Heterotrimeric G-protein Complex Research Articles

Generation of Comprehensive GPCR-Transducer-Deficient Cell Lines to Dissect the Complexity of GPCR Signaling.

G-protein-coupled receptors (GPCRs) compose the largest family of transmembrane receptors and are targets of approximately one-third of Food and Drug Administration-approved drugs owing to their involvement in almost all physiologic processes. GPCR signaling occurs through the activation of heterotrimeric G-protein complexes and β-arrestins, both of which serve as transducers, resulting in distinct cellular responses. Despite seeming simple at first glance, accumulating evidence indicates that activation of either transducer is not a straightforward process as a stimulation of a single molecule has the potential to activate multiple signaling branches. The complexity of GPCR signaling arises from the aspects of G-protein-coupling selectivity, biased signaling, interpathway crosstalk, and variable molecular modifications generating these diverse signaling patterns. Numerous questions relative to these aspects of signaling remained unanswered until the recent development of CRISPR genome-editing technology. Such genome editing technology presents opportunities to chronically eliminate the expression of G-protein subunits, β-arrestins, G-protein-coupled receptor kinases (GRKs), and many other signaling nodes in the GPCR pathways at one's convenience. Here, we review the practicality of using CRISPR-derived knockout (KO) cells in the experimental contexts of unraveling the molecular details of GPCR signaling mechanisms. To mention a few, KO cells have revealed the contribution of β-arrestins in ERK activation, Gα protein selectivity, GRK-based regulation of GPCRs, and many more, hence validating its broad applicability in GPCR studies. SIGNIFICANCE STATEMENT: This review emphasizes the practical application of G-protein-coupled receptor (GPCR) transducer knockout (KO) cells in dissecting the intricate regulatory mechanisms of the GPCR signaling network. Currently available cell lines, along with accumulating KO cell lines in diverse cell types, offer valuable resources for systematically elucidating GPCR signaling regulation. Given the association of GPCR signaling with numerous diseases, uncovering the system-based signaling map is crucial for advancing the development of novel drugs targeting specific diseases.

Agronomic potential of plant-specific Gγ proteins.

The vascular plant-specific type III Gγ proteins have emerged as important targets for biotechnological applications. These proteins are exemplified by Arabidopsis AGG3, rice Grain Size 3 (GS3), Dense and Erect Panicle 1 (DEP1), and GGC2 and regulate plant stature, seed size, weight and quality, nitrogen use efficiency, and multiple stress responses. These Gγ proteins are an integral component of the plant heterotrimeric G-protein complex and differ from the canonical Gγ proteins due to the presence of a long, cysteine-rich C-terminal region. Most cereal genomes encode three or more of these proteins, which have similar N-terminal Gγ domains but varying lengths of the C-terminal domain. The C-terminal domain is hypothesized to give specificity to the protein function. Intriguingly, many accessions of cultivated cereals have natural deletion of this region in one or more proteins, but the mechanistic details of protein function remain perplexing. Distinct, sometimes contrasting, effects of deletion of the C-terminal region have been reported in different crops or under varying environmental conditions. This review summarizes the known roles of type III Gγ proteins, the possible action mechanisms, and a perspective on what is needed to comprehend their full agronomic potential.

Emerging Trends in Big Data Analysis in Computational Biology and Bioinformatics in Health Informatics: A Case Study on Epilepsy and Seizures.

