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Heterotrimeric G proteins composed of Gα, Gβ and Gγ subunits are evolutionarily conserved signaling modules involved in diverse biological processes in plants and animals. The role and action of Gα remain largely enigmatic in plant innate immunity. We have recently demonstrated that Arabidopsis Gα (GPA1) is a key component of a new immune signaling pathway activated by bacteria-secreted proteases. Here we show that GPA1 is also involved in the signaling network of Arabidopsis in response to the bacterial flagellin epitope flg22. Specifically, GPA1 plays a pivotal role in an immune pathway involving the flg22 receptor FLS2, co-receptor BAK1, Regulator of G Signaling 1 (RGS1), and Arabidopsis Gβ (AGB1), in which flg22 elicits GPA1/AGB1 dissociation from the FLS2/BAK1/RGS1 receptor complex. Consequently, we observed flg22-induced degradation of FLS2, BAK1 and RGS1 but not GPA1 or AGB1. We also found that GPA1 constitutively interacts with the NADPH oxidase RbohD to potentiate flg22-induced ROS burst independently of the central cytoplasmic kinase BIK1. Taken together, our work sheds multiple novel insights into the functions and regulatory mechanisms of GPA1 in Arabidopsis innate immunity.

Heterotrimeric G proteins play central roles in many signaling pathways, including the phototransduction cascade in animals. However, the degree of involvement of the G protein subunit Gαq is not clear since animals with previously reported strong loss-of-function mutations remain responsive to light stimuli. We recovered a new allele of Gαq in Drosophila that abolishes light response in a conventional electroretinogram assay, and reduces sensitivity in whole-cell recordings of dissociated cells by at least five orders of magnitude. In addition, mutant eyes demonstrate a rapid rate of degeneration in the presence of light. Our new allele is likely the strongest hypomorph described to date. Interestingly, the mutant protein is produced in the eyes but carries a single amino acid change of a conserved hydrophobic residue that has been assigned to the interface of interaction between Gαq and its downstream effector, PLC. Our study has thus uncovered possibly the first point mutation that specifically affects this interaction in vivo.

Chemotaxis, which is chemoattractant-guided directional cell migration, plays major roles in recruitment of neutrophils, the metastasis of cancer cells, and the development of the model organism Dictyostelium discoideum. These cells share remarkable similarities in the signaling pathways by which they control chemotaxis. They all use a G protein-coupled receptor (GPCR)-mediated signal transduction pathway to sense the chemotactic gradient to guide cell migration. Diverse chemokines activate Rac through conserved GPCR signaling pathways. ELMO proteins are an evolutionarily conserved, essential component of the ELMO/Dock complex, which functions as a guanine nucleotide exchange factor (GEF) for small G protein Rac activation. The linkages between the GPCR-initiated gradient sensing compass and the Rac-mediated migrating machinery have long been missing. Here, we summarize recent findings on ELMO proteins that directly interact with G protein and transduce GPCR signaling to control the reorganization of actin-based cytoskeleton through regulating Rac activation during chemotaxis, first in D. discoideum and then in mammalian cancer cells. This represents an evolutionarily conserved signaling shortcut from GPCR to the actin cytoskeleton.

G proteins classically associated with the 7TM or serpentine receptors generally exist as a heterotrimeric complex consisting of alpha, beta and gamma subunits. These proteins serve as transducers of extracellular signal from plasma membrane to cellular interior. Binding of G protein-coupled receptor (GPCR) with a ligand leads to their activation, followed by that of G alpha, which then dissociates from G beta gamma subunits and initiate downstream effects through plasma membrane-localized effectors. This plasma membrane-restricted view of G proteins was challenged when they were found to be present in various intracellular locations such as endomembranes, cytoskeleton, mitochondria, and nucleus thereby opening up newer spatial domains for the action of these signaling molecules. Many recent studies have addressed the spatiotemporal dynamics underlying this atypical distribution and the G proteins have been shown to undergo activation-dependent as well as activation-independent relocalization. This spatially `directed' targeting of G proteins provides them rapid access to intracellular communication network without relying on diffusible second messengers. Here, we present a consolidated review of the existing knowledge about the presence and physiological roles for G proteins in these atypical locations, along with the mechanistic knowhow presently known about underlying processes.

Heterotrimeric guanine nucleotide-binding protein (G protein) signaling is an evolutionarily conserved mechanism in diverse eukaryotic organisms. In the past half century, five Nobel Prizes are awarded to researchers working in the G protein field, these seminal studies make the G protein pathway become the best understood signaling transmit system in the world. In animals, heterotrimeric G proteins contains α , β and γ subunits, perceives extracellular stimuli through G protein-coupled receptors (GPCR), and transmit signals to ion channels, enzymes and other effectors to affect numerous cellular behaviors. In recent years, much has been learned about the diversity of signal transduction through plant G proteins thanks to the identification and mutation of genes in Arabidopsis and rice. Plant cells have most of the core elements found in animal G signaling, such as α , β and γ subunits. However, plant G proteins transmit signals by atypical mechanisms. The plant G α subunit spontaneously releases GDP and forms a stable GTP bound state in vitro , and thus are self-activating. The self-activating characteristic also means that plant G protein paradigm do not need and therefore do not have typical animal GPCR. Indeed, the current evidence supports that no typical GPCR exists in plant. In most plants, the role of GPCR is performed by GTPase-accelerating protein (GAP), such as Arabidopsis regulator of G protein signaling 1 (AtRGS1). Interestingly, there is no ortholog of AtRGS1 in rice genome. Recently, an important advance has been proved that up-regulation of COLD1 is positively correlated with chilling tolerance in rice. COLD1 has nine-transmembrane domains, and localize to plasma membrane (PM) and endoplasmic reticulum (ER). The COLD1 physically interacts with RGA1 and acts as RGS protein that senses low temperature signals and accelerates G-protein GTPase activity. These results indicate that non-GPCR proteins in plant can topologically and functionally resemble animal GPCRs. In plants, the repertoire of the heterotrimeric G protein is much simpler than that in mammalian, but G γ subunits exhibit an extraordinary level of structural diversity and show significant differences from their animal counterparts. The non-canonical G γ subunits in plant kingdom have a large cysteine domain on C terminal, can be four times larger than the average mammalian G γ subunits size. The non-canonical G γ subunits present an inhibitory effects of N terminal on C terminal. Recent studies have shown that the manipulation of two non-canonical G γ subunits, GS3 (grain size 3) and DEP1 (dense and erect panicle 1), represents new strategy to simultaneously increase grain yield and nitrogen use efficiency in rice breeding. In this review, we focus on the recent insights of G protein network derived from the research about the model plant rice and Arabidopsis , the difference of G protein signal between plants and animals, and the potential of G protein to improve the crops productivity.

