GRK specificity and Gßγ dependency determines the potential of a GPCR for arrestin-biased agonism.

G protein-coupled receptors (GPCRs) are mainly regulated by GPCR kinase (GRK) phosphorylation and subsequent ß-arrestin recruitment. The ubiquitously expressed GRKs are classified into cytosolic GRK2/3 and membrane-tethered GRK5/6 subfamilies. GRK2/3 interact with activated G protein ßγ-subunits to translocate to the membrane. Yet, this need was not linked as a factor for bias, influencing the effectiveness of ß-arrestin-biased agonist creation. Using multiple approaches such as GRK2/3 mutants unable to interact with Gßγ, membrane-tethered GRKs and G protein inhibitors in GRK2/3/5/6 knockout cells, we show that G protein activation will precede GRK2/3-mediated ß-arrestin2 recruitment to activated receptors. This was independent of the source of free Gßγ and observable for Gs-, Gi- and Gq-coupled GPCRs. Thus, ß-arrestin interaction for GRK2/3-regulated receptors is inseparably connected with G protein activation. We outline a theoretical framework of how GRK dependence on free Gßγ can determine a GPCR's potential for biased agonism. Due to this inherent cellular mechanism for GRK2/3 recruitment and receptor phosphorylation, we anticipate generation of ß-arrestin-biased ligands to be mechanistically challenging for the subgroup of GPCRs exclusively regulated by GRK2/3, but achievable for GRK5/6-regulated receptors, that do not demand liberated Gßγ. Accordingly, GRK specificity of any GPCR is foundational for developing arrestin-biased ligands.

Role of the V2R-ßarrestin-Gßγ complex in promoting G protein translocation to endosomes.

Classically, G protein-coupled receptors (GPCRs) promote signaling at the plasma membrane through activation of heterotrimeric Gαßγ proteins, followed by the recruitment of GPCR kinases and ßarrestin (ßarr) to initiate receptor desensitization and internalization. However, studies demonstrated that some GPCRs continue to signal from internalized compartments, with distinct cellular responses. Both ßarr and Gßγ contribute to such non-canonical endosomal G protein signaling, but their specific roles and contributions remain poorly understood. Here, we demonstrate that the vasopressin V2 receptor (V2R)-ßarr complex scaffolds Gßγ at the plasma membrane through a direct interaction with ßarr, enabling its transport to endosomes. Gßγ subsequently potentiates Gαs endosomal translocation, presumably to regenerate an endosomal pool of heterotrimeric Gs. This work shines light on the mechanism underlying G protein subunits translocation from the plasma membrane to the endosomes and provides a basis for understanding the role of ßarr in mediating sustained G protein signaling.

Arabidopsis AGB1 participates in salinity response through bZIP17-mediated unfolded protein response.

BACKGROUND: Plant heterotrimeric G proteins respond to various environmental stresses, including high salinity. It is known that Gß subunit AGB1 functions in maintaining local and systemic Na+/K+ homeostasis to accommodate ionic toxicity under salt stress. However, whether AGB1 contributes to regulating gene expression for seedling's survival under high salinity remains unclear. RESULTS: We showed that AGB1-Venus localized to nuclei when facing excessive salt, and the induction of a set of bZIP17-dependent salt stress-responsive genes was reduced in the agb1 mutant. We confirmed both genetic and physical interactions of AGB1 and bZIP17 in plant salinity response by comparing salt responses in the single and double mutants of agb1 and bzip17 and by BiFC assay, respectively. In addition, we show that AGB1 depletion decreases nuclei-localization of transgenic mRFP-bZIP17 under salt stress, as shown in s1p s2p double mutant in the Agrobacteria-mediated transient mRFP-bZIP17 expression in young seedlings. CONCLUSIONS: Our results indicate that AGB1 functions in S1P and/or S2P-mediated proteolytic processing of bZIP17 under salt stress to regulate the induction of salinity-responsive gene expression.

G protein ß subunits regulate Cav3.3 T-type channel activity and current kinetics via interaction with the Cav3.3 C-terminus.

