G-protein Alpha Research Articles

Involvement of G-protein alpha subunit in soybean cyst nematode chemotaxis

The soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is a significant agricultural pest that causes extensive damage to soybean production worldwide. Second-stage juveniles (J2s) of the SCN migrate through the soil and infest the roots of host plants in response to certain chemical substances secreted from the host roots. Therefore, controlling SCN chemotaxis could be an effective strategy for its management.In the present study, we identified the Hg-gpa-3d gene, which encodes the G protein alpha subunit, as a key regulator of SCN chemotaxis. Gene silencing of Hg-gpa-3d reduced the attraction of SCN J2s to host roots, as well as to nitrate ions, a chemoattractant recognized through a mechanism different from that of host recognition. However, silencing of Hg-gpa-3d did not affect avoidance behavior towards unpleasant temperatures or stylet protrusion. These results suggest that Hg-gpa-3d is a crucial gene in the regulation of SCN chemotaxis and provide new insights into the chemotactic mechanisms of the SCN.

Mechanism of sensory perception unveiled by simultaneous measurement of membrane voltage and intracellular calcium

Measuring neuronal activity is important for understanding neuronal function. Ca2+ imaging by genetically encoded calcium indicators (GECIs) is a powerful way to measure neuronal activity. Although it revealed important aspects of neuronal function, measuring the neuronal membrane voltage is important to understand neuronal function as it triggers neuronal activation. Recent progress of genetically encoded voltage indicators (GEVIs) enabled us fast and precise measurements of neuronal membrane voltage. To clarify the relation of the membrane voltage and intracellular Ca2+, we analyzed neuronal activities of olfactory neuron AWA in Caenorhabditis elegans by GCaMP6f (GECI) and paQuasAr3 (GEVI) responding to odorants. We found that the membrane voltage encodes the stimuli change by the timing and the duration by the weak semi-stable depolarization. However, the change of the intracellular Ca2+ encodes the strength of the stimuli. Furthermore, ODR-3, a G-protein alpha subunit, was shown to be important for stabilizing the membrane voltage. These results suggest that the combination of calcium and voltage imaging provides a deeper understanding of the information in neural circuits.

G-Protein-Coupled Receptor Kinase 2 Inhibition Induces Meiotic Arrest by Disturbing Ca2+ Release in Porcine Oocytes.

G-protein-coupled receptor kinase 2 (GRK2) interacts with Gβγ and Gαq, subunits of G-protein alpha, to regulate cell signalling. The second messenger inositol trisphosphate, produced by activated Gαq, promotes calcium release from the endoplasmic reticulum (ER) and regulates maturation-promoting factor (MPF) activity. This study aimed to investigate the role of GRK2 in MPF activity during the meiotic maturation of porcine oocytes. A specific inhibitor of GRK2 (βi) was used in this study. The present study showed that GRK2 inhibition increased the percentage of oocyte arrest at the metaphase I (MI) stage (control: 13.84 ± 0.95%; βi: 31.30 ± 4.18%), which resulted in the reduction of the maturation rate (control: 80.36 ± 1.94%; βi: 65.40 ± 1.14%). The level of phospho-GRK2 decreased in the treated group, suggesting that GRK2 activity was reduced upon GRK2 inhibition. Furthermore, the addition of βi decreased Ca2+ release from the ER. The protein levels of cyclin B and cyclin-dependent kinase 1 were higher in the treatment group than those in the control group, indicating that GRK2 inhibition prevented a decrease in MPF activity. Collectively, GRK2 inhibition induced meiotic arrest at the MI stage in porcine oocytes by preventing a decrease in MPF activity, suggesting that GRK2 is essential for oocyte meiotic maturation in pigs.

The G-protein alpha subunit AaGA1 positively regulates vegetative growth, appressorium-like formation, and pathogenicity in Alternaria alternata.

The Gα subunit is a major component of heterotrimeric G proteins, which play a crucial role in the development and pathogenicity of several model fungi. However, its detailed function in the causal agent of pear black spot (Alternaria alternata) is unclear. Our aim was to understand the characteristics and functions of AaGA1 in A. alternata. AaGA1 was cloned from A. alternata in this study, which encodes 353 amino acids and has a "G-alpha" domain. Mutant ΔAaGA1 resulted in reduced vegetative growth, conidiation, and spore germination. Especially, mutant ΔAaGA1 produced only fewer conidia on the V8A medium, and spore formation-related genes AbaA, BrlA, and WetA were significantly downregulated. More tolerance against cell wall-inhibiting agents was observed after the deletion of AaGA1. Moreover, AaGA1 deletion led to a significant reduction in melanin and toxin production. Interestingly, deletion of AaGA1 resulted in defective appressorium-like formations, complete loss of the ability to penetrate cellophane, and decreased infection on non-wound inoculated tobacco leaves. Cell wall-degrading enzyme-related genes PME, CL, Cut2, and LC were significantly downregulated in mutant ΔAaGA1 mutant, significantly reducing virulence on wound-inoculated pear fruits. The G protein alpha subunit AaGA1 is indispensable for fungal development, appressorium-like formations, and pathogenicity in A. alternata.