The directional movement toward extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

Frizzleds (FZDs) are unconventional G protein-coupled receptors that belong to the class Frizzled. They are bound and activated by the Wingless/Int-1 lipoglycoprotein (WNT) family of secreted lipoglycoproteins. To date, mechanisms of signal initiation and FZD-G protein coupling remain poorly understood. Previously, we showed that FZD(6) assembles withG alpha(i1)/G alpha(q) (but not withG alpha(s), G alpha(o) and G alpha(12/13)), and that these inactive-state complexes are dissociated by WNTs and regulated by the phosphoprotein Dishevelled (DVL). Here, we investigated the inactive-state assembly of heterotrimeric G proteins with FZD(4), a receptor important in retinal vascular development and frequently mutated in Norrie disease or familial exudative vitreoretinopathy. Live-cell imaging experiments using fluorescence recovery after photobleaching show that human FZD(4) assembles-in a DVL-independent manner-with G alpha(12/13) but not representatives of other heterotrimeric G protein subfamilies, such as G alpha(i1), G alpha(o), G alpha(s), andG alpha(q). The FZD(4)-Gprotein complex dissociates upon stimulation with WNT-3A, WNT-5A, WNT-7A, and WNT-10B. In addition, WNT-induced dynamic mass redistribution changes in untransfected and, even more so, in FZD(4) green fluorescent protein-transfected cells depend on G alpha(12/13). Furthermore, expression of FZD(4) and G alpha(12) or G alpha(13) in human embryonic kidney 293 cells induces WNT-dependent membrane recruitment of p115-RHOGEF (RHO guanine nucleotide exchange factor, molecular weight 115 kDa), a direct target of G alpha(12/13) signaling, underlining the functionality of an FZD(4)-G alpha(12/13)-RHO signaling axis. In summary, G alpha(12/13)-mediatedWNT/FZD(4) signaling through p115-RHOGEF offers an intriguing and previously unappreciated mechanistic link of FZD(4) signaling to cytoskeletal rearrangements and RHO signaling with implications for the regulation of angiogenesis during embryonic and tumor development.

Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1.

Interactions between proteins and cell membranes are critical for biological processes such as transmembrane signaling, and specific components of the membrane may play roles in helping to organize or mandate particular conformations of both integral and peripheral membrane proteins. One example of a signaling enzyme whose function is dependent on membrane binding and whose activity is affected by specific lipid components is G protein-coupled receptor (GPCR) kinase 2 (GRK2). Efficient GRK2-mediated phosphorylation of activated GPCRs is dependent not only on its recruitment to the membrane by heterotrimeric Gβγ subunits but also on the presence of highly negatively charged lipids, in particular phosphatidylinositol 4',5'-bisphosphate (PIP2). We hypothesized that PIP2 may favor a distinct orientation of the GRK2-Gβγ complex on the membrane that is more optimal for function. In this study, we compared the possible orientations of the GRK2-Gβγ complex and Gβγ alone on model cell membranes prepared with various anionic phospholipids as deduced from sum frequency generation vibrational and attenuated total reflectance Fourier transform infrared spectroscopic methods. Our results indicate that PIP2 affects the membrane orientation of the GRK2-Gβ1γ2 complex but not that of complexes formed with anionic phospholipid binding deficient mutations in the GRK2 pleckstrin homology (PH) domain. Gβ1γ2 exhibits a similar orientation on the lipid bilayer regardless of its lipid composition. The PIP2-induced orientation of the GRK2-Gβ1γ2 complex is therefore most likely caused by specific interactions between PIP2 and the GRK2 PH domain. Thus, PIP2 not only helps recruit GRK2 to the membrane but also "fine tunes" the orientation of the GRK2-Gβγ complex so that it is better positioned to phosphorylate activated GPCRs.

