Heterotrimeric G-protein Subunits Research Articles

SymRK Regulates G-Protein Signaling During Nodulation in Soybean (Glycine max) by Modifying RGS Phosphorylation and Activity.

Molecular interspecies dialogue between leguminous plants and nitrogen-fixing rhizobia results in the development of symbiotic root nodules. This is initiated by several nodulation-related receptors present on the surface of root hair epidermal cells. We have shown previously that specific subunits of heterotrimeric G-proteins and their associated regulator of G-protein signaling (RGS) proteins act as molecular links between the receptors and downstream components during nodule formation in soybeans. Nod factor receptor 1 (NFR1) interacts with and phosphorylates RGS proteins to regulate the G-protein cycle. Symbiosis receptor-like kinases (SymRK) phosphorylate Gα to make it inactive and unavailable for Gβγ. We now show that like NFR1, SymRK also interacts with the RGS proteins to phosphorylate them. Phosphorylated RGS has higher activity for accelerating guanosine triphosphate (GTP) hydrolysis by Gα, which favors conversion of active Gα to its inactive form. Phosphorylation of RGS proteins is physiologically relevant, as overexpression of a phospho-mimic version of the RGS protein enhances nodule formation in soybean. These results reveal an intricate fine-tuning of the G-protein signaling during nodulation, where a negative regulator (Gα) is effectively deactivated by RGS due to the concerted efforts of several receptor proteins to ensure adequate nodulation. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Gαi2 Protein Inhibition Blocks Chemotherapy- and Anti-Androgen-Induced Prostate Cancer Cell Migration.

We have previously shown that heterotrimeric G-protein subunit alphai2 (Gαi2) is essential for cell migration and invasion in prostate, ovarian and breast cancer cells, and novel small molecule inhibitors targeting Gαi2 block its effects on migratory and invasive behavior. In this study, we have identified potent, metabolically stable, second generation Gαi2 inhibitors which inhibit cell migration in prostate cancer cells. Recent studies have shown that chemotherapy can induce the cancer cells to migrate to distant sites to form metastases. In the present study, we determined the effects of taxanes (docetaxel), anti-androgens (enzalutamide and bicalutamide) and histone deacetylase (HDAC) inhibitors (SAHA and SBI-I-19) on cell migration in prostate cancer cells. All treatments induced cell migration, and simultaneous treatments with new Gαi2 inhibitors blocked their effects on cell migration. We concluded that a combination treatment of Gαi2 inhibitors and chemotherapy could blunt the capability of cancer cells to migrate and form metastases.

TIPE proteins control directed migration of human T cells by directing GPCR and lipid second messenger signaling.

Tissue infiltration by circulating leukocytes via directed migration (also referred to as chemotaxis) is a common pathogenic mechanism of inflammatory diseases. G protein-coupled receptors (GPCRs) are essential for sensing chemokine gradients and directing the movement of leukocytes during immune responses. The tumor necrosis factor α-induced protein 8-like (TIPE or TNFAIP8L) family of proteins are newly described pilot proteins that control directed migration of murine leukocytes. However, how leukocytes integrate site-specific directional cues, such as chemokine gradients, and utilize GPCR and TIPE proteins to make directional decisions are not well understood. Using both gene knockdown and biochemical methods, we demonstrated here that 2 human TIPE family members, TNFAIP8 and TIPE2, were essential for directed migration of human CD4+ T cells. T cells deficient in both of these proteins completely lost their directionality. TNFAIP8 interacted with the Gαi subunit of heterotrimeric (α, β, γ) G proteins, whereas TIPE2 bound to PIP2 and PIP3 to spatiotemporally control immune cell migration. Using deletion and site-directed mutagenesis, we established that Gαi interacted with TNFAIP8 through its C-terminal amino acids, and that TIPE2 protein interacted with PIP2 and PIP3 through its positively charged amino acids on the α0 helix and at the grip-like entrance. We also discovered that TIPE protein membrane translocation (i.e. crucial for sensing chemokine gradients) was dependent on PIP2. Collectively, our work describes a new mechanistic paradigm for how human T cells integrate GPCR and phospholipid signaling pathways to control directed migration. These findings have implications for therapeutically targeting TIPE proteins in human inflammatory and autoimmune diseases.