Ca2+ influx through Cav3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Cav3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Cav3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gß2 as an interaction partner. Subsequent assays revealed that the interaction of Gß2 subunit was specific to the Cav3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789-1810) as the Gß2 interaction site. Coexpression studies of rat Cav3.3 with various Gßγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gß2γ2 reduced Cav3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gß2γ2, but were mimicked by Gß2 alone as well as other Gßγ dimers, with similar potencies. Deletion of the Gß2 interaction site abolished the effects of Gß2γ2. Importantly, these Gß2 effects were reproduced in human Cav3.3. Overall, our findings provide evidence that Gß(γ) complexes inhibit Cav3.3 channel activity and accelerate the inactivation kinetics through the Gß interaction with the Cav3.3 C-terminus.

Generation of a patient-specific hiPS cell line with heterozygous GNB2 mutation (UKMi003-A) causative for human sinus node dysfunction and a corresponding CRISPR/Cas9-corrected isogenic control (UKMi004-A).

The heterozygous mutation c.155G > T in GNB2 clinically leads to sinus bradycardia and sinus node dysfunction. Here, patient-specific skin fibroblasts of the mutation carrier were used for Sendai virus reprogramming into human induced-pluripotent stem cells (hiPSC). For generating the isogenic control cell line, a CRISPR/Cas9-mediated HDR-repair of the hiPSCs was carried out. Both generated cell lines (GNB2 SV5528, GNB2 K26) maintained a normal karyotype, cell morphology, pluripotency in immunofluoresence and RT-qPCR analysis. Both hiPSC-lines showed differentiation potential into all three germ layers. Differentiated cardiomyocytes of this isogenic set may pave the way for investigating pharmacological rescue strategies for sinus node dysfunction.

[GmAGB1 plays a positive regulatory role in soybean defense responses].

Heterotrimeric GTP-binding protein (G-proteins) complex, which consists of Gα, Gß and Gγ subunits, plays critical roles in defense signaling. Arabidopsis genome contains only a single Gß-encoding gene, AGB1. Loss function of AGB1 in Arabidopsis results in enhanced susceptibility to a wide range of pathogens. However, the function of soybean AGB1 in immunity has not been previously interrogated. Bioinformatic analysis indicated that there are four GmAGB1 homologous genes in soybean genome, sharing homology of 86%-97%. To overcome the functional redundancy of these GmAGB1 homologs, virus-induced gene silencing (VIGS) mediated by the bean pod mottle virus (BPMV) was used to silence these four genes simultaneously. As expected, these four GmAGB1 homologous genes were indeed silenced by a single BPMV-VIGS vector carrying a conserved fragments among these four genes. A dwarfed phenotype was observed in GmAGB1s-silenced soybean plants, suggesting that GmAGB1s play a crucial role in growth and development. Disease resistance analysis indicated that silencing GmAGB1s significantly compromised the resistance of soybean plants against Xanthomonas campestris pv. glycinea (Xag). This reduced resistance was correlated with the decreased accumulation of pathogen-induced reactive oxygen species (ROS) and the reduced activation of GmMPK3 in response to flg22, a conserved N-terminal peptide of flagellin protein. These results indicate that GmAGB1 functions as a positive regulator in disease resistance and GmAGB1 is indispensable for the ROS production and GmMPK3 activation induced by pathogen infection. Yeast two hybrid assay showed that GmAGB1 interacted with GmAGG1, suggesting that an evolutionary conserved heterotrimeric G protein complex similarly functions in soybean.

GNB1 Encephalopathy: Clinical Case Report and Literature Review.

GNB1 encephalopathy is a rare genetic disease caused by pathogenic variants in the G Protein Subunit Beta 1 (GNB1) gene, with only around 68 cases documented worldwide. Although most cases had been caused by de novo germline mutations, in this case, the pathogenic variant was inherited from patient's mother, indicating an autosomal dominant inheritance pattern. The patient presented at 25 years of age with mild developmental delay and cognitive impairment, prominent generalized dystonia, and horizontal nystagmus which are all characterizing symptoms of GNB1 encephalopathy. Electroencephalography (EEG) showed no epileptiform patterns, and magnetic resonance imaging (MRI) revealed hypointensities in globus pallidus and dentate nucleus areas. The main theory for GNB1 encephalopathy pathogenesis is neuronal hyperexcitability caused by impaired ion channel regulation. Due to low specificity of symptoms, diagnosis relies on genetic testing. As there are no standardized GNB1 encephalopathy treatment guidelines, evaluation of different treatment options is based on anecdotal cases. Reviewing different treatment options, deep brain stimulation and intrathecal baclofen pump, as well as some other medications still in preclinical trials, seem to be the most promising.