Plants lack 7-transmembrane, G-protein coupled receptors (GPCRs) because the G alpha subunit of the heterotrimeric G protein complex is “self-activating”—meaning that it spontaneously exchanges bound GDP for GTP without the need of a GPCR. In lieu of GPCRs, most plants have a seven transmembrane receptor-like regulator of G-protein signaling (RGS) protein, a component of the complex that keeps G-protein signaling in its non-activated state. The addition of glucose physically uncouples AtRGS1 from the complex through specific endocytosis leaving the activated G protein at the plasma membrane. The complement of proteins in the AtRGS1/G-protein complex over time from glucose-induced endocytosis was profiled by immunoprecipitation coupled to mass spectrometry (IP-MS). A total of 119 proteins in the AtRGS1 complex were identified. Several known interactors of the complex were identified, thus validating the approach, but the vast majority (93/119) were not known previously. AtRGS1 protein interactions were dynamically modulated by d -glucose. At low glucose levels, the AtRGS1 complex is comprised of proteins involved in transport, stress and metabolism. After glucose application, the AtRGS1 complex rapidly sheds many of these proteins and recruits other proteins involved in vesicular trafficking and signal transduction. The profile of the AtRGS1 components answers several questions about the type of coat protein and vesicular trafficking GTPases used in AtRGS1 endocytosis and the function of endocytic AtRGS1.

Regulator of G protein signaling (RGS) proteins interact with activated Gα subunits via their RGS domains and accelerate the hydrolysis of GTP. Although the R4 subfamily of RGS proteins generally accepts both Gαi/o and Gαq/11 subunits as substrates, the R7 and R12 subfamilies select against Gαq/11. In contrast, only one RGS protein, RGS2, is known to be selective for Gαq/11. The molecular basis for this selectivity is not clear. Previously, the crystal structure of RGS2 in complex with Gαq revealed a non-canonical interaction that could be due to interfacial differences imposed by RGS2, the Gα subunit, or both. To resolve this ambiguity, the 2.6 A crystal structure of RGS8, an R4 subfamily member, was determined in complex with Gαq. RGS8 adopts the same pose on Gαq as it does when bound to Gαi3, indicating that the non-canonical interaction of RGS2 with Gαq is due to unique features of RGS2. Based on the RGS8-Gαq structure, residues in RGS8 that contact a unique α-helical domain loop of Gαq were converted to those typically found in R12 subfamily members, and the reverse substitutions were introduced into RGS10, an R12 subfamily member. Although these substitutions perturbed their ability to stimulate GTP hydrolysis, they did not reverse selectivity. Instead, selectivity for Gαq seems more likely determined by whether strong contacts can be maintained between α6 of the RGS domain and Switch III of Gαq, regions of high sequence and conformational diversity in both protein families.

The mechanisms by which G proteins modulate voltage-gated Ca2+ channel currents (CaV), particularly CaV2.2 and CaV2.3, are voltage dependent (VD) or voltage independent (VI). VD pathways are typically mediated by Gαi/o and GαS subfamilies. On the other hand, VI inhibition modulation is coupled to the Gαq subfamily and signaling pathways downstream of phospholipase C stimulation. In most studies, this latter pathway has been shown to be linked to Gαq and/or Gα11 protein subunits. However, there are no studies that have examined whether natively expressed Gα14 subunits (Gαq subfamily member) couple G protein-coupled receptors (GPCR) with CaV2.2 channels. We report that Gα14 subunits functionally couple the substance P (SP)/neurokinin-1 (NK-1) receptor pathway to CaV2.2 channels in acutely dissociated rat celiac-superior mesenteric ganglion (CSMG) neurons. Exposure of CSMG neurons to SP blocked the CaV2.2 currents in a predominantly VD manner that was pertussis toxin and cholera toxin resistant, as well as Gαq/11 independent. However, silencing Gα14 subunits significantly attenuated the SP-mediated Ca2+ current block. In another set of experiments, exposure of CSMG neurons to SP led to the inhibition of KCNQ K+ M-currents. The SP-mediated M-current block was significantly reduced in neurons transfected with Gα14 small-interference RNA. Finally, overexpression of the GTP-bound Gαq/11 binding protein RGS2 did not alter the block of M-currents by SP but significantly abolished the oxotremorine methiodide-mediated M-current inhibition. Taken together, these results provide evidence of a new Gα14-coupled signaling pathway that modulates CaV2.2 and M-currents via SP-stimulated NK-1 receptors in CSMG neurons.

The heterotrimeric G protein complex, comprising Gα, Gγ and Gγ subunits, is an evolutionarily conserved signaling molecular machine that transmits signals from transmembrane receptors to downstream target proteins. Plants conserved the core G protein elements, while developing their own regulatory systems differently from animals. Genetic evidence supports the conclusion that the heterotrimeric G proteins regulate shoot, root and epidermis development, as well as sugar sensing, hormone responsiveness and abiotic and biotic stress tolerance. This review is a compendium of the known morphological changes conferred by loss- and gain-of-function mutations of the G protein subunit genes across three higher land plant models, namely Arabidopsis, rice and maize.

Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the “Gα-binding and -activating (GBA) motif”. So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subun...

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.

G protein coupled receptors regulate numerous cellular functions primarily via coupling to heterotrimeric G proteins and subsequent regulation of effector proteins such as ion channels, enzymes or GTP exchange factors for small G proteins. The dynamics of interactions between G protein subunits and effectors have been difficult to study particularly in a cellular context. The introduction of Förster resonance energy transfer methods into the field of GPCR research led to interesting insights into the temporal patterns of interactions between G protein subunits and their effectors. In this review we specifically focus on the interaction of Gαi subunits with adenylyl cyclases and of Gαq subunits with p63RhoGEF or G protein coupled receptor kinases type 2. Comparing the dynamics of these interactions revealed remarkable differences between different G protein effectors regarding the ability to be modulated by members of the regulator of G protein signalling protein family as well as the sensitivity towards receptor activation.

