Gγ Subunits Research Articles

EXTRA-LARGE G PROTEINs Interact with E3 Ligases PUB4 and PUB2 and Function in Cytokinin and Developmental Processes.

Heterotrimeric GTP-binding proteins (G proteins) composed of Gα, Gβ, and Gγ subunits are conserved signal transduction molecules in animals and plants. In Arabidopsis (Arabidopsis thaliana), there are three Gα-like proteins named EXTRA-LARGE G PROTEINs (XLGs) in addition to the canonical Gα protein GPA1. XLGs have been reported to be implicated in multiple pathways, although the underlying mechanisms of their action remain elusive. Here, we report that all three XLGs interact with two closely related plant U-box (PUB) E3 ligases, PUB2 and PUB4. Three XLGs are predominantly localized at the plasma membrane, whereas XLG2 and XLG3 also show nuclear localization. The interactions between PUB2/4 and XLGs suggest that they might function in the same pathways. Indeed, we found that a newly obtained xlg1/2/3 triple knockout mutant, the pub4 mutant, and the pub2/4 double mutant all exhibited defects in cytokinin responses, stamen development, tapetum development, and male fertility. However, the xlg single mutants and the pub2 mutant did not exhibit an obvious defect in these processes, which suggests functional redundancy among the three XLGs and between PUB2 and PUB4. Overexpressing ARR10 to enhance the cytokinin response in pub4 or in the xlg1/2/3 triple mutant partially restored several phenotypes caused by the pub4 and xlg1/2/3 mutations. Our findings reveal that the XLGs and PUB2/4 are components in the complex cytokinin signaling networks regulating many developmental and physiological processes.

The structural basis of the dominant negative phenotype of the Gαi1β1γ2 G203A/A326S heterotrimer

Aim:Dominant negative mutant G proteins have provided critical insight into the mechanisms of G protein-coupled receptor (GPCR) signaling, but the mechanisms underlying the dominant negative characteristics are not completely understood. The aim of this study was to determine the structure of the dominant negative Gαi1β1γ2 G203A/A326S complex (Gi-DN) and to reveal the structural basis of the mutation-induced phenotype of Gαi1β1γ2.Methods:The three subunits of the Gi-DN complex were co-expressed with a baculovirus expression system. The Gi-DN heterotrimer was purified, and the structure of its complex with GDP was determined through X-ray crystallography.Results:The Gi-DN heterotrimer structure revealed a dual mechanism underlying the dominant negative characteristics. The mutations weakened the hydrogen bonding network between GDP/GTP and the binding pocket residues, and increased the interactions in the Gα-Gβγ interface. Concomitantly, the Gi-DN heterotrimer adopted a conformation, in which the C-terminus of Gαi and the N-termini of both the Gβ and Gγ subunits were more similar to the GPCR-bound state compared with the wild type complex. From these structural observations, two additional mutations (T48F and D272F) were designed that completely abolish the GDP binding of the Gi-DN heterotrimer.Conclusion:Overall, the results suggest that the mutations impede guanine nucleotide binding and Gα-Gβγ protein dissociation and favor the formation of the G protein/GPCR complex, thus blocking signal propagation. In addition, the structure provides a rationale for the design of other mutations that cause dominant negative effects in the G protein, as exemplified by the T48F and D272F mutations.

Dopamine Receptor Signaling in MIN6 β-Cells Revealed by Fluorescence Fluctuation Spectroscopy

Insulin secretion defects are central to the development of type II diabetes mellitus. Glucose stimulation of insulin secretion has been extensively studied, but its regulation by other stimuli such as incretins and neurotransmitters is not as well understood. We investigated the mechanisms underlying the inhibition of insulin secretion by dopamine, which is synthesized in pancreatic β-cells from circulating L-dopa. Previous research has shown that this inhibition is mediated primarily by activation of the dopamine receptor D3 subtype (DRD3), even though both DRD2 and DRD3 are expressed in β-cells. To understand this dichotomy, we investigated the dynamic interactions between the dopamine receptor subtypes and their G-proteins using two-color fluorescence fluctuation spectroscopy (FFS) of mouse MIN6 β-cells. We show that proper membrane localization of exogenous G-proteins depends on both the Gβ and Gγ subunits being overexpressed in the cell. Triple transfections of the dopamine receptor subtype and Gβ and Gγ subunits, each labeled with a different-colored fluorescent protein (FP), yielded plasma membrane expression of all three FPs and permitted an FFS evaluation of interactions between the dopamine receptors and the Gβγ complex. Upon dopamine stimulation, we measured a significant decrease in interactions between DRD3 and the Gβγ complex, which is consistent with receptor activation. In contrast, dopamine stimulation did not cause significant changes in the interactions between DRD2 and the Gβγ complex. These results demonstrate that two-color FFS is a powerful tool for measuring dynamic protein interactions in living cells, and show that preferential DRD3 signaling in β-cells occurs at the level of G-protein release.