GNB1 and obesity: Evidence for a correlation between haploinsufficiency and syndromic obesity.

Most patients with GNB1 encephalopathy have developmental delay and/or intellectual disability, brain anomalies and seizures. Recently, two cases with GNB1 encephalopathy caused by haploinsufficiency have been reported that also show a Prader-Willi-like phenotype of childhood hypotonia and severe obesity. Here we present three new cases from our expert centre for genetic obesity in which GNB1 truncating and splice variants, probably leading to haploinsufficiency, were identified. They all have obesity, hyperphagia and intellectual deficit. The clinical cases and their weight courses are presented, together with a review of all 68 published cases with GNB1 encephalopathy. Information on weight was not mentioned in most of these articles, so we contacted authors for additional clinical information on weight status and hyperphagia. Of the 42 patients whose weight status we could determine, obesity was present in 8 patients (19%). Obesity is significantly over-represented in the group with truncating and splicing variants. In this group, we see an obesity prevalence of 75%. Since GNB1 has been linked to several key genes in the hypothalamic leptin-melanocortin pathway, which regulates satiety and energy expenditure, our data support the potential association between GNB1 haploinsufficiency and genetic obesity. We also suggest GNB1 is a candidate gene for the known obesity phenotype of the 1p36 microdeletion syndrome given this chromosomal region includes the GNB1 gene. Knowledge of an additional obesity phenotype is important for prognosis, early interventions against obesity and awareness when prescribing weight-inducing medication.

The association of GNB5 with Alzheimer disease revealed by genomic analysis restricted to variants impacting gene function.

Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.

Inheritance of c.628-6G>A GNB5 hypomorphic allele uncovers another challenge in the pathogenic prediction of genomic variants.

We studied a patient with a severe phenotype carrying two GNB5 variants: c.514delT from the unaffected heterozygous mother and c.628-6G>A from the unaffected homozygous father. Functional genomics studies showed that parents express 50% (nonsense-mediated decay, NMD) of the RNA/protein while the patient does not produce enough protein for normal development.

Arabidopsis G-protein ß subunit AGB1 represses abscisic acid signaling via attenuation of the MPK3-VIP1 phosphorylation cascade.

Heterotrimeric G proteins play key roles in cellular processes. Although phenotypic analyses of Arabidopsis Gß (AGB1) mutants have implicated G proteins in abscisic acid (ABA) signaling, the AGB1-mediated modules involved in ABA responses remain unclear. We found that a partial AGB1 protein was localized to the nucleus where it interacted with ABA-activated VirE2-interacting protein 1 (VIP1) and mitogen-activated protein kinase 3 (MPK3). AGB1 acts as an upstream negative regulator of VIP1 activity by initiating responses to ABA and drought stress, and VIP1 regulates the ABA signaling pathway in an MPK3-dependent manner in Arabidopsis. AGB1 outcompeted VIP1 for interaction with the C-terminus of MPK3, and prevented phosphorylation of VIP1 by MPK3. Importantly, ABA treatment reduced AGB1 expression in the wild type, but increased in vip1 and mpk3 mutants. VIP1 associates with ABA response elements present in the AGB1 promoter, forming a negative feedback regulatory loop. Thus, our study defines a new mechanism for fine-tuning ABA signaling through the interplay between AGB1 and MPK3-VIP1. Furthermore, it suggests a common G protein mechanism to receive and transduce signals from the external environment.

Plasma methylated GNB4 and Riplet as a novel dual-marker panel for the detection of hepatocellular carcinoma.

Early detection of hepatocellular carcinoma (HCC) can greatly improve the survival rate of patients. We aimed to develop a novel marker panel based on cell-free DNA (cfDNA) methylation for the detection of HCC. The differentially methylated CpG sites (DMCs) specific for HCC blood diagnosis were selected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, then validated by the whole genome bisulphite sequencing (WGBS) of 12 paired HCC and paracancerous tissues. The clinical performance of the panel was evaluated using tissue samples [32 HCC, chronic liver disease (CLD), and healthy individuals] and plasma cohorts (173 HCC, 199 CLD, and 98 healthy individuals). The combination of G protein subunit beta 4 (GNB4) and Riplet had the optimal area under the curve (AUC) in seven candidates through TCGA, GEO, and WGBS analyses. In tissue validation, the GNB4 and Riplet showed an AUC of 100% with a sensitivity and specificity of 100% for detecting any-stage HCC. In plasma, it demonstrated a high sensitivity of 84.39% at 91.92% specificity, with an AUC of 92.51% for detecting any-stage HCC. The dual-marker panel had a higher sensitivity of 78.26% for stage I HCC than alpha-fetoprotein (AFP) of 47.83%, and a high sensitivity of 70.27% for detecting a single tumour (size ≤3 cm). In conclusion, we developed a novel dual-marker panel that demonstrates high accuracy in detecting HCC, surpassing the performance of AFP testing.