Heterotrimeric G proteins are evolutionarily conserved protein complexes in eukaryotes, which are formed by α, β and γ subunits. In animals, they are activated by G protein-coupled receptors (GPCRs). Past genetic analysis of individual subunits of plant heterotrimeric G proteins revealed their functions in many different biological processes. Recent studies suggest that unlike in animals, plant G proteins are not likely activated through GPCRs. Instead, receptor-like kinases probably serve as upstream receptors for heterotrimeric G proteins-mediated signaling. This review summarizes the relevance of heterotrimeric G proteins in plant responses to environmental stresses and discusses our current understanding of the mechanisms of how they regulate these processes.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) regulate a multitude of signaling pathways in mammalian cells by transducing signals from G protein-coupled receptors (GPCRs) to effectors, which in turn regulate cellular function. In the myocardium, G protein signaling occurs in all cardiac cell types and is centrally involved in the regulation of heart rate, pump function, and vascular tone and in the response to hemodynamic stress and injury. Perturbations in G protein-mediated signaling are well known to contribute to cardiac hypertrophy, failure, and arrhythmias. Most of the currently used drugs for cardiac and other diseases target GPCR signaling. In the canonical G protein signaling paradigm, G proteins that are located at the cytoplasmic surface of the plasma membrane become activated after an agonist-induced conformational change of GPCRs, which then allows GTP-bound Gα and free Gβγ subunits to activate or inhibit effector proteins. Research over the past two decades has markedly broadened the original paradigm with a GPCR-G protein-effector at the cell surface at its core by revealing novel binding partners and additional subcellular localizations for heterotrimeric G proteins that facilitate many previously unrecognized functional effects. In this review, we focus on non-canonical and epigenetic-related mechanisms that regulate heterotrimeric G protein expression, activation, and localization and discuss functional consequences using cardiac examples where possible. Mechanisms reviewed involve microRNAs, histone deacetylases, chaperones, alternative modes of G protein activation, and posttranslational modifications. Some of these newly characterized mechanisms may be further developed into novel strategies for the treatment of cardiac disease and beyond.

In animals, heterotrimeric G proteins, comprising Gα, Gβ, and Gγ subunits, are molecular switches whose function tightly depends on Gα and Gβγ interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gβγ, but not Gα. We report here that the Gβγ dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gβ, and Gγ are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gβγ functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gβ-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gβγ dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Gα subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gβγ dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Gα subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Kir3 (or GIRK) channels have been known for nearly three decades to be activated by direct interactions with the βγ subunits of heterotrimeric G (Gαβγ) proteins in a membrane-delimited manner. Gα also interacts with GIRK channels and since PTX-sensitive Gα subunits show higher affinity of interaction they confer signaling specificity to G Protein- Coupled Receptors (GPCRs) that normally couple to these G protein subunits. In heterologous systems, overexpression of non PTX-sensitive Gα subunits scavenges the available Gβγ and biases GIRK activation through GPCRs that couple to these Gα subunits. Moreover, all Kir channels rely on their direct interactions with the phospholipid PIP2 to maintain their activity. Thus, signals that activate phospholipase C (e.g. through Gq signaling) to hydrolyze PIP2 result in inhibition of Kir channel activity. In this review, we illustrate with experiments performed in Xenopus oocytes that Kir channels can be used efficiently as reporters of GPCR function through Gi, Gs or Gq signaling. The membrane-delimited nature of this expression system makes it highly efficient for constructing dose-response curves yielding highly reproducible apparent affinities of different ligands for each GPCR tested.

Fluorescence polarization (FP) is a simple and sensitive method allowing for the quantification of interactions between proteins and fluorescently tagged small molecules like peptides. Heterotrimeric G proteins are critical signal transducing molecules and their activity is controlled by a complex network of regulatory proteins. Some of these regulators have defined short motifs (40 amino acids) that are sufficient to bind G proteins and subsequently modulate their activity. For these cases, FP represents a robust and quantitative method to characterize the G protein regulator interaction. Here we describe FP assays in a 384-well plate format to quantify interactions between Gα subunits of heterotrimeric G proteins and peptides corresponding to the Gα binding and activating (GBA) or GoLoco motifs, which are present in some proteins with guanine nucleotide exchange factor (GEF) (e.g., GIV/Girdin) or guanine nucleotide dissociation inhibitor (GDI) (e.g., RGS12) activity, respectively. This assay can be used to determine equilibrium dissociation constants, characterize the impact of single amino acid point mutations on the Gα-peptide interaction, and is suitable for high-throughput screening.

Gβγ dimers are one of the essential signaling units of activated G protein coupled receptors (GPCRs). There are five Gβ and twelve Gγ subunits in humans; numerous studies have demonstrated that different Gβ and Gγ subunits selectively interact to form unique Gβγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gβγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple reaction monitoring (MRM) studies of Gβ and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified and compared their individual protein levels. In this study, we have developed a quantitative MRM method to not only quantify but also compare the protein abundance of neuronal Gβ and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gβ and Gγ subunits and their subcellular localizations. For example, Gβ1 was mostly localized at the membrane while Gβ2 was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gβ and Gγ subunits may affect the Gβγ dimerization and Gβγ-effector interactions. This study offers not only a new tool to quantify and compare Gβ and Gγ subunits, but also new insights into the in vivo distribution of Gβ and Gγ subunits, and Gβγ dimer assembly in normal brain function.

Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein–coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ1 revealed that Gβ1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ1-GST protein, and PP1c co-immunoprecipitated with Gβ1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα−/− murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ1 complexes with myristoylated Gβ1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ1 protein enlists PP1c to modulate GPCR signaling in platelets.