Heterotrimeric G-proteins facilitate resistance to plant pathogenic viruses in Arabidopsis thaliana (L.) Heynh

ABSTRACTHeterotrimeric G-proteins, consisting of Gα, Gβ and Gγ subunits, are important signal transducers in eukaryotes. In plants, G-protein-mediated signaling contributes to defense against a range of fungal and bacterial pathogens. Here we studied response of G-protein-deficient mutants to ssRNA viruses representing 2 different families: Cucumber mosaic virus (CMV) (Bromoviridae) and Turnip mosaic virus (TuMV) (Potyviridae). We found that development of spreading necrosis on infected plants was suppressed in the Gβ-deficient mutant (agb1-2) compared to wild type and Gα-deficient mutant (gpa1-4). In accordance, ion leakage caused by viral infection was also significantly reduced in agb1-2 compared to wild type and gpa1-4. Nevertheless, both viruses replicated better in agb1-2 plants, while gpa1-4 was similar to wild type. Analysis of pathogenesis-related genes showed that Gβ negatively regulated salicylic acid, jasmonic acid and abscisic acid marker genes during CMV and TuMV infections. Interestingly, analysis of salicylic acid deficient transgenic plants indicated that salicylic acid did not affect resistance against these viruses and did not influence the Gβ-mediated defense response. We conclude that heterotrimeric G-proteins play a positive role in defense against viral pathogens probably by promoting cell death.

Interaction of Heterotrimeric G-Protein Components with Receptor-like Kinases in Plants: An Alternative to the Established Signaling Paradigm?

Heterotrimeric G proteins comprising Gα, Gβ, and Gγ subunits (hereafter termed G proteins) mediate important signaling processes in all eukaryotes. As per the established paradigm, during resting phase the Gα is guanosine diphosphate (GDP)-bound and the three subunits form an inactive trimeric complex GDP⋅Gαβγ. Signal perception occurs at the G-protein coupled receptors (GPCRs), which act as guanine nucleotide exchange factors (GEFs) and facilitate the exchange of GDP for guanosine triphosphate (GTP) on Gα.

Gγ7 proteins contribute to coupling of nociceptin/orphanin FQ peptide (NOP) opioid receptors and voltage-gated Ca2+ channels in rat stellate ganglion neurons

The nociceptin/orphanin FQ peptide (NOP) opioid receptors regulate neurotransmitter release via inhibition of voltage-gated Ca2+ channels (CaV2.2) in sympathetic and sensory neurons. Stimulation of NOP receptors by its endogenous agonist, nociception (Noc), leads to membrane-delimited, voltage-dependent (VD) block of CaV2.2 channel currents mediated by Gβγ protein subunits. Previously we reported that the pertussis toxin-sensitive Gαi1 and Gβ2/β4 isoforms mediate the functional coupling of NOP opioid receptors with CaV channels in rat stellate ganglion (SG) sympathetic neurons. In the present report we extended our studies by identifying the Gγ subunit that forms the heterotrimer within this signaling pathway. Small interference RNA (or siRNA) was employed to silence the expression of the natively expressed Gγ subunits. Initial PCR assays indicated that SG neurons expressed seven Gγ subunits. Silencing Gγ3 subunits did not alter signaling between NOP receptors and Ca2+ channels. However, after Gγ7 isoforms were silenced, the Noc-mediated inhibition of CaV channels was significantly decreased when compared to SG neurons transfected with scrambled siRNA. We observed that Gγ10 and Gγ11 mRNA levels increased 2.5- and 2.7-fold, respectively, after Gγ7 subunits were silenced. However, this compensatory increase in mRNA expression did not appear to fully rescue the NOP receptor coupling efficiency. Additionally, both Gγ2 and Gγ5 levels increased 50 and 75%, respectively, while Gγ3 and Gγ4 expression levels remained relatively unchanged. Taken together, our findings suggest that the Gαi1/Gβ2(β4)/Gγ7 heterotrimeric G protein complex determines the NOP receptor-mediated modulation of CaV channels in SG neurons.

The role of PLDα1 in providing specificity to signal-response coupling by heterotrimeric G-protein components in Arabidopsis.