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

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

Heterotrimeric G Protein-Mediated Signaling Is Involved in Stress-Mediated Growth Inhibition in Arabidopsis thaliana.

Heterotrimeric G protein-mediated signaling plays a vital role in physiological and developmental processes in eukaryotes. On the other hand, because of the absence of a G protein-coupled receptor and self-activating mechanism of the Gα subunit, plants appear to have different regulatory mechanisms, which remain to be elucidated, compared to canonical G protein signaling established in animals. Here we report that Arabidopsis heterotrimeric G protein subunits, such as Gα (GPA1) and Gß (AGB1), regulate plant growth under stress conditions through the analysis of heterotrimeric G protein mutants. Flg22-mediated growth inhibition in wild-type roots was found to be caused by a defect in the elongation zone, which was partially blocked in agb1-2 but not gpa1-4. These results suggest that AGB1 may negatively regulate plant growth under biotic stress conditions. In addition, GPA1 and AGB1 exhibited genetically opposite effects on FCA-mediated growth inhibition under heat stress conditions. Therefore, these results suggest that plant G protein signaling is probably related to stress-mediated growth regulation for developmental plasticity in response to biotic and abiotic stress conditions.

Atypical clinical course in two patients with GNB1 variants who developed acute encephalopathy.

INTRODUCTION: Variants in the GNB1 gene, which encodes the ß1 subunit of a trimeric G protein, can cause moderate to severe psychomotor retardation. Acute encephalopathies have also been observed in patients with central nervous system abnormalities; however, severe neurological sequelae have not previously been reported. CASE PRESENTATIONS: Patient 1 was a Japanese female with a de novo GNB1 variant (c.284 T > C). At 8 months old she contracted influenza A and developed generalized convulsions. In the acute phase, brain magnetic resonance imaging (MRI) findings indicated acute encephalopathy; diffuse cerebral atrophy was present 1 month later. Although multidisciplinary treatment was administered, she had severe neurological sequelae including spastic tetraplegia, severe intellectual disabilities, and refractory epilepsy. Patient 2 was a Japanese male with a de novo GNB1 variant (c.239 T > C). He experienced an unexplained respiratory arrest aged 17 years; refractory convulsions developed. Brain MRI at 1 month showed bilateral basal ganglia high intensities; at 3 months, diffuse cerebral cortex and white matter atrophy was observed. Despite multidisciplinary treatment, he developed severe spastic tetraplegia and mental regression. DISCUSSION: We report two patients with GNB1 variants who had acute lesions on brain MRI and unexpected disease courses. In such patients with acute neurological deterioration, multidisciplinary treatment is required; patients should also be carefully observed for progression to acute encephalopathy.

Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G protein subunits.

G-protein-coupled receptors (GPCRs) mediate neuromodulation through the activation of heterotrimeric G proteins (Gαßγ). Classical models depict that G protein activation leads to a one-to-one formation of Gα-GTP and Gßγ species. Each of these species propagates signaling by independently acting on effectors, but the mechanisms by which response fidelity is ensured by coordinating Gα and Gßγ responses remain unknown. Here, we reveal a paradigm of G protein regulation whereby the neuronal protein GINIP (Gα inhibitory interacting protein) biases inhibitory GPCR responses to favor Gßγ over Gα signaling. Tight binding of GINIP to Gαi-GTP precludes its association with effectors (adenylyl cyclase) and, simultaneously, with regulator-of-G-protein-signaling (RGS) proteins that accelerate deactivation. As a consequence, Gαi-GTP signaling is dampened, whereas Gßγ signaling is enhanced. We show that this mechanism is essential to prevent the imbalances of neurotransmission that underlie increased seizure susceptibility in mice. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.