Chemokine receptor signaling is a tightly regulated process which was for a long time exclusively attributed to heterotrimeric G proteins. β-Arrestins constitute a separable signaling arm from classical heterotrimeric G proteins, in addition to their well-established roles in receptor desensitization and endocytosis. In order to clearly dissect β-arrestin- from G protein-dependent effects we forced the recruitment of β-arrestin to CXCR4 and CCR5 independently of agonist-promoted receptor activation through chemically-induced dimerization. Targeting β-arrestins to receptors at the plasma membrane prior to chemokine stimulation attenuated G protein-mediated calcium release. Association of β-arrestins to the receptors was sufficient to induce their internalization in the absence of ligand and this effect could be further enhanced by translocation of a constitutively active β-arrestin 1 variant. CXCR4 and CCR5 were targeted to different intracellular compartments upon chemical-induced dimerization with β-arrestins and reproduced the intracellular distribution of receptors after activation with their respective ligands. Our data further provide evidence for direct β-arrestin-mediated signaling via MAP kinases ERK 1/2. These results provide clear evidence that CXCR4- or CCR5-β-arrestin complexes induce receptor endocytosis and signaling in the absence of G protein coupling and ligand-induced conformational changes of the receptor.

Heterotrimeric guanine nucleotide-binding proteins (G? proteins) are intracellular nanomachines deputed to signal transduction. The switch-on process requires the release of bound GDP from a site at the interface between GTPase and helical domains. Nucleotide release is catalyzed by G protein Coupled Receptors (GPCRs). Here we investigate the functional dynamics of wild type (WT) and six constitutively active mutants (CAMs) of the G? protein transducin (Gt) by combining atomistic molecular dynamics (MD) simulations with Maxwell-Demod discrete MD (MDdMD) simulations of the receptor-catalyzed transition between GDP-bound and nucleotide-free states. Compared to the WT, Gt CAMs increase the overall fluctuations of nucleotide and its binding site. This is accompanied by weakening of native links involving GDP, ?1, the G boxes, ?1-?3, and ?5. Collectively, constitutive activation by the considered mutants seems to associate with weakening of the interfaces between ?5 and the surrounding portions and the interface between GTPase (G) and helical (H) domains. These mutational effects associate with increases in the overall fluctuations of the G and H domains, which reflect on the collective motions of the protein. Gt CAMs, with prominence to G56P, T325A, and F332A, prioritize collective motions of the H domain overlapping with the collective motions associated with receptor-catalyzed nucleotide release. In spite of different local perturbations, the mechanisms of nucleotide exchange catalyzed by activating mutations and by receptor are expected to employ similar molecular switches in the nucleotide binding site and to share the detachment of the H domain from the G domain.

Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR–G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR–G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

Resistance to inhibitors of cholinesterase (Ric-8)A and Ric-8B are essential genes that encode positive regulators of heterotrimeric G protein α subunits. Controversy persists surrounding the precise way(s) that Ric-8 proteins affect G protein biology and signaling. Ric-8 proteins chaperone nucleotide-free Gα-subunit states during biosynthetic protein folding prior to G protein heterotrimer assembly. In organisms spanning the evolutionary window of Ric-8 expression, experimental perturbation of Ric-8 genes results in reduced functional abundances of G proteins because G protein α subunits are misfolded and degraded rapidly. Ric-8 proteins also act as Gα-subunit guanine nucleotide exchange factors (GEFs) in vitro. However, Ric-8 GEF activity could strictly be an in vitro phenomenon stemming from the ability of Ric-8 to induce partial Gα unfolding, thereby enhancing GDP release. Ric-8 GEF activity clearly differs from the GEF activity of G protein–coupled receptors (GPCRs). G protein βγ is inhibitory to Ric-8 action but obligate for receptors. It remains an open question whether Ric-8 has dual functions in cells and regulates G proteins as both a molecular chaperone and GEF. Clearly, Ric-8 has a profound influence on heterotrimeric G protein function. For this reason, we propose that Ric-8 proteins are as yet untested therapeutic targets in which pharmacological inhibition of the Ric-8/Gα protein–protein interface could serve to attenuate the effects of disease-causing G proteins (constitutively active mutants) and/or GPCR signaling. This minireview will chronicle the understanding of Ric-8 function, provide a comparative discussion of the Ric-8 molecular chaperoning and GEF activities, and support the case for why Ric-8 proteins should be considered potential targets for development of new therapies.

Heterotrimeric G-proteins are the immediate downstream effectors of G-protein coupled receptors (GPCRs). Endogenous protein guanine nucleotide dissociation inhibitors (GDIs) like AGS3/4 and RGS12/14 function through GPR/Goloco GDI domains. Extensive characterization of GPR domain peptides indicate they function as selective GDIs for Gαi by competing for the GPCR and Gβγ and preventing GDP release. We modified a GPR consensus peptide by testing FGF and TAT leader sequences to make the peptide cell permeable. FGF modification inhibited GDI activity while TAT preserved GDI activity. TAT-GPR suppresses G-protein coupling to the receptor and completely blocked α2-adrenoceptor (α2AR) mediated decreases in cAMP in HEK293 cells at 100 nM. We then sought to discover selective small molecule inhibitors for Gαi. Molecular docking was used to identify potential molecules that bind to and stabilize the Gαi–GDP complex by directly interacting with both Gαi and GDP. Gαi–GTP and Gαq-GDP were used as a computational counter screen and Gαq-GDP was used as a biological counter screen. Thirty-seven molecules were tested using nucleotide exchange. STD NMR assays with compound 0990, a quinazoline derivative, showed direct interaction with Gαi. Several compounds showed Gαi specific inhibition and were able to block α2AR mediated regulation of cAMP. In addition to being a pharmacologic tool, GDI inhibition of Gα subunits has the advantage of circumventing the upstream component of GPCR-related signaling in cases of overstimulation by agonists, mutations, polymorphisms, and expression-related defects often seen in disease.