Heterotrimeric G-proteins comprised of Gα, Gβ and Gγ subunits are important signal transducers in all eukaryotes. In plants, G-proteins affect multiple biotic and abiotic stress responses, as well as many developmental processes, even though their repertoire is significantly limited compared with that in metazoan systems. One canonical and three extra-large Gα, 1Gβ and 3Gγ proteins represent the heterotrimeric G-protein complex in Arabidopsis, and a single regulatory protein, RGS1, is one of the few known biochemical regulators of this signaling complex. This quantitative disparity between the number of signaling components and the range of processes they influence is rather intriguing. We now present evidence that the phospholipase Dα1 protein is a key component and modulator of the G-protein complex in affecting a subset of signaling pathways. We also show that the same G-protein subunits and their modulators exhibit distinct physiological and genetic interactions depending on specific signaling and developmental pathways. Such developmental plasticity and interaction specificity likely compensates for the lack of multiplicity of individual subunits, and helps to fine tune the plants' responses to constantly changing environments.

The specificity of Gβγ subunits regulating exocytosis through the Gi/o‐coupled α2a adrenergic receptor

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

GprotPRED: Annotation of Gα, Gβ and Gγ subunits of G-proteins using profile Hidden Markov Models (pHMMs) and application to proteomes

Heterotrimeric G-proteins form a major protein family, which participates in signal transduction. They are composed of three subunits, Gα, Gβ and Gγ. The Gα subunit is further divided in four distinct families Gs, Gi/o, Gq/11 and G12/13. The goal of this work was to detect and classify members of the four distinct families, plus the Gβ and the Gγ subunits of G-proteins from sequence alone. To achieve this purpose, six specific profile Hidden Markov Models (pHMMs) were built and checked for their credibility. These models were then applied to ten (10) proteomes and were able to identify all known G-protein and classify them into the distinct families. In a separate case study, the models were applied to twenty seven (27) arthropod proteomes and were able to give more credible classification in proteins with uncertain annotation and in some cases to detect novel proteins. An online tool, GprotPRED, was developed that uses these six pHMMs. The sensitivity and specificity for all pHMMs were equal to 100% with the exception of the Gβ case, where sensitivity equals to 100%, while specificity is 99.993%. In contrast to Pfam's pHMM which detects Gα subunits in general, our method not only detects Gα subunits but also classifies them into the appropriate Gα-protein family and thus could become a useful tool for the annotation of G-proteins in newly discovered proteomes. GprotPRED online tool is publicly available for non-commercial use at http://bioinformatics.biol.uoa.gr/GprotPRED and, also, a standalone version of the tool at https://github.com/vkostiou/GprotPRED.

Heterotrimeric G-Protein Alpha (α) Subunit from a. Thaliana Forms Trimeric Structures in Solution

The heterotrimeric guanine nucleotide-binding proteins (G-proteins) mediate transmission of signals from G protein coupled receptors (GPCRs) to effector systems including ion channels, enzymes and intracellular second messengers in yeast, mammals, and plants. The complex is comprised of alpha (Gα), beta (Gβ) and gamma (Gγ) subunits; Gα has GTP binding and hydrolysis activity, and Gβ and Gγ interact with downstream effectors as a dimeric complex. Although the structure and activation mechanism for the mammalian complex are well known, these are still not fully understood in plants. In our group subunits of the heterotrimeric complexes from Arabidopsis thaliana and Oryza sativa (rice) are heterologously expressed in yeast and bacteria respectively. The recombinant proteins are then purified for biochemical characterization and structural investigations. Our overall aim is to gain insight into the activation and signaling mechanisms of the G-protein complex in plants through structural studies on the individual subunits as well as the in vitro reconstituted complex. The work in this thesis is undertaken with the primary aim of optimizing the purification protocol of the A. thaliana Gα subunit, GPA1, expressed in yeast to obtain sufficient quantities of homogeneous recombinant protein for biochemical, biophysical, and structural analyses. A further goal is the recombinant production of a truncated version of the wild-type GPA1, which lacks the N-terminal 36 amino acids (GPA1t) in E. coli. GPA1t is commonly used in structural studies due to its stability. Results show that the optimized purification procedure has improved the yield to 5.5 mg GPA1 from 0.5 liters P. pastoris culture which represents a fivefold increase compared to earlier results in the group. Nucleotide (GTP, GDP, GTPγS) binding to the recombinant GPA1 is confirmed by absorbance spectroscopy and circular dichroism spectropolorimetry (CD). Results indicate that the secondary structure elements of GPA1 are more stable when it binds GTPγS as compared to GDP-bound form. Dynamic light scattering (DLS) results and Native-PAGE analyses combined with small angle X-ray scattering (SAXS) measurements reveal that GPA1 has a tendency to form trimers in solution. SAXS data also shows that GPA1-GDP has a globular structure with flexible regions extending from the protein. We cloned GPA1t gene in BL21 cells using the pQE80-L vector and a purification procedure was developed for the isolation of the recombinant protein. Despite the low protein yield, preliminary biochemical and biophysical characterizations were carried out on this protein. DLS measurements confirmed the smaller/more compact size of the GPA1t compared to GPA1. According to the CD analyses its thermal stability is higher than that of GPA1. In future studies conditions for the co-existence of the two species (monomer and trimer) and its physiological significance need to be investigated. Moreover, shape models for both species need to be developed to understand AtGPA1 interactions with other components in the heterotrimer.