N-terminal intrinsic disorder is an ancestral feature of Gγ subunits that influences the balance between different Gßγ signaling axes in yeast.

Activated G protein-coupled receptors promote the dissociation of heterotrimeric G proteins into Gα and Gßγ subunits that bind to effector proteins to drive intracellular signaling responses. In yeast, Gßγ subunits coordinate the simultaneous activation of multiple signaling axes in response to mating pheromones, including MAP kinase (MAPK)-dependent transcription, cell polarization, and cell cycle arrest responses. The Gγ subunit in this complex contains an N-terminal intrinsically disordered region that governs Gßγ-dependent signal transduction in yeast and mammals. Here, we demonstrate that N-terminal intrinsic disorder is likely an ancestral feature that has been conserved across different Gγ subtypes and organisms. To understand the functional contribution of structural disorder in this region, we introduced precise point mutations that produce a stepwise disorder-to-order transition in the N-terminal tail of the canonical yeast Gγ subunit, Ste18. Mutant tail structures were confirmed using circular dichroism and molecular dynamics and then substituted for the wildtype gene in yeast. We find that increasing the number of helix-stabilizing mutations, but not isometric mutation controls, has a negative and proteasome-independent effect on Ste18 protein levels as well as a differential effect on pheromone-induced levels of active MAPK/Fus3, but not MAPK/Kss1. When expressed at wildtype levels, we further show that mutants with an alpha-helical N terminus exhibit a counterintuitive shift in Gßγ signaling that reduces active MAPK/Fus3 levels whilst increasing cell polarization and cell cycle arrest. These data reveal a role for Gγ subunit intrinsically disordered regions in governing the balance between multiple Gßγ signaling axes.

Specific regulation of mechanical nociception by Gß5 involves GABA-B receptors.

Mechanical, thermal, and chemical pain sensation is conveyed by primary nociceptors, a subset of sensory afferent neurons. The intracellular regulation of the primary nociceptive signal is an area of active study. We report here the discovery of a Gß5-dependent regulatory pathway within mechanical nociceptors that restrains antinociceptive input from metabotropic GABA-B receptors. In mice with conditional knockout (cKO) of the gene that encodes Gß5 (Gnb5) targeted to peripheral sensory neurons, we demonstrate the impairment of mechanical, thermal, and chemical nociception. We further report the specific loss of mechanical nociception in Rgs7-Cre+/- Gnb5fl/fl mice but not in Rgs9-Cre+/- Gnb5fl/fl mice, suggesting that Gß5 might specifically regulate mechanical pain in regulator of G protein signaling 7-positive (Rgs7+) cells. Additionally, Gß5-dependent and Rgs7-associated mechanical nociception is dependent upon GABA-B receptor signaling since both were abolished by treatment with a GABA-B receptor antagonist and since cKO of Gß5 from sensory cells or from Rgs7+ cells potentiated the analgesic effects of GABA-B agonists. Following activation by the G protein-coupled receptor Mrgprd agonist ß-alanine, enhanced sensitivity to inhibition by baclofen was observed in primary cultures of Rgs7+ sensory neurons harvested from Rgs7-Cre+/- Gnb5fl/fl mice. Taken together, these results suggest that the targeted inhibition of Gß5 function in Rgs7+ sensory neurons might provide specific relief for mechanical allodynia, including that contributing to chronic neuropathic pain, without reliance on exogenous opioids.

Rod-cone dystrophy in an adult with GNB1-related disorder: An expansion of the phenotype and natural history.

GNB1-related disorder is characterized by intellectual disability, abnormal tone, and other variable neurologic and systemic features. GNB1 encodes the ß1 subunit of the heterotrimeric G-protein, a complex with a key role in signal transduction. Consistent with its particularly high expression in rod photoreceptors, Gß1 forms a subunit of retinal transducin (Gαtß1γ1 ), which mediates phototransduction. In mice, GNB1 haploinsufficiency has been associated with retinal dystrophy. In humans, however, although vision and eye movement abnormalities are common in individuals with GNB1-related disorder, rod-cone dystrophy is not yet an established feature of this condition. We expand the phenotype of GNB1-related disorder with the first confirmed report of rod-cone dystrophy in an affected individual, and contribute to a further understanding of the natural history of this condition in a mildly affected 45-year-old adult.