Our laboratory has identified a number of small molecules that bind to G protein βγ subunits (Gβγ) by competing for peptide binding to the Gβγ "hot spot." M119/Gallein were identified as inhibitors of Gβγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gβγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca(2+) release, and activated other downstream targets of Gβγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gβγ in vitro from Gαi1β1γ2 heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gβγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gβγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner.

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian Gα subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A2B receptor (hA2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: Gαi, Gαs, Gαq, and Gα12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the Gα subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103(3.50) and I107(3.54) are vital too in G protein-coupling and the activation of the hA2BR, whereas L213(IL3) is more important in G protein inactivation. Substitution of S235(6.36) to alanine provided the most divergent G protein-coupling profile. Finally, L236(6.37) substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.

The heterotrimeric G protein Gα q is a central player in signal transduction, relaying signals from activated G-protein-coupled receptors (GPCRs) to effectors and other proteins to elicit changes in intracellular Ca 2+ , the actin cytoskeleton, and gene transcription. Gα q functions at the intracellular surface of the plasma membrane, as do its best-characterized targets, phospholipase C-β, p63RhoGEF, and GPCR kinase 2 (GRK2). Recent insights into the structure and function of these signaling complexes reveal several recurring themes, including complex multivalent interactions between Gα q , its protein target, and the membrane, that are likely essential for allosteric control and maximum efficiency in signal transduction. Thus, the plasma membrane is not only a source of substrates but also a key player in the scaffolding of Gα q -dependent signaling pathways.

G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs. Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation.

Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gαi1, which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine “finger.” We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gαi1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gαi1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gαi1·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.

Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding. DOI: http://dx.doi.org/10.7554/eLife.19238.001

Activators of G protein signaling (AGS), initially discovered in the search for receptor-independent activators of G protein signaling, define a broad panel of biologic regulators that influence signal transfer from receptor to G-protein, guanine nucleotide binding and hydrolysis, G protein subunit interactions, and/or serve as alternative binding partners for Gα and Gβγ independently of the classic heterotrimeric Gαβγ. AGS proteins generally fall into three groups based upon their interaction with and regulation of G protein subunits: group I, guanine nucleotide exchange factors (GEF); group II, guanine nucleotide dissociation inhibitors; and group III, entities that bind to Gβγ. Group I AGS proteins can engage all subclasses of G proteins, whereas group II AGS proteins primarily engage the Gi/Go/transducin family of G proteins. A fourth group of AGS proteins with selectivity for Gα16 may be defined by the Mitf-Tfe family of transcription factors. Groups I-III may act in concert, generating a core signaling triad analogous to the core triad for heterotrimeric G proteins (GEF + G proteins + effector). These two core triads may function independently of each other or actually cross-integrate for additional signal processing. AGS proteins have broad functional roles, and their discovery has advanced new concepts in signal processing, cell and tissue biology, receptor pharmacology, and system adaptation, providing unexpected platforms for therapeutic and diagnostic development.

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and Gi heterotrimer (CB1-Gi), guided by the x-ray structure of β2-adrenergic receptor (β2AR) in complex with Gs (β2AR-Gs), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-2183.54, Tyr-224IC2, Asp-3386.30, Arg-3406.32, Leu-3416.33, and Thr-3446.36, as potential key contacts with the extreme C-terminal helix α5 of Gαi. Ala mutations of these residues at the receptor-Gi interface resulted in little G protein coupling activity, consistent with the present model of the CB1-Gi complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α5 of Gαi. The model also suggests that unique conformational changes in the extreme C-terminal helix α5 of Gα play a crucial role in the receptor-mediated G protein activation. Background: The molecular basis of CB1 coupling to its cognate G protein is unknown. Results: Using an approach combining mutagenesis and molecular dynamics simulations, we identified CB1 residues critical for G protein signaling. Conclusion: Tight interactions between CB1 and the C-terminal helix α5 of Gαi are crucial for G protein signaling. Significance: This is the first reported molecular description of CB1 receptor coupling at the receptor-Gi interface.

It was, until recently, accepted that the two classes of acetylcholine (ACh) receptors are distinct in an important sense: muscarinic ACh receptors signal via heterotrimeric GTP binding proteins (G proteins), whereas nicotinic ACh receptors (nAChRs) open to allow flux of Na+, Ca2+, and K+ ions into the cell after activation. Here we present evidence of direct coupling between G proteins and nAChRs in neurons. Based on proteomic, biophysical, and functional evidence, we hypothesize that binding to G proteins modulates the activity and signaling of nAChRs in cells. It is important to note that while this hypothesis is new for the nAChR, it is consistent with known interactions between G proteins and structurally related ligand-gated ion channels. Therefore, it underscores an evolutionarily conserved metabotropic mechanism of G protein signaling via nAChR channels.