TIPE proteins control directional migration of human T cells via GPCR and lipid second messenger signaling

Abstract Infiltration of specific tissues by circulating leukocytes is a common pathogenic mechanism of inflammatory diseases. G-protein-coupled receptors (GPCR) are essential for sensing chemokine gradients by leukocytes during immune responses. However, how leukocytes integrate site-specific directional cues such as chemokine gradients and make directional decisions in human immune cells is largely unknown. The TNFa-induced protein 8-Like (TIPE or TNFAIP8) family proteins are newly described pilot proteins that control directional migration (chemotaxis) of leukocytes to targeted tissues in mice. Without TIPE proteins, leukocytes become directionless, but the underlying mechanism remains elusive. Using siRNA and biochemical methods, we demonstrated here that two TIPE family members, TNFAIP8 and TIPE2, synergistically controlled directional migration (chemotaxis) in human CD4 T cells: TNFAIP8, through interacting with the Ga subunit of heterotrimeric G-proteins, and TIPE2, through utilizing lipid second messenger signaling by binding to phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3). Using site-directed mutagenesis, we identified the region of Ga that interacts with TNFAIP8, and the amino acids of TIPE2 protein crucial for its interaction with PIP2 and PIP3. We also discovered that TIPE protein membrane translocation was dependent on PIP2. Collectively, our work describes a new mechanistic paradigm of human T cells integrating GPCR and phospholipid signaling pathways to control directional migration of human immune cells. Additionally, our findings have implications for therapeutic targeting of TIPE proteins in human inflammatory and autoimmune diseases. NIH 5R01AI136945

3.6.5.2 Small monomeric GTPases in GtoPdb v.2023.1

Small G-proteins, are a family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate (GTP). They are a type of G-protein found in the cytosol that are homologous to the alpha subunit of heterotrimeric G-proteins, but unlike the alpha subunit of G proteins, a small GTPase can function independently as a hydrolase enzyme to bind to and hydrolyze a guanosine triphosphate (GTP) to form guanosine diphosphate (GDP). The best-known members are the Ras GTPases and hence they are sometimes called Ras subfamily GTPases.

Effective Strategies for Heterologous Expression of Plant Heterotrimeric G-protein γ Subunits without Gβ Subunit Partners.

In plants, heterotrimeric G-protein (Gγ) subunits are diverse, and they have structural plasticity to provide functional selectivity to the heterotrimer. Although the Gβ and Gγ subunits dimerize to function in the signaling pathway, the interaction mechanism of various Gγ subunits with the Gβ subunit partners is still elusive. To better understand the interaction mechanism, one approach is to separate the subunits for the re-assembly in vitro. Hence, developing a reliable method for achieving the efficient production and purification of these proteins has become necessary. In this study, Gγ1 and Gγ2 proteins from Oryza sativa and Arabidopsis thaliana were successfully identified, cloned, expressed in bacteria, and purified as recombinant proteins with the fusion tags. Highly expressed recombinant Gγ subunits in E. coli were digested by proteases, which were also produced in the presented study. Preliminary structural characterization studies without the Gβ partners showed that Gγ1 proteins have disordered structures with coiled-coil, α-helix extensions, and loops, whereas the Gγ2 protein has a more dominant β-sheet and turns structure. Finally, computational analyses performed on Gγ genes have laid the foundation of new targets for biotechnological purposes. The proposed optimized expression and purification protocol can contribute to investigations on the Gβγ binding mechanism in plant G-protein signaling. The investigations on selective binding are critical to shed light on the role(s) of different plant Gγ subunit types in biological processes.