GprC of the nematode-trapping fungus Arthrobotrys flagrans activates mitochondria and reprograms fungal cells for nematode hunting

Initiation of development requires differential gene expression and metabolic adaptations. Here we show in the nematode-trapping fungus, Arthrobotrys flagrans, that both are achieved through a dual-function G-protein-coupled receptor (GPCR). A. flagrans develops adhesive traps and recognizes its prey, Caenorhabditis elegans, through nematode-specific pheromones (ascarosides). Gene-expression analyses revealed that ascarosides activate the fungal GPCR, GprC, at the plasma membrane and together with the G-protein alpha subunit GasA, reprograms the cell. However, GprC and GasA also reside in mitochondria and boost respiration. This dual localization of GprC in A. flagrans resembles the localization of the cannabinoid receptor CB1 in humans. The C. elegans ascaroside-sensing GPCR, SRBC66 and GPCRs of many fungi are also predicted for dual localization, suggesting broad evolutionary conservation. An SRBC64/66-GprC chimaeric protein was functional in A. flagrans, and C. elegans SRBC64/66 and DAF38 share ascaroside-binding sites with the fungal GprC receptor, suggesting 400-million-year convergent evolution.

Agonist-selective activation of individual G-proteins by muscarinic receptors

Selective activation of individual subtypes of muscarinic receptors is a promising way to safely alleviate a wide range of pathological conditions in the central nervous system and the periphery as well. The flexible G-protein interface of muscarinic receptors allows them to interact with several G-proteins with various efficacy, potency, and kinetics. Agonists biased to the particular G-protein mediated pathway may result in selectivity among muscarinic subtypes and, due to the non-uniform expression of individual G-protein alpha subunits, possibly achieve tissue specificity. Here, we demonstrate that novel tetrahydropyridine-based agonists exert specific signalling profiles in coupling with individual G-protein α subunits. These signalling profiles profoundly differ from the reference agonist carbachol. Moreover, coupling with individual Gα induced by these novel agonists varies among subtypes of muscarinic receptors which may lead to subtype selectivity. Thus, the novel tetrahydropyridine-based agonist can contribute to the elucidation of the mechanism of pathway-specific activation of muscarinic receptors and serve as a starting point for the development of desired selective muscarinic agonists.

Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

The concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity. In this study we demonstrate that mechanical stimulation can induce β2-adrenoceptor agonist-independent Gs-mediated cAMP signalling that is sensitive to inhibition by inverse agonists such as ICI-118551 and propranolol. The size of the mechano-sensitive response is dependent on the cell surface receptor expression level in HEK293G cells, is still observed in a ligand-binding deficient D113A mutant β2-adrenoceptor and can be attenuated by site-directed mutagenesis of the extracellular N-glycosylation sites on the N-terminus and second extracellular loop of the β2-adrenoceptor. Similar mechano-sensitive agonist-independent responses are observed in HEK293G cells overexpressing the A2A-adenosine receptor. These data provide new insights into how agonist-independent constitutive receptor activity can be enhanced by mechanical stimulation and regulated by inverse agonists.

Investigating the interaction between some of Bipolaris sorokiniana’s toxins and the Gα Subunit of the Wheat G-Protein using bioinformatics tools

Spot blotch disease of wheat, caused by the fungus Bipolaris sorokiniana (Sacc.) Shoem., produces several toxins like: prehelminthosporol, helminthosporol, helminthosporic acid, sorokinianin, Bipolaroxin. These toxins interact with the plant and thereby increase the symptoms of small dark lesions and huge yield losses in different regions around the world so there is an urgent need to decipher the molecular interaction between wheat and those toxins for in-depth understanding of host–pathogen interactions. In the present study, we have modeledthe three-dimensional structure of G-protein alpha subunit from Triticum aestivum as G-protein was shown that it is an important player behind the resistance to many plant diseases. Molecular docking studies were performed using the active site of the modeled G-protein alpha subunit from T. aestivumand some of fungus’s toxins followed by molecular dynamics (MD) simulation studies to explore the stability, conformational flexibility, and dynamic behavior. Protein-ligand interaction study revealed one H-bond formed by Lys302 and hydrophobic contacts formed by Tyr159, Gly162, Val167, Asp256, Gln257 and Ile298 with prehelminthosporol, Protein-ligand interaction study revealed H-bond formed three H-bonds formed by Tyr159, Gly162, and Asp256 and hydrophobic interactions formed by Ser160, Cys161, Ser162, Ile298, Lys302 and Val306 with helminthosporic acid. Protein-ligand interaction study revealed two H-bond formed by His172, Arg301 and hydrophobic interactions by Tyr159, Pro168, Asp169, Ile298 and Lys302 with sorokinianin. Protein-ligand interaction study revealed H-bond formed six H-bonds mainly formed by Glu29, Ser30, Lys32, and Ala177. In addition to H-bonds, hydrophobic contacts formed by Gly28, Gly31, Ser33, Thr34, Arg78, Val179, Thr181 and Gly209 with Bipolaroxin were also observed.

The RNA-Binding and RNA-Melting Activities of the Multifunctional Protein Nucleobindin 1

Nucleobindin 1 (NUCB1) is a ubiquitous multidomain protein that belongs to the EF-hand Ca2+-binding superfamily. NUCB1 interacts with Galphai3 protein, cyclooxygenase, amyloid precursor protein, and lipids. It is involved in stress response and human diseases. In addition, this protein is a transcription factor that binds to the DNA E-box motif. Using surface plasmon resonance and molecular beacon approaches, we first showed the RNA binding and RNA melting activities of NUCB1. We suggest that NUCB1 could induce local changes in structured RNAs via binding to the GGAUAU loop sequence. Our results demonstrate the importance of the multidomain structure of NUCB1 for its RNA-chaperone activity in vitro.

Elucidating the role of key physio-biochemical traits and molecular network conferring heat stress tolerance in cucumber.

Cucumber is an important vegetable crop grown worldwide and highly sensitive to prevailing temperature condition. The physiological, biochemical and molecular basis of high temperature stress tolerance is poorly understood in this model vegetable crop. In the present study, a set of genotypes with contrasting response under two different temperature stress (35/30°C and 40/35°C) were evaluated for important physiological and biochemical traits. Besides, expression of the important heat shock proteins (HSPs), aquaporins (AQPs), photosynthesis related genes was conducted in two selected contrasting genotypes at different stress conditions. It was established that tolerant genotypes were able to maintain high chlorophyll retention, stable membrane stability index, higher retention of water content, stability in net photosynthesis, high stomatal conductance and transpiration in combination with less canopy temperatures under high temperature stress conditions compared to susceptible genotypes and were considered as the key physiological traits associated with heat tolerance in cucumber. Accumulation of biochemicals like proline, protein and antioxidants like SOD, catalase and peroxidase was the underlying biochemical mechanisms for high temperature tolerance. Upregulation of photosynthesis related genes, signal transduction genes and heat responsive genes (HSPs) in tolerant genotypes indicate the molecular network associated with heat tolerance in cucumber. Among the HSPs, higher accumulation of HSP70 and HSP90 were recorded in the tolerant genotype, WBC-13 under heat stress condition indicating their critical role. Besides, Rubisco S, Rubisco L and CsTIP1b were upregulated in the tolerant genotypes under heat stress condition. Therefore, the HSPs in combination with photosynthetic and aquaporin genes were the underlying important molecular network associated with heat stress tolerance in cucumber. The findings of the present study also indicated negative feedback of G-protein alpha unit and oxygen evolving complex in relation to heat stress tolerance in cucumber. These results indicate that the thermotolerant cucumber genotypes enhanced physio-biochemical and molecular adaptation under high-temperature stress condition. This study provides foundation to design climate smart genotypes in cucumber through integration of favorable physio-biochemical traits and understanding the detailed molecular network associated with heat stress tolerance in cucumber.

LRP6-mediated phosphorylation of connexin43 in myocardial infarction

Ventricular tachycardia (VT) and ventricular fibrillation are most causes of early death in patients with acute myocardial infarction (AMI). Conditional cardiac-specific low-density lipoprotein receptor-related protein 6 (LRP6)-knockout mice with connexin 43 (Cx43) reduction triggered the lethal ventricular arrhythmias. Thus, it is necessary for exploring whether LRP6 and its upstream genes circRNA1615 mediate the phosphorylation of Cx43 in VT of AMI. Here, we showed that circRNA1615 regulated the expression of LRP6 mRNA through sponge adsorption of miR-152-3p. Importantly, LRP6 interference fragments aggravated hypoxia injury of Cx43, while overexpression of LRP6 improved the phosphorylation of Cx43. Subsequently, interference with G-protein alpha subunit (Gαs) downstream of LRP6 further inhibited the phosphorylation of Cx43, along with increasing VT. Our results demonstrated that LRP6 upstream genes circRNA1615 controlled the damage effect and VT in AMI, and LRP6 mediated the phosphorylation of Cx43 via Gαs which played a role in VT of AMI.

ODP484 Hyperthyroidism During Prostaglandin I2 Treatment in Pulmonary Hypertension and Its Molecular Mechanism

Abstract Continuous intravenous infusion of prostaglandin I2 (PGI2) analog, epoprostenol, has improved the survival rate for severe pulmonary hypertension, but longer treatment with PGI2 sometimes occurs hyperthyroidism. For the hyperthyroidism during PGI2 treatment, two molecular mechanisms are speculated; the direct effect of PGI2 on thyroid follicular cells via PGI2 receptor which is coupled with G-protein alpha subunit like TSH receptor, and the indirect effect of PGI2 on activated T helper 17 cells which are associated with autoimmune disease (1). Here, we experienced three different cases of hyperthyroidism during PGI2 treatment in pulmonary hypertension. In case 1, epoprostenol