Heterotrimeric G-proteins, composed of Gα, Gβ, and Gγ subunits, regulate many fundamental processes in plants. In animals, ligand binding to seven transmembrane (7TM) cell surface receptors designated G-protein coupled receptors (GPCR) leads to heterotrimeric G-protein activation. Because the plant G-protein complex is constitutively active, the exact role of plant 7TM proteins in this process is unclear. Members of the mildew resistance locus O (MLO) family represent the best-characterized 7TM plant proteins. Although genetic ablation of either MLO2 or G-proteins alters susceptibility to pathogens in Arabidopsis thaliana, it is unknown whether G-proteins directly couple signaling through MLO2. Here, we exploited two well-documented phenotypes of mlo2 mutants, broad-spectrum powdery mildew resistance and spontaneous callose deposition in leaf mesophyll cells, to assess the relationship of MLO2 proteins to the G-protein complex. Although our data reveal modulation of antifungal defense responses by Gβ and Gγ subunits and a role for the Gγ1 subunit in mlo2-conditioned callose deposition, our findings overall are inconsistent with a role of MLO2 as a canonical GPCR. We discovered that mutants lacking the Gβ subunit show delayed accumulation of a subset of defense-associated genes following exposure to the microbe-associated molecular pattern flg22. Moreover, Gβ mutants were found to be hypersusceptible to spray inoculation with the bacterial pathogen Pseudomonas syringae.

Heterotrimeric G proteins, composed of α, β, and γ subunits, can transduce a variety of signals from seven-transmembrane-type receptors to intracellular effectors. By whole-exome sequencing and subsequent mutation screening, we identified de novo heterozygous mutations in GNAO1, which encodes a Gαo subunit of heterotrimeric G proteins, in four individuals with epileptic encephalopathy. Two of the affected individuals also showed involuntary movements. Somatic mosaicism (approximately 35% to 50% of cells, distributed across multiple cell types, harbored the mutation) was shown in one individual. By mapping the mutation onto three-dimensional models of the Gα subunit in three different complexed states, we found that the three mutants (c.521A>G [p.Asp174Gly], c.836T>A [p.Ile279Asn], and c.572_592del [p.Thr191_Phe197del]) are predicted to destabilize the Gα subunit fold. A fourth mutant (c.607G>A), in which the Gly203 residue located within the highly conserved switch II region is substituted to Arg, is predicted to impair GTP binding and/or activation of downstream effectors, although the p.Gly203Arg substitution might not interfere with Gα binding to G-protein-coupled receptors. Transient-expression experiments suggested that localization to the plasma membrane was variably impaired in the three putatively destabilized mutants. Electrophysiological analysis showed that Gαo-mediated inhibition of calcium currents by norepinephrine tended to be lower in three of the four Gαo mutants. These data suggest that aberrant Gαo signaling can cause multiple neurodevelopmental phenotypes, including epileptic encephalopathy and involuntary movements.

Heterotrimeric G proteins play an essential role in cellular signaling; however, the mechanism regulating their synthesis and assembly remains poorly understood. A line of evidence indicates that the posttranslational processing of G protein β subunits begins inside the protein-folding chamber of the chaperonin containing t-complex protein 1. This process is facilitated by the ubiquitously expressed phosducin-like protein (PhLP), which is thought to act as a CCT co-factor. Here we demonstrate that alternative splicing of the PhLP gene gives rise to a transcript encoding a truncated, short protein (PhLPs) that is broadly expressed in human tissues but absent in mice. Seeking to elucidate the function of PhLPs, we expressed this protein in the rod photoreceptors of mice and found that this manipulation caused a dramatic translational and posttranslational suppression of rod heterotrimeric G proteins. The investigation of the underlying mechanism revealed that PhLPs disrupts the folding of Gβ and the assembly of Gβ and Gγ subunits, events normally assisted by PhLP, by forming a stable and apparently inactive tertiary complex with CCT preloaded with nascent Gβ. As a result, the cellular levels of Gβ and Gγ, which depends on Gβ for stability, decline. In addition, PhLPs evokes a profound and rather specific down-regulation of the Gα transcript, leading to a complete disappearance of the protein. This study provides the first evidence of a generic mechanism, whereby the splicing of the PhLP gene could potentially and efficiently regulate the cellular levels of heterotrimeric G proteins.

Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses. A key event in this signalling system is the direct and dynamic interaction of Gαi1 subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between Gαi1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity resulting in a left-shift of the concentration–response curve by approximately one order of magnitude. Furthermore, Gi1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gαi1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

Voltage gated calcium channels (Ca2+ channels) are key mediators of depolarization induced calcium influx into excitable cells, and thereby play pivotal roles in a wide array of physiological responses. This review focuses on the inhibition of CaV2 (N- and P/Q-type) Ca2+-channels by G protein coupled receptors (GPCRs), which exerts important autocrine/paracrine control over synaptic transmission and neuroendocrine secretion. Voltage-dependent inhibition is the most widespread mechanism, and involves direct binding of the G protein βγ dimer (Gβγ) to the α1 subunit of CaV2 channels. GPCRs can also recruit several other distinct mechanisms including phosphorylation, lipid signaling pathways, and channel trafficking that result in voltage-independent inhibition. Current knowledge of Gβγ-mediated inhibition is reviewed, including the molecular interactions involved, determinants of voltage-dependence, and crosstalk with other cell signaling pathways. A summary of recent developments in understanding the voltage-independent mechanisms prominent in sympathetic and sensory neurons is also included. This article is part of a Special Issue entitled: Calcium channels.