Advanced technology innovations allow cost-effective, high-throughput profiling of biological systems. It enabled genome sequencing in days using advanced technologies (e.g., next-generation sequencing, microarrays, and mass spectrometry). Since technology has been developed, massive biological data (e.g., genomics, proteomics) has been produced cheaply, allowing the "big data" era to create new opportunities to solve medical and biological complications in many disciplines-preventive medicine, biology, Personalized Medicine, gene sequencing, healthcare, and industry. Computational biology and bioinformatics are interdisciplinary fields that develop and apply computational methods (e.g., analytical methods, mathematical modeling, and simulation) to analyze large collections of biological data, such as genetic sequences, cell populations, or protein samples, to make new predictions or discover new biology. Biological data storage, mining, and analysis have challenges because data is much more heterogeneous. In this study, the big data resources of genomics, proteomics, and metabolomics have been explored to solve biological problems using big data analysis approaches. The goal is to build a network of relationship-based gene-disease associations to prioritize phenotypes common to epilepsy and seizure disease. Through network analysis, The 10 seed genes, 22 associated genes, 132 microRNAs, and 38 transcription factors have been identified that have a direct effect on all forms of epilepsy and seizures. The majority of seed genes, according to the results of a functional analysis of seed genes, are involved in the acetylcholine-gated channel complex (10%) and the heterotrimeric G-protein complex (10%) pathways related to cellular components, followed by a role in the regulation of action potential (20%) and positive regulation of vascular endothelial growth factor production (20%) in Epilepsy and Seizures pathways related to biological processes. This study might provide insight into the workings of the disease and shows the importance of continued research into epilepsy and other conditions that can trigger seizure activity.

Overexpression of a Plant Specific Gγ Protein, AGG3, in the Model Monocot Setaria viridis Confers Tolerance to Heat Stress.

The vascular plant-specific, cysteine-rich group III Gγ proteins, which are integral components of the heterotrimeric G protein complex, play crucial roles in regulating a multitude of plant processes, including those related to crop yield and responses to abiotic stresses. The presence of multiple copies of group III Gγ proteins in most plants and a propensity of the presence of specific truncated alleles in many cultivated crops, presents an ambiguous picture of their roles in modulating specific responses. AGG3 is a canonical group III Gγ protein of Arabidopsis, and its overexpression in additional model crops offers the opportunity to directly evaluate the effects of protein expression levels on plant phenotypes. We have shown that AGG3 overexpression in the monocot model Setaria viridis leads to an increase in seed yield. In this study, we have investigated the response of the S. viridis plants overexpressing AGG3 to heat stress (HS), one of the most important abiotic stresses affecting crops worldwide. We show that a short span of HS at a crucial developmental time point has a significant effect on plant yield in the later stages. We also show that plants with higher levels of AGG3 are more tolerant to HS. This is attributed to a higher upregulation of stress-responsive genes and improved modulation of the photosynthetic efficiency during the stress. Overall, our results confirm that AGG3 plays a crucial role in regulating plant responses to unfavorable environmental conditions and may contribute positively to avoiding crop yield losses.

Microarray and in silico analysis of DNA repair genes between human testis of patients with nonobstructive azoospermia and normal cells.

DNA repair processes are critical to maintaining genomic integrity. As a result, dysregulation of repair genes is likely to be linked with health implications, such as an increased prevalence of infertility and an accelerated rate of aging. We evaluated all the DNA repair genes (322 genes) by microarray. This study has provided insight into the connection between DNA repair genes, including RAD23B, OBFC2A, PMS1, UBE2V1, ERCC5, SMUG1, RFC4, PMS2L5, MMS19, SHFM1, INO80, PMS2L1, CHEK2, TRIP13, and POLD4. The microarray analysis of six human cases with different nonobstructive azoospermia revealed that RAD23B, OBFC2A, PMS1, UBE2V1, ERCC5, SMUG1, RFC4, PMS2L5, MMS19, SHFM1, and INO80 were upregulated, and expression of PMS2L1, CHEK2, TRIP13, and POLD4 was downregulated versus the normal case. For this purpose, Enrich Shiny GO, STRING, and Cytoscape online evaluation was applied to predict proteins' functional and molecular interactions and then performed to recognize the master pathways. Functional enrichment analysis revealed that the biological process (BP) terms "base-excision repair, AP site formation,""nucleotide-excision repair, DNA gap filling," and "nucleotide-excision repair, preincision complex assembly" was significantly overexpressed in upregulated differentially expressed genes (DEGs). BP analysis of downregulated DEGs highlighted "histone phosphorylation,""DNA damage response, detection DNA response,""mitotic cell cycle checkpoint signaling,"and "double-strand break repair."Overrepresented molecular function (MF) terms in upregulated DEGs included "Oxidized base lesion DNA N-glycosylase activity,""uracil DNA N-glycosylase activity,""bubble DNA binding" and "DNA clamp loader activity."Interestingly, MF investigation of downregulated DEGs showed overexpression in "heterotrimeric G-protein complex,""5'-deoxyribose-5-phosphate lyase activity,""minor groove of adenine-thymine-rich DNA binding," and "histone kinase activity."Our findings suggest that these genes and their interacting hub proteins could help determine the pathophysiology of germ cell abnormalities and infertility.