Differential Expression Profile of Sphingosine‐1‐Phosphate Receptors in Human Brain Vascular Smooth Muscle and Endothelial Cells Following Hypoxia Plus Glucose Deprivation

Sphingosine‐1‐phosphate receptors (S1PRs) and their signaling pathways play an important role in mediating vascular health and function. Upon ligand activation, S1PRs 1‐5 couple with diverse heterotrimeric G‐protein subunits (Gαi, Gαq/11, Gα12/13) initiating multimodal downstream signaling pathways which result in various physiological outcomes in the vasculature, including cell proliferation and migration, barrier integrity preservation or loss, contraction, and inflammation. Specifically, S1PR2 activation has been linked to endothelial activation, barrier integrity loss, and inflammation, whereas S1PR1 activation contributes to barrier integrity preservation, vasodilation, and anti‐inflammatory properties. Although the role of S1PRs during pathophysiological conditions such as acute ischemic stroke is under current investigation, S1PR expression in the brain vasculature following ischemic injury has not yet been investigated. Therefore, the present study characterized the expression profile of S1PRs 1‐5 in human brain microvascular endothelial cells (ECs) and brain vascular smooth muscle cells (VSMCs) following hypoxia plus glucose deprivation (HGD). Male ECs and VSMCs were exposed to either HGD (1% O2) or normoxia (21% O2) for 3h. Semi‐quantitative RT‐PCR was used to examine gene expression and concomitantly, protein levels were assessed with standard western blot and/or FIJI‐mediated ICC analysis. We hypothesized that ECs and VSMCs differentially express S1PRs 1‐5 both basally and during ischemia‐like conditions. At the mRNA level, S1PRs 1‐5 gene expression was observed in VSMCs with S1PR4 and 5 being just above the level of detection. S1PRs 1‐4 expression was detected in ECs with S1PR4 having the lowest expression. During normoxic conditions, we observed that S1PRs 1 and 3 mRNA levels were most prominently expressed in both the ECs and VSMCs. In ECs, HGD exposure did not alter the gene expression of S1PRs 1‐3. At the protein level, we observed that HGD increased S1PR1 levels, however S1PR2 and 3 protein levels were unaltered. In VSMCs, HGD increased both mRNA and protein levels of S1PR2 but did not alter S1PRs 1 and 3 mRNA expression and protein levels. In conclusion, acute exposure to HGD appears to differentially regulate S1PRs in VSMCs and ECs at the transcriptional and translational level, respectively. In VSMCs, HGD increased S1PR2 gene expression and protein levels, whereas in ECs, HGD increased S1PR1 protein levels. The differential expression in S1PRs both basally and following HGD exposure may suggest differential signaling mechanisms within the two brain vascular cell types. Furthermore, differential expression of S1PR1 and 2 highlight the potential beneficial and/or detrimental involvement of S1PRs in the pathophysiology of ischemic injury within the cerebrovasculature. Further investigation will be necessary to determine the signaling role of S1PRs and their impact on cerebrovascular cell function following acute ischemic injury in the brain.

Role of Two G-Protein α Subunits in Vegetative Growth, Cell Wall Integrity, and Virulence of the Entomopathogenic Fungus Metarhizium robertsii.

Heterotrimeric G-proteins are crucial for fungal growth and differentiation. The α subunits of heterotrimeric G-proteins play an essential role in controlling signal transduction. However, the function of G-protein α subunits in entomopathogenic fungi remains poorly understood. Two group II Gα subunits (MrGPA2 and MrGPA4) were characterized in the entomopathogenic fungus, Metarhizium robertsii. Bioinformatics analysis showed that the relationship between MrGPA2 and MrGPA4 was closer than that of other MrGPAs. Both green fluorescent protein (GFP)-tagged MrGPA2 and MrGPA4 were localized at the cytoplasm. Furthermore, ∆MrGpa2∆MrGpa4 double mutants showed remarkably reduced vegetative growth compared to the wild-type and single-mutant strains, which was accompanied by the downregulation of several growth-related genes, such as ssk2, pbs2, stuA, hog1, and ac. Only the ∆MrGpa2∆MrGpa4 double mutant was sensitive to Congo red stress. The insect bioassay demonstrated significantly attenuated virulence for the ∆MrGpa2∆MrGpa4 double mutant compared to the wild-type and single-mutant strains. Further analysis indicated that double deletion of MrGpa2 and MrGpa4 had no effect on appressorium formation but suppressed the expression levels of several virulence-related genes in the insect hemocoel. These findings demonstrate that MrGpa2 and MrGpa4 exhibit functional redundancy and contribute to the vegetative growth, stress tolerance, and pest control potential in M. robertsii.