TASK-2 (K2P5.1) is a background K(+) channel opened by extra- or intracellular alkalinisation that plays a role in renal bicarbonate handling, central chemoreception and cell volume regulation. Here, we present results that suggest that TASK-2 is also modulated by Gβγ subunits of heterotrimeric G protein. TASK-2 was strongly inhibited when GTP-γ-S was used as a replacement for intracellular GTP. No inhibition was present using GDP-β-S instead. Purified Gβγ introduced intracellularly also inhibited TASK-2 independently of whether GTP or GDP-β-S was present. The effects of GTP-γ-S and Gβγ subunits were abolished by neutralisation of TASK-2 C terminus double lysine residues K257-K258 or K296-K297. Use of membrane yeast two hybrid (MYTH) experiments and immunoprecipitation assays using tagged proteins gave evidence for a physical interaction between Gβ1 and Gβ2 subunits and TASK-2, in agreement with expression of these subunits in proximal tubule cells. Co-immunoprecipitation was impeded by mutating C terminus K257-K258 (but not K296-K297) to alanines. Gating by extra- or intracellular pH was unaltered in GTP-γ-S-insensitive TASK-2-K257A-K258A mutant. Shrinking TASK-2-expressing cells in hypertonic solution decreased the current to 36 % of its initial value. The same manoeuvre had a significantly diminished effect on TASK-2-K257A-K258A- or TASK-2-K296-K297-expressing cells, or in cells containing intracellular GDP-β-S. Our data are compatible with the concept that TASK-2 channels are modulated by Gβγ subunits of heterotrimeric G protein. We propose that this modulation is a novel way in which TASK-2 can be tuned to its physiological functions.

The cytosolic protein Ric-8A acts as a guanine nucleotide exchange factor for Gα subunits of the Gi, Gq, and G12/13 classes of heterotrimeric G protein in vitro, and is also known to increase the amounts of these Gα proteins in vivo. The mechanism whereby Ric-8 regulates Gα content, however, has not been fully understood. Here we show that Ric-8 Astabilizes Gαi2 and Gαq by preventing their ubiquitination. Ric-8A interacts with and stabilizes Gαi2, Gαq, Gα12, but not Gαs, when expressed in COS-7 cells. The protein levels of Gαi2 and Gαq appear to be controlled via the ubiquitin-proteasome degradation pathway, because these Gα subunits undergo polyubiquitination and are stabilized with the proteasome inhibitor MG132. The ubiquitination of Gαi2 and Gαq is suppressed by expression of Ric-8A. The suppression likely requires Ric-8A interaction with these Gα proteins; the C-terminal truncation of Gαq and Gαi2 completely abrogates their interaction with Ric-8A, their stabilization by Ric-8A, and Ric-8A-mediated inhibition of Gα ubiquitination.

G-protein β subunits perform essential neuronal functions as part of G-protein βγ and Gβ5-regulators of G-protein signaling (RGS) complexes. Both Gβγ and Gβ5-RGS are obligate dimers that are thought to require the assistance of the cytosolic chaperonin CCT and a cochaperone, phosducin-like protein 1 (PhLP1) for dimer formation. To test this hypothesisin vivo, we deleted thePhlp1gene in mouse (Mus musculus) retinal rod photoreceptor cells and measured the effects on G-protein biogenesis and visual signal transduction. In the PhLP1-depleted rods, Gβγ dimer formation was decreased 50-fold, resulting in a >10-fold decrease in light sensitivity. Moreover, a 20-fold reduction in Gβ5and RGS9–1 expression was also observed, causing a 15-fold delay in the shutoff of light responses. These findings conclusively demonstratein vivothat PhLP1 is required for the folding and assembly of both Gβγ and Gβ5-RGS9.

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which consist of an α-, a β- and a γ-subunit, have crucial roles as molecular switches in the regulation of the downstream effector molecules of multiple G protein-coupled receptor signalling pathways, such as phospholipase C and adenylyl cyclase. According to the structural and functional similarities of their α-subunits, G proteins can be divided into four subfamilies: Gαs, Gαi/o, Gαq/11 and Gα12/13. Most of the α- and the βγ-subunits are abundantly expressed on the surface of immune cells. Recent studies have demonstrated that G proteins are a group of important immunomodulatory factors that regulate the migration, activation, survival, proliferation, differentiation and cytokine secretion of immune cells. In this review, we summarise the recent findings on the functions of G proteins in immune regulation and autoimmunity.

Heterotrimeric G proteins couple external signals to the activation of intracellular signal transduction pathways. Agonist-stimulated guanine nucleotide exchange activity of G-protein-coupled receptors results in the exchange of G-protein-bound GDP to GTP and the dissociation and activation of the complex into Gα-GTP and a Gβγ dimer. In Dictyostelium , a basal chemotaxis pathway consisting of heterotrimeric and monomeric G proteins is sufficient for chemotaxis. Symmetry breaking and amplification of chemoattractant sensing occurs between heterotrimeric G protein signaling and Ras activation. In a pull-down screen coupled to mass spectrometry, with Gα proteins as bait, we have identified resistant to inhibitors of cholinesterase 8 (Ric8) as a nonreceptor guanine nucleotide exchange factor for Gα-protein. Ric8 is not essential for the initial activation of heterotrimeric G proteins or Ras by uniform chemoattractant; however, it amplifies Gα signaling, which is essential for Ras-mediated symmetry breaking during chemotaxis and development.

The heterotrimeric G protein Gαq is a key regulator of blood pressure, and excess Gαq signaling leads to hypertension. A specific inhibitor of Gαq is the GTPase activating protein (GAP) known as regulator of G protein signaling 2 (RGS2). The molecular basis for how Gαq/11 subunits serve as substrates for RGS proteins and how RGS2 mandates its selectivity for Gαq is poorly understood. In crystal structures of the RGS2-Gαq complex, RGS2 docks to Gαq in a different orientation from that observed in RGS-Gαi/o complexes. Despite its unique pose, RGS2 maintains canonical interactions with the switch regions of Gαq in part because its α6 helix adopts a distinct conformation. We show that RGS2 forms extensive interactions with the α-helical domain of Gαq that contribute to binding affinity and GAP potency. RGS subfamilies that do not serve as GAPs for Gαq are unlikely to form analogous stabilizing interactions.