A Model of Potassium‐Assisted Olfactory Sensory Neuron Response to Odorant

Odorants interact with specialized G‐protein coupled receptors lining the cilia of the olfactory sensory neuron initiating an intracellular cascade of signal transduction events resulting in cAMP‐gated ion channel opening and generation of ion currents. These ion currents polarize the cilia membrane resulting in the voltage transition across the olfactory epithelium, which can be recorded as an electroolfactogram (EOG). The observed dose‐dependent change of the EOG amplitude after stimulation of olfactory epithelium with the mixture of odorant and electron donors and acceptors indicates a potential involvement of electrons in the initial events of signal transduction. Our model of signal transduction consists of a dimer of the G‐protein coupled olfactory receptors positioned vertically in the phospholipid bilayer between olfactory extracellular mucus layer and the intracellular cytoplasm, within the cilia of the olfactory sensory neuron. In this model, two identical receptors of the dimer form an odorant activated potassium ion channel. Prior to odorant binding at the receptor, the ion channel gate is closed. Each receptor has a single biding site for the odorant molecule on the extracellular end located within the mucus layer, and a binding site for the heterotrimeric G‐protein complex within the cytoplasm. The brief binding of odorant molecules to the mucosal part of the receptors causes the ion channel to open and pass intracellular potassium ions to the extracellular mucus layer. The potassium‐coupled electrons polarize the cytoplasmic portion of the receptors and facilitate activation of the bound G‐protein complex thereby influencing the subsequent signal cascade.

Genome‑wide characterization of the Gα subunit gene family in Rosaceae and expression analysis of PbrGPAs under heat stress.

The Gα subunit is an important component of the heterotrimeric G-protein complex and an integral component of several signal transduction pathways. It plays crucial roles in the diverse processes of plant growth and development, including the response to abiotic stress, regulation of root development, involvement in stomatal movement, and participation in hormone responses, which have been well investigated in many species. However, no comprehensive analysis has identified and explored the evolution, expression pattern characteristics and heat stress response of the Gα subunit genes in Rosaceae. In this study, 52 Gα subunit genes were identified in eight Rosaceae species; these genes were divided into three subfamilies (I, II, and III) based on their phylogenetic, conserved motif, and structural characteristics. Whole genome and dispersed duplication events were found to have contributed significantly to the expansion of the Gα subunit gene family, and purifying selection to have played a key role in the evolution of Gα subunit genes. An expression analysis identified some PbrGPA genes that were highly expressed in leaf, root, and fruit, and exhibited diverse spatiotemporal expression models in pear. Under abiotic stress conditions, the mRNA transcript levels of PbrGPA genes were up-regulated in response to high temperature treatment in leaves. Furthermore, three Gα subunit genes were shown to be located in the plasma membrane and nucleus in pear. In conclusion, the study of the Gα subunit gene family will help us to better understand its evolutionary history and expression patterns, while facilitating further investigations into the function of the Gα subunit gene in response to heat stress.

The stress induced caleosin, RD20/CLO3, acts as a negative regulator of GPA1 in Arabidopsis.