GNB1, a novel diagnostic and prognostic potential biomarker of head and neck and liver hepatocellular carcinoma

Background: Nucleotide-binding protein beta 1 (GNB1) encodes for heterotrimeric G-protein subunit beta (β) that is involved in various transmembrane signaling systems. A little has been reported earlier regarding GNB1 dysregulation in human cancers. Therefore, we carried out this study to investigate GNB1 expression levels and explore its prognostic values in distinct human cancers through a multi-layered bioinformatics approach. Methods: GNB1 expression and promoter methylation levels across distinct cancers were analyzed using UALCAN, GENT2, and MEXPRESS databases, whereas its potential prognostic values were evaluated through Kaplan–Meier plotter. Then, cBioPortal was utilized to analyze the GNB1-related genetic mutations and copy number variations (CNVs). However, GNB1-related pathways were explored using DAVID. Moreover, the correlational analysis between GNB1 expression and CD8+ T immune cell infiltration and a gene–drug interaction network analysis were performed using TIMER, CDT, and Cytoscape. Results: GNB1 was found commonly upregulated in 23 major subtypes of human cancers and its upregulation was significantly correlated with the reduced overall survival of only head and neck squamous cell carcinoma (HNSC) and liver hepatocellular carcinoma (LIHC). This implies that GNB1 plays a significant role in the development and progression of these cancers. The GNB1 was also found to be upregulated in HNSC and LIHC patients of different clinicopathological features. Pathways enrichment analysis revealed the involvement of GNB1-enriched genes in several diverse pathways, whereas a few interesting correlations were also documented between GNB1 expression and promoter methylation level, CD8+ T immune cell infiltration, and CNVs. In addition, we have also predicted few drugs that could regulate the GNB1 expression. Conclusions: Our findings suggested GNB1 as a potential diagnostic and prognostic biomarker of HNSC and LIHC.

Variants in GNAI1 cause a syndrome associated with variable features including developmental delay, seizures, and hypotonia

PurposeNeurodevelopmental disorders (NDDs) encompass a spectrum of genetically heterogeneous disorders with features that commonly include developmental delay, intellectual disability, and autism spectrum disorders. We sought to delineate the molecular and phenotypic spectrum of a novel neurodevelopmental disorder caused by variants in the GNAI1 gene. MethodsThrough large cohort trio-based exome sequencing and international data-sharing, we identified 24 unrelated individuals with NDD phenotypes and a variant in GNAI1, which encodes the inhibitory Gαi1 subunit of heterotrimeric G-proteins. We collected detailed genotype and phenotype information for each affected individual. ResultsWe identified 16 unique variants in GNAI1 in 24 affected individuals; 23 occurred de novo and 1 was inherited from a mosaic parent. Most affected individuals have a severe neurodevelopmental disorder. Core features include global developmental delay, intellectual disability, hypotonia, and epilepsy. ConclusionThis collaboration establishes GNAI1 variants as a cause of NDDs. GNAI1-related NDD is most often characterized by severe to profound delays, hypotonia, epilepsy that ranges from self-limiting to intractable, behavior problems, and variable mild dysmorphic features.

Antifungal Activity and Molecular Mechanisms of Partial Purified Antifungal Proteins from Rhinacanthus nasutus against Talaromyces marneffei.

Antifungal proteins (AFPs) are able to inhibit a wide spectrum of fungi without significant toxicity to the hosts. This study examined the antifungal activity of AFPs isolated from a Thai medicinal plant, Rhinacanthus nasutus, against the human pathogenic fungus Talaromyces marneffei. This dimorphic fungus causes systemic infections in immunocompromised individuals and is endemic in Southeast Asian countries. The R. nasutus crude protein extract inhibited the growth of T. marneffei. The anti-T. marneffei activity was completely lost when treated with proteinase K and pepsin, indicating that the antifungal activity was dependent on a protein component. The total protein extract from R. nasutus was partially purified by size fractionation to ≤10, 10–30, and ≥30 kDa fractions and tested for the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). All fractions showed anti-T. marneffei activity with the MIC and MFC values of 32 to 128 μg/mL and >128 μg/mL, respectively. In order to determine the mechanism of inhibition, all fractions were tested with T. marneffei mutant strains affected in G-protein signaling and cell wall integrity pathways. The anti-T. marneffei activity of the 10–30 kDa fraction was abrogated by deletion of gasA and gasC, the genes encoding alpha subunits of heterotrimeric G-proteins, indicating that the inhibitory effect is related to intracellular signaling through G-proteins. The work demonstrates that antifungal proteins isolated from R. nasutus represent sources for novel drug development.