Plant Morphology of Heterotrimeric G Protein Mutants.

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.

Chapter Two - Heterotrimeric G Protein Ubiquitination as a Regulator of G Protein Signaling.

Ubiquitin-mediated regulation of G proteins has been known for over 20 years as a result of discoveries made independently in yeast and vertebrate model systems for pheromone and photoreception, respectively. Since that time, several details underlying the cause and effect of G protein ubiquitination have been determined-including the initiating signals, responsible enzymes, trafficking pathways, and their effects on protein structure, function, interactions, and cell signaling. The collective body of evidence suggests that Gα subunits are the primary targets of ubiquitination. However, longstanding and recent results suggest that Gβ and Gγ subunits are also ubiquitinated, in some cases impacting cell polarization-a process essential for chemotaxis and polarized cell growth. More recently, evidence from mass spectrometry (MS)-based proteomics coupled with advances in PTM bioinformatics have revealed that protein families representing G protein subunits contain several structural hotspots for ubiquitination-most of which have not been investigated for a functional role in signal transduction. Taken together, our knowledge and understanding of heterotrimeric G protein ubiquitination as a regulator of G protein signaling-despite 20 years of research-is still emerging.

Type B Heterotrimeric G Protein γ-Subunit Regulates Auxin and ABA Signaling in Tomato.

Heterotrimeric G proteins composed of α, β, and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein-SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato.

Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean.

Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gβ, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gβ and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants.

Heterotrimeric G protein subunits differentially respond to endoplasmic reticulum stress in Arabidopsis

Canonical heterotrimeric G proteins in eukaryotes are major components that localize at plasma membrane and transmit extracellular stimuli into the cell. Genome of a seed plant Arabidopsis thaliana encodes at least one Gα (GPA1), one Gβ (AGB1), and 3 Gγ (AGG1, AGG2 and AGG3) subunits. The loss-of-function mutations of G protein subunit(s) cause multiple defects in development as well as biotic and abiotic stress responses. However, it remains elusive how these subunits differentially express these defects. Here, we report that Arabidopsis heterotrimeric G protein subunits differentially respond to the endoplasmic reticulum (ER) stress. An isolated homozygous mutant of AGB1, agb1-3, was more sensitive to the tunicamycin-induced ER stress compared to the wild type and the other loss-of-function mutants of G protein subunits. Moreover, ER stress responsive genes were highly expressed in the agb1-3 plant. Our results indicate that AGB1 positively contributes to ER stress tolerance in Arabidopsis.

FgRIC8 is involved in regulating vegetative growth, conidiation, deoxynivalenol production and virulence in Fusarium graminearum

Proteins of the resistance to inhibitors of cholinesterase 8 (Ric8) group act as guanine nucleotide exchange factors (GEFs) and play important roles in regulating G-protein signaling in animals. In filamentous fungi, putative Ric8 orthologs have so far been identified in Magnaporthe oryzae, Neurospora crassa, Aspergillus nidulans and Aspergillus fumigatus. Here, we report the functional investigation of a potential RIC8 ortholog (FgRIC8) in the wheat head blight pathogen Fusarium graminearum. Targeted gene deletion mutants of FgRIC8 exhibited a significant reduction in vegetative growth, conidiation, pigment production as well as deoxynivalenol (DON) biosynthesis. Pathogenicity assays using a point-inoculated spikelet approach showed that the mutants were severely impaired in virulence on flowering wheat heads. Quantitative RT-PCR analysis revealed that genes encoding F. graminearum Gα (FgGpa1 and FgGpa3), Gβ (FgGpb1) and Gγ (FgGpg1) subunits were significantly down-regulated in Fgric8 mutants. Moreover, we showed that FgRic8 physically interacts with both FgGpa1 and FgGpa3, but not FgGpa2, in yeast two-hybrid assays. The intracellular cAMP levels in Fgric8 mutants were significantly decreased compared to the isogenic wild-type strain. Taken together, our results indicate that FgRic8 plays critical roles in fungal development, secondary metabolism and virulence in F. graminearum and may act as a regulator of G protein alpha subunits.