A stress induced calcium-binding protein, RD20/CLO3 interacts with the alpha subunit of the heterotrimeric G-protein complex in Arabidopsis and affects etiolation and leaf morphology. Heterotrimeric G proteins and calcium signaling have both been shown to play a role in the response to environmental abiotic stress in plants; however, the interaction between calcium-binding proteins and G-protein signaling molecules remains elusive. We investigated the interaction between the alpha subunit of the heterotrimeric G-protein complex, GPA1, of Arabidopsis thaliana with the calcium-binding protein, the caleosin RD20/CLO3, a gene strongly induced by drought, salt and abscisic acid. The proteins were found to interact in vivo by bimolecular fluorescent complementation (BiFC); the interaction was localized to the endoplasmic reticulum and to oil bodies within the cell. The constitutively GTP-bound GPA1 (GPA1QL) also interacts with RD20/CLO3 as well as its EF-hand mutant variations and these interactions are localized to the plasma membrane. The N-terminal portion of RD20/CLO3 was found to be responsible for the interaction with GPA1 and GPA1QL using both BiFC and yeast two-hybrid assays. RD20/CLO3 contains a single calcium-binding EF-hand in the N-terminal portion of the protein; disruption of the calcium-binding capacity of the protein obliterates interaction with GPA1 in in vivo assays and decreases the interaction between the caleosin and the constitutively active GPA1QL. Analysis of rd20/clo3 mutants shows that RD20/CLO3 plays a key role in the signaling pathway controlling hypocotyl length in dark grown seedlings and in leaf morphology. Our findings indicate a novel role for RD20/CLO3 as a negative regulator of GPA1.

Deep transcriptome sequencing of subgenual anterior cingulate cortex reveals cross-diagnostic and diagnosis-specific RNA expression changes in major psychiatric disorders

Despite strong evidence of heritability and growing discovery of genetic markers for major mental illness, little is known about how gene expression in the brain differs across psychiatric diagnoses, or how known genetic risk factors shape these differences. Here we investigate expressed genes and gene transcripts in postmortem subgenual anterior cingulate cortex (sgACC), a key component of limbic circuits linked to mental illness. RNA obtained postmortem from 200 donors diagnosed with bipolar disorder, schizophrenia, major depression, or no psychiatric disorder was deeply sequenced to quantify expression of over 85,000 gene transcripts, many of which were rare. Case–control comparisons detected modest expression differences that were correlated across disorders. Case–case comparisons revealed greater expression differences, with some transcripts showing opposing patterns of expression between diagnostic groups, relative to controls. The ~250 rare transcripts that were differentially-expressed in one or more disorder groups were enriched for genes involved in synapse formation, cell junctions, and heterotrimeric G-protein complexes. Common genetic variants were associated with transcript expression (eQTL) or relative abundance of alternatively spliced transcripts (sQTL). Common genetic variants previously associated with disease risk were especially enriched for sQTLs, which together accounted for disproportionate fractions of diagnosis-specific heritability. Genetic risk factors that shape the brain transcriptome may contribute to diagnostic differences between broad classes of mental illness.

A novel polymorphism in the 5′ UTR of HvDEP1 is associated with grain length and 1000-grain weight in barley (Hordeum vulgare)

The gene HvDEP1, on barley (Hordeum vulgare L.) chromosome 5H, encodes a γ-subunit of the heterotrimeric G-protein complex and was previously determined to be a candidate gene underlying a major quantitative trait locus for grain length. In the present study, we identified a 9 bp indel (insertion–deletion mutation) at position –84 bp from the start codon within a reported upstream open reading frame located in the 5′ UTR (untranslated region) and developed a diagnostic molecular marker. We also identified a 13 bp indel (–514 bp) in linkage disequilibrium that bridges an important regulatory motif. Using a doubled-haploid population and a barley diversity panel, we were able to show that the effects of these indels were environmentally stable and consistently delineated phenotypic groups based on grain length and 1000-grain weight. Genotypes represented by deletions at these two positions relative to the reference cv. Morex had consistently shorter grains, by 3.69–3.96%, and lower 1000-grain weight, by 2.38–4.21%, in a doubled-haploid population studied. Additionally, a diversity panel was tested but consistent differences were observed only for grain length, reinforcing literature indicating the importance of this gene for grain-length regulation. The frequency of the longer and heavier grained reference allele was higher in modern cultivars, suggesting that indirect selection for longer grain may have occurred through direct selection for grain yield via grain-weight improvement. These results indicate that grain length and 1000-grain weight in barley can be manipulated by targeting variation in gene promoters through marker-assisted selection.

The Nucleotide-Dependent Interactome of Rice Heterotrimeric G-Protein α -Subunit.