Small Molecule Inhibitors Targeting Gαi2 Protein Attenuate Migration of Cancer Cells.

Heterotrimeric G-proteins are ubiquitously expressed in several cancers, and they transduce signals from activated G-protein coupled receptors. These proteins have numerous biological functions, and they are becoming interesting target molecules in cancer therapy. Previously, we have shown that heterotrimeric G-protein subunit alphai2 (Gαi2) has an essential role in the migration and invasion of prostate cancer cells. Using a structure-based approach, we have synthesized optimized small molecule inhibitors that are able to prevent specifically the activation of the Gαi2 subunit, keeping the protein in its inactive GDP-bound state. We observed that two of the compounds (13 and 14) at 10 μΜ significantly inhibited the migratory behavior of the PC3 and DU145 prostate cancer cell lines. Additionally, compound 14 at 10 μΜ blocked the activation of Gαi2 in oxytocin-stimulated prostate cancer PC3 cells, and inhibited the migratory capability of DU145 cells overexpressing the constitutively active form of Gαi2, under basal and EGF-stimulated conditions. We also observed that the knockdown or inhibition of Gαi2 negatively regulated migration of renal and ovarian cancer cell lines. Our results suggest that small molecule inhibitors of Gαi2 have potential as leads for discovering novel anti-metastatic agents for attenuating the capability of cancer cells to spread and invade to distant sites.

Regulator of G-Protein Signaling (RGS) Protein Modulation of Opioid Receptor Signaling as a Potential Target for Pain Management.

Opioid drugs are the gold standard for the management of pain, but their use is severely limited by dangerous and unpleasant side effects. All clinically available opioid analgesics bind to and activate the mu-opioid receptor (MOR), a heterotrimeric G-protein-coupled receptor, to produce analgesia. The activity of these receptors is modulated by a family of intracellular RGS proteins or regulators of G-protein signaling proteins, characterized by the presence of a conserved RGS Homology (RH) domain. These proteins act as negative regulators of G-protein signaling by serving as GTPase accelerating proteins or GAPS to switch off signaling by both the Gα and βγ subunits of heterotrimeric G-proteins. Consequently, knockdown or knockout of RGS protein activity enhances signaling downstream of MOR. In this review we discuss current knowledge of how this activity, across the different families of RGS proteins, modulates MOR activity, as well as activity of other members of the opioid receptor family, and so pain and analgesia in animal models, with particular emphasis on RGS4 and RGS9 families. We discuss inhibition of RGS proteins with small molecule inhibitors that bind to sensitive cysteine moieties in the RH domain and the potential for targeting this family of intracellular proteins as adjuncts to provide an opioid sparing effect or as standalone analgesics by promoting the activity of endogenous opioid peptides. Overall, we conclude that RGS proteins may be a novel drug target to provide analgesia with reduced opioid-like side effects, but that much basic work is needed to define the roles for specific RGS proteins, particularly in chronic pain, as well as a need to develop newer inhibitors.

3.6.5.2 Small monomeric GTPases (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Small G-proteins, are a family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate (GTP). They are a type of G-protein found in the cytosol that are homologous to the alpha subunit of heterotrimeric G-proteins, but unlike the alpha subunit of G proteins, a small GTPase can function independently as a hydrolase enzyme to bind to and hydrolyze a guanosine triphosphate (GTP) to form guanosine diphosphate (GDP). The best-known members are the Ras GTPases and hence they are sometimes called Ras subfamily GTPases.

A somatic missense mutation in GNAQ causes capillary malformation.