Rice G-protein subunits qPE9-1 and RGB1 play distinct roles in abscisic acid responses and drought adaptation.

Heterotrimeric GTP-binding protein (G-protein)-mediated abscisic acid (ABA) and drought-stress responses have been documented in numerous plant species. However, our understanding of the function of rice G-protein subunits in ABA signalling and drought tolerance is limited. In this study, the function of G-protein subunits in ABA response and drought resistance in rice plants was explored. It was found that the transcription level of qPE9-1 (rice Gγ subunit) gradually decreased with increasing ABA concentration and the lack of qPE9-1 showed an enhanced drought tolerance in rice plants. In contrast, mRNA levels of RGB1 (rice Gβ subunit) were significantly upregulated by ABA treatment and the lack of RGB1 led to reduced drought tolerance. Furthermore, the results suggested that qPE9-1 negatively regulates the ABA response by suppressing the expression of key transcription factors involved in ABA and stress responses, while RGB1 positively regulates ABA biosynthesis by upregulating NCED gene expression under both normal and drought stress conditions. Taken together, it is proposed that RGB1 is a positive regulator of the ABA response and drought adaption in rice plants, whereas qPE9-1 is modulated by RGB1 and functions as a negative regulator in the ABA-dependent drought-stress responses.

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling

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.

The putative Gγ subunit gene MGG1 is required for conidiation, appressorium formation, mating and pathogenicity in Magnaporthe oryzae.

Heterotrimeric G-proteins play key roles in the transduction of extracellular signals to various downstream effectors in eukaryotes. In our previous study, a T-DNA insertional mutant A1-412, in which the promoter of a putative Gγ subunit gene MGG1 was disrupted, was impaired in asexual/sexual sporulation, appressorium formation, and pathogenicity in Magnaporthe oryzae. Here the roles of MGG1 in regulating fungal development and plant infection were further investigated and verified using a gene deletion strategy. Targeted gene deletion mutants of MGG1 exhibited similar phenotypes to those of A1-412. The Δmgg1 mutants were unable to differentiate appressorium on hydrophobic surfaces and nonpathogenic to susceptible hosts. The defects of the Δmgg1 mutants in appressorium formation were partially restored by adding exogenous cAMP or IBMX (a phosphodiesterase inhibitor), although the induced appressoria were still nonfunctional. Expressing Mgg1-GFP fusion protein in an Δmgg1 mutant could complement all phenotypes of the mutant, and bright GFP fluorescence was observed at the periphery of fungal cells, indicating that Mgg1 mainly localizes to plasma membrane. Quantitative RT-PCR analysis revealed that deletion of MGG1 resulted in a significant reduction in mRNA levels of the genes encoding Gα (MagA, MagB, and MagC), Gβ (Mgb1), and adenylate cyclase (Mac1). Moreover, intracellular cAMP accumulation was significantly reduced in Δmgg1 mutants compared to that in the wild-type strain. Taken together, our results suggested that Gγ subunit Mgg1 might act upstream of cAMP signaling pathway and play critical roles in regulation of conidiation, appressorium formation, mating, and plant infection in M. oryzae.

Gβ4γ1 as a modulator of M3 muscarinic receptor signalling and novel roles of Gβ1 subunits in the modulation of cellular signalling

Much is known about the how Gβγ subunits regulate effectors in response to G protein-coupled receptor stimulation. However, there is still a lot we don't know about how specific combinations of Gβ and Gγ are wired into different signalling pathways. Here, using an siRNA screen for different Gβ and Gγ subunits, we examined an endogenous M3 muscarinic receptor signalling pathway in HEK 293 cells. We observed that Gβ4 subunits were critical for calcium signalling and a downstream surrogate measured as ERK1/2 MAP kinase activity. A number of Gγ subunits could partner with Gβ4 but the best coupling was seen via Gβ4γ1. Intriguingly, knocking down Gβ1 actually increased signalling through the M3-mAChR most likely via an increase in Gβ4 levels. We noted that Gβ1 occupies the promoter of Gβ4 and may participate in maturation of its mRNA. This highlights a new role for Gβγ signalling beyond their canonical roles in cellular signalling.