The rice heterotrimeric G-protein complex, a guanine-nucleotide-dependent on-off switch, mediates vital cellular processes and responses to biotic and abiotic stress. Exchange of bound GDP (resting state) for GTP (active state) is spontaneous in plants including rice and thus there is no need for promoting guanine nucleotide exchange in vivo as a mechanism for regulating the active state of signaling as it is well known for animal G signaling. As such, a master regulator controlling the G-protein activation state is unknown in plants. Therefore, an ab initio approach is taken to discover candidate regulators. The rice Gα subunit (RGA1) is used as bait to screen for nucleotide-dependent protein partners. A total of 264 proteins are identified by tandem mass spectrometry of which 32 were specific to the GDP-bound inactive state and 22 specific to the transition state. Approximately, 10% are validated as previously identified G-protein interactors.

Clinical Characteristics of GNB1 and GNAS Mutations in an Unselected Cohort of 6,343 Patients with Hematologic Abnormalities

Genes encoding the alpha (GNAS) and beta (GNB1) subunits of the heterotrimeric G-protein complex are recurrently mutated in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Alterations in G-protein coupled receptors can affect signaling via the PI3K/AKT/mTOR and RAS/MAPK pathways, suggesting that GNB1/GNAS mutations may function similarly to mutations in RAS and tyrosine kinases in myeloid disease progression. However, unlike RAS mutations, GNB1/GNAS mutations are among the most commonly affected genes in clonal hematopoiesis of indeterminate potential (CHIP), suggestive of a distinct functional role in myeloid disease initiation.We evaluated 6343 unique patients who received gene panel sequencing at our institution as part of a diagnostic evaluation of known or suspected hematologic malignancy. We identified 68 patients that had at least 1 sample with a mutation in GNB1 (N =42), GNAS (N=24) or both (N=2) (Figure 1). Twenty-six patients had multiple samples (range 2-10) obtained at serial timepoints during the course of progression and treatment. GNB1 mutations affected codon 57 (K57E/M/N/T) in 38 out of 42 cases; the remaining were G53E, D76G, I81V, and K89R. GNAS mutations affected codon 201 (R201H/C) in 23 out of 24 cases.Forty patients had active myeloid neoplasms [AML (N=10), MDS (N=13), MPN (N=11), MDS/MPN (N=5), BPDCN (N=1)]. The distribution of myeloid diagnoses was similar among patients with GNB1 or GNAS mutations. Analysis of serial samples showed that GNB1/GNAS mutations were frequently acquired at the time of leukemic transformation and clinical progression. Among 5 patients with secondary AML, 2 patients acquired GNB1/GNAS mutations at the time of transformation from MDS to AML, and 3 patients were found to have GNB1/GNAS mutations in high-risk MDS prior to subsequent transformation to AML. After induction chemotherapy for AML, two patients had expanded or newly acquired GNB1 mutations at time of treatment failure.Notably, 15 out of the 40 (37.5%) patients with active myeloid disease had co-mutations in the MPN-associated genes JAK2, CALR, and MPL. Eleven of these patients had a clinical diagnosis of MPN, including myelofibrosis (n=5), essential thrombocytosis (n=3), polycythemia vera (n=2), and systemic mastocytosis (n=1). In the context of underlying oncogenic kinase alterations including JAK2 V617F, GNB1 mutations have been shown to promote resistance to kinase inhibitors in vitro. Fourteen out of fifteen patients had no history of ruxolitinib therapy, suggesting that these mutations arise during the course of disease progression in the absence of therapeutic selection pressures. The impact of GNB1/GNAS mutations on the clinical response to JAK inhibitors has not been evaluated.Twenty-eight patients in the cohort (41%) had no evidence of active myeloid disease, including 14 patients with a lymphoid malignancy [mature B-cell neoplasms (n=8), mature T cell neoplasms (n=3), plasma cell neoplasms (n=2), acute lymphoblastic leukemia (n=1)], 6 patients with AML in complete remission, and 8 patients without a clinical diagnosis of hematologic malignancy. Four of the AML patients had a GNAS or GNB1 mutation that was newly acquired or persistent at the time of complete remission following induction chemotherapy. All 4 patients underwent allogeneic stem cell transplant (HSCT) based on disease risk at diagnosis and are alive with median follow up of 2.5 years. In 1 patient, a new GNB1 mutation was identified immediately after HSCT in the context of 100% donor chimerism, suggesting donor-engrafted CHIP. All 8 patients without hematologic malignancy were evaluated for cytopenias or cytoses at the time of the identification of the GNB1/GNAS mutation.GNB1 and GNAS are recurrently mutated in diverse clinical and genetic contexts. In patients with active myeloid disease, MPN-associated mutations were common and GNB1/GNAS mutations were newly acquired at the time of clinical progression, suggesting a functional role similar to other gene mutations that activate mitogenic signaling pathways. However, many GNB1 and GNAS mutations are seen as sole mutations in the absence of active myeloid disease, suggesting that unlike other signaling mutations they can drive initiation of clonal hematopoiesis. The diverse clinical spectrum of patients with GNB1/GNAS mutations suggests that these mutations have distinct, context dependent effects in hematopoietic cells. [Display omitted] DisclosuresKim:Aushon Biosciences: Consultancy; LabCorp, Inc.: Consultancy; Papgene, Inc: Consultancy. Lane:N-of-one: Consultancy; Stemline Therapeutics: Research Funding.

Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β-subunit of the heterotrimeric G-protein.

Arabidopsis heterotrimeric G-protein complex modulates pathogen-associated molecular pattern-triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ- (agb1-2) or Gγ-subunits have an altered wall composition compared with wild-type plants. Here we performed a mutant screen to identify suppressors of agb1-2 (sgb) that restore susceptibility to pathogens to wild-type levels. Out of the four sgb mutants (sgb10-sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant-specific polysaccharide O-acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1-7) of ESK1 restore to wild-type levels the enhanced susceptibility of agb1-2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1-2 esk1-7 double mutant was not the result of the re-activation of deficient PTI responses in agb1-2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan-derived metabolites, and the accumulation of disease resistance-related secondary metabolites and different osmolites. These esk1-mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1-2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI-mediated resistance is partially compensated by the activation of specific cell-wall-triggered immune responses.

Phosphatidic acid binding inhibits RGS1 activity to affect specific signaling pathways in Arabidopsis.

Modulation of the active versus inactive forms of the Gα protein is critical for the signaling processes mediated by the heterotrimeric G-protein complex. We have recently established that in Arabidopsis, the regulator of G-protein signaling (RGS1) protein and a lipid-hydrolyzing enzyme, phospholipase Dα1 (PLDα1), both act as GTPase-activity accelerating proteins (GAPs) for the Gα protein to attenuate its activity. RGS1 and PLDα1 interact with each other, and RGS1 inhibits the activity of PLDα1 during regulation of a subset of responses. In this study, we present evidence that this regulation is bidirectional. Phosphatidic acid (PA), a second messenger typically derived from the lipid-hydrolyzing activity of PLDα1, is a molecular target of RGS1. PA binds and inhibits the GAP activity of RGS1. A conserved lysine residue in RGS1 (Lys259 ) is directly involved in RGS1-PA binding. Introduction of this RGS1 protein variant in the rgs1 mutant background makes plants hypersensitive to a subset of abscisic acid-mediated responses. Our data point to the existence of negative feedback loops between these two regulatory proteins that precisely modulate the level of active Gα, consequently generating a highly controlled signal-response output.

A Mutation in the G-Protein Gene GNB2 Causes Familial Sinus Node and Atrioventricular Conduction Dysfunction.

