investigate the molecular basis for this interaction, prior studies performed by our group utilizing a “peptide mapping” approach have shown that both the N‐terminal and the C‐terminal domains of SNAP‐25 are essential for the interaction of Gβγ with SNAP‐25. Here, utilizing a similar approach, we demonstrate that the N‐terminal Habc domain is essential for the interaction of Gβγ with Stx1A. The interaction is mediated through two positively‐charged residues: K55 and R125. Mutation of these residues to Ala abolishes Gβγ binding in vitro, while retaining the ability to bind Syt1. We anticipate that these studies will enhance our understanding of the GPCR‐mediated regulatory mechanisms of exocytosis.

The Roles of Gβγ and Gα in Gating and Regulation of GIRK Channels.

G protein-gated K(+) (GIRK, or Kir3) channels mediate inhibitory neurotransmission via G protein-coupled receptors (GPCRs) in heart and brain. The signaling cascade involves activation of GPCR by an agonist, activation of a G protein followed by rearrangement or dissociation of activated Gα(GTP) from Gβγ, and activation of GIRK by Gβγ. Gβγ is the main transducer of GPCR activating signal to the GIRK channel. It promotes channel opening by direct binding to GIRK's cytosolic domain formed by the N- and C-terminal segments of the GIRK's four subunits. Gβγ's interaction with, and activation of, the GIRK channels are well understood and reviewed elsewhere; however, several important details involving distal parts of the cytosolic domain remain incompletely understood. Gα(i/o) also binds to GIRKs and has been implicated in regulating channel's gating, in concert with Gβγ. Among known functions of Gα, best-described (though not well understood) are selectivity of signaling (only G(i/o) proteins normally couple to GIRKs) and regulation of the basal activity of GIRKs (I(basal)). A role for a direct effect of the activated Gα(i/o)(GTP) in GIRK gating has also been proposed but remains elusive. This chapter discusses the mechanisms of signaling within the essential cascade, from GPCR to the heterotrimeric G protein and to the channel. The focus is on the role of Gα and on the relationships between Gα and Gβγ in channel regulation, their role in specific signaling from GPCRs to GIRKs, and the role of stoichiometry and cooperativity of G protein-GIRK interactions in channel's function.

Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction.

BackgroundAssembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood.ResultsHere, we report that Gβγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. While Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gβγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gβγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gβγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells.ConclusionsAltogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-014-0132-4) contains supplementary material, which is available to authorized users.

Dual regulation of G proteins and the G-protein–activated K + channels by lithium

Lithium (Li(+)) is widely used to treat bipolar disorder (BPD). Cellular targets of Li(+), such as glycogen synthase kinase 3β (GSK3β) and G proteins, have long been implicated in BPD etiology; however, recent genetic studies link BPD to other proteins, particularly ion channels. Li(+) affects neuronal excitability, but the underlying mechanisms and the relevance to putative BPD targets are unknown. We discovered a dual regulation of G protein-gated K(+) (GIRK) channels by Li(+), and identified the underlying molecular mechanisms. In hippocampal neurons, therapeutic doses of Li(+) (1-2 mM) increased GIRK basal current (Ibasal) but attenuated neurotransmitter-evoked GIRK currents (Ievoked) mediated by Gi/o-coupled G-protein-coupled receptors (GPCRs). Molecular mechanisms of these regulations were studied with heterologously expressed GIRK1/2. In excised membrane patches, Li(+) increased Ibasal but reduced GPCR-induced GIRK currents. Both regulations were membrane-delimited and G protein-dependent, requiring both Gα and Gβγ subunits. Li(+) did not impair direct activation of GIRK channels by Gβγ, suggesting that inhibition of Ievoked results from an action of Li(+) on Gα, probably through inhibition of GTP-GDP exchange. In direct binding studies, Li(+) promoted GPCR-independent dissociation of Gαi(GDP) from Gβγ by a Mg(2+)-independent mechanism. This previously unknown Li(+) action on G proteins explains the second effect of Li(+), the enhancement of GIRK's Ibasal. The dual effect of Li(+) on GIRK may profoundly regulate the inhibitory effects of neurotransmitters acting via GIRK channels. Our findings link between Li(+), neuronal excitability, and both cellular and genetic targets of BPD: GPCRs, G proteins, and ion channels.

Gβγ interacts with mTOR and promotes its activation

Diverse G protein-coupled receptors depend on Gβγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gβγ. Thus, we tested the hypothesis that Gβγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gβγ. Cell stimulation with serum modulates Gβγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gβγ heterodimers containing different Gβ subunits, except Gβ4. Both, mTORC1 and mTORC2 complexes interact with Gβ1γ2 which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gβγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gβγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gβγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gβγ signaling interfaces.

Interaction of G-Protein βγ Complex with Chromatin Modulates GPCR-Dependent Gene Regulation

Heterotrimeric G-protein signal transduction initiated by G-protein-coupled receptors (GPCRs) in the plasma membrane is thought to propagate through protein-protein interactions of subunits, Gα and Gβγ in the cytosol. In this study, we show novel nuclear functions of Gβγ through demonstrating interaction of Gβ2 with integral components of chromatin and effects of Gβ2 depletion on global gene expression. Agonist activation of several GPCRs including the angiotensin II type 1 receptor specifically augmented Gβ2 levels in the nucleus and Gβ2 interacted with specific nucleosome core histones and transcriptional modulators. Depletion of Gβ2 repressed the basal and angiotensin II-dependent transcriptional activities of myocyte enhancer factor 2. Gβ2 interacted with a sequence motif that was present in several transcription factors, whose genome-wide binding accounted for the Gβ2-dependent regulation of approximately 2% genes. These findings suggest a wide-ranging mechanism by which direct interaction of Gβγ with specific chromatin bound transcription factors regulates functional gene networks in response to GPCR activation in cells.

Inhibition of the Ethanol-induced Potentiation of α1 Glycine Receptor by a Small Peptide That Interferes with Gβγ Binding

Gβγ interaction with GlyR is an important determinant in ethanol potentiation of this channel. A small peptide, RQH(C7), can inhibit ethanol potentiation of GlyR currents. Results with RQH(C7) indicate that ethanol mediated potentiation of GlyR is in part by Gβγ activation. Molecular interaction between Gβγ and GlyR could be used as a target for pharmacological modification of ethanol effects. Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through Gβγ interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQH(C7)), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of Gβγ on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQH(C7) displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with Gβγ or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ~350Å(2) involving aspartic acids 186, 228, and 246 in Gβγ where we propose that RQH(C7) binds and exerts its blocking action on the effect of ethanol in GlyR.

Gβγ Inhibits Exocytosis via Interaction with Critical Residues on SolubleN-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 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.