Capillary malformations, the most common type of vascular malformation, are caused by a somatic mosaic mutation in GNAQ, which encodes the Gαq subunit of heterotrimeric G-proteins. How the single amino acid change - predicted to activate Gαq - causes capillary malformations is not known but recent advances are helping to unravel the mechanisms. The GNAQ R183Q mutation is present not only in endothelial cells isolated from skin and brain capillary malformations but also in brain tissue underlying the capillary malformation, raising questions about the origin of capillary malformation-causing cells. Insights from computational analyses shed light on the mechanisms of constitutive activation and new basic science shows Gαq plays roles in sensing shear stress and in regulating cerebral blood flow. Several studies confirm the GNAQ R183Q mutation in 90% of nonsyndromic and Sturge-Weber syndrome (SWS) capillary malformations. The mutation is enriched in endothelial cells and blood vessels isolated from skin, brain, and choroidal capillary malformations, but whether the mutation resides in other cell types must be determined. Further, the mechanisms by which the R183Q mutation alters microvascular architecture and blood flow must be uncovered to develop new treatment strategies for SWS in particular, a devastating disease for which there is no cure.

APP-Mediated Signaling Prevents Memory Decline in Alzheimer’s Disease Mouse Model

Amyloid precursor protein (APP) and its metabolites play key roles in Alzheimer's disease (AD) pathophysiology. Whereas short amyloid-β (Aβ) peptides derived from APP are pathogenic, the APP holoprotein serves multiple purposes in the nervous system through its cell adhesion and receptor-like properties. Our studies focused on the signaling mediated by the APP cytoplasmic tail. We investigated whether sustained APP signaling during brain development might favor neuronal plasticity and memory process through a direct interaction with the heterotrimeric G-protein subunit GαS (stimulatory G-protein alpha subunit). Our results reveal that APP possesses autonomous regulatory capacity within its intracellular domain that promotes APP cell surface residence, precludes Aβ production, facilitates axodendritic development, and preserves cellular substrates of memory. Altogether, these events contribute to strengthening cognitive functions and are sufficient to modify the course of AD pathology.

Heterotrimeric G-protein subunit Gαi2 contributes to agonist-sensitive apoptosis and degranulation in murine platelets.

Gα i2, a heterotrimeric G‐protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet‐expressed Gα i2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gα i2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen‐related peptide (CoRP), which are further known to enhance degranulation and activation of α II b β3‐integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gα i2 and their wild‐type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 μg/mL or 5 μg/mL) significantly upregulated PS‐exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P‐selectin, and α II b β3‐integrin activation. These molecular alterations were significantly less pronounced in Gα i2‐deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gα i2 signaling in governing platelet activation and apoptosis.

The G-protein γ subunit of Phytophthora infestans is involved in sporangial development

The oomycete Phytophthora infestans is a notorious plant pathogen with potato and tomato as its primary hosts. Previous research showed that the heterotrimeric G-protein subunits Gα and Gβ have a role in zoospore motility and virulence, and sporangial development, respectively. Here, we present analyses of the gene encoding a Gγ subunit in P. infestans, Pigpg1. The overall similarity of PiGPG1 with non-oomycete Gγ subunits is low, with only the most conserved amino acids maintained, but similarity with its homologs in other oomycetes is high. Pigpg1 is expressed in all life stages and shows a similar expression profile as the gene encoding the Gβ subunit, Pigpb1. To elucidate its function, transformants were generated in which Pigpg1 is silenced or overexpressed and their phenotypes were analyzed. Pigpg1-silenced lines produce less sporangia, which are malformed. Altogether, the results show that PiGPG1 is crucial for proper sporangia development and zoosporogenesis. PiGPG1 is a functional Gγ, and likely forms a dimer with PiGPB1 that mediates signaling.

Correction: A FRET-based biosensor for measuring Gα13 activation in single cells.

[This corrects the article DOI: 10.1371/journal.pone.0193705.].

A FRET-based biosensor for measuring Gα13 activation in single cells.

Förster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human Gα13 subunit, aiming to develop a FRET-based Gα13 activation biosensor. Three fluorescently tagged Gα13 variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged Gα13 subunits were used as FRET donor and combined with cp173Venus fused to the Gγ2 subunit, as the acceptor. We constructed Gα13 biosensors by generating a single plasmid that produces Gα13-mTurquoise2, Gβ1 and cp173Venus-Gγ2. The Gα13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were exposed to thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the Gα13 sensor can be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the Gα13 biosensor is a valuable tool for live-cell measurements that probe spatiotemporal aspects of Gα13 activation.