Familial sinus node and atrioventricular conduction dysfunction is a rare disorder that leads to paroxysmal dizziness, fatigue, and syncope because of a temporarily or permanently reduced heart rate. To date, only a few genes for familial sinus and atrioventricular conduction dysfunction are known, and the majority of cases remain pathogenically unresolved. We aim to identify the disease gene in a large 3-generation family (n=25) with autosomal dominant sinus node dysfunction (SND) and atrioventricular block (AVB) and to characterize the mutation-related pathomechanisms in familial SND+AVB. Genome-wide linkage analysis mapped the SND+AVB disease locus to chromosome 7q21.1-q31.1 (2-point logarithm of the odds score: 4.64; θ=0); in this region, targeted exome sequencing identified a novel heterozygous mutation (p.Arg52Leu) in the GNB2 gene that strictly cosegregated with the SND+AVB phenotype. GNB2 encodes the β2 subunit (Gβ2) of the heterotrimeric G-protein complex that is being released from G-protein-coupled receptors on vagal stimulation. In 2 heterologous expression systems (HEK-293T cells and Xenopus laevis oocytes), an enhanced activation of the G-protein-activated K+ channel (GIRK; Kir3.1/Kir3.4) was shown when mutant Gβ2 was coexpressed with Gγ2; this was in contrast to coexpression of mutant Gβ2-Gγ2 with other cardiac ion channels (HCN4, HCN2, and Cav1.2). Molecular dynamics simulations suggested a reduced binding property of mutant Gβ2 to cardiac GIRK channels when compared with native Gβ2. A GNB2 gene mutation is associated with familial SND+AVB and leads to a sustained activation of cardiac GIRK channels, which is likely to hyperpolarize the myocellular membrane potential and thus reduces their spontaneous activity. Our findings describe for the first time a role of a mutant G-protein in the nonsyndromic pacemaker disease because of GIRK channel activation.

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.

A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells.

G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gαi1, Gαi2 and Gαi3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gαi subunit, and cp173Venus fused to the Gγ2 subunit as acceptor. The Gαi FRET biosensors constructs are expressed together with Gβ1 from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gαi FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gαi FRET sensor in single cells upon stimulation of several GPCRs, including the LPA2, M3 and BK2 receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gαi1, Gαi2 and Gαi3 activation will be valuable for live-cell measurements that probe Gαi activation.

Heterotrimeric G-proteins in Picea abies and their regulation in response to Heterobasidion annosum s.l. infection.

BackgroundHeterotrimeric G-proteins are important signalling switches, present in all eukaryotic kingdoms. In plants they regulate several developmental functions and play an important role in plant-microbe interactions. The current knowledge on plant G-proteins is mostly based on model angiosperms and little is known about the G-protein repertoire and function in other lineages. In this study we investigate the heterotrimeric G-protein subunit repertoire in Pinaceae, including phylogenetic relationships, radiation and sequence diversity levels in relation to other plant linages. We also investigate functional diversification of the G-protein complex in Picea abies by analysing transcriptional regulation of the G-protein subunits in different tissues and in response to pathogen infection.ResultsA full repertoire of G-protein subunits in several conifer species were identified in silico. The full-length P. abies coding regions of one Gα-, one Gβ- and four Gγ-subunits were cloned and sequenced. The phylogenetic analysis of the Gγ-subunits showed that PaGG1 clustered with A-type-like subunits, PaGG3 and PaGG4 clustered with C-type-like subunits, while PaGG2 and its orthologs represented a novel conifer-specific putative Gγ-subunit type. Gene expression analyses by quantitative PCR of P. abies G-protein subunits showed specific up-regulation of the Gα-subunit gene PaGPA1 and the Gγ-subunit gene PaGG1 in response to Heterobasidion annosum sensu lato infection.ConclusionsConifers possess a full repertoire of G-protein subunits. The differential regulation of PaGPA1 and PaGG1 indicates that the heterotrimeric G-protein complex represents a critical linchpin in Heterobasidion annosum s.l. perception and downstream signaling in P. abies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0676-1) contains supplementary material, which is available to authorized users.

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.

Pathogen-secreted proteases activate a novel plant immune pathway.

Mitogen-Activated Protein Kinase (MAPK) cascades play central roles in innate immune signaling networks in plants and animals1,2. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive1. We report that pathogen-secreted proteases activate a previously unknown signaling pathway in Arabidopsis thaliana involving the Gα, Gβ and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of a MAPK cascade. In this pathway, Receptor for Activated C Kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G protein signaling to downstream activation of a MAPK cascade. The protease-G protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signaling pathways such as the one elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to a MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the novel protease-mediated immune signaling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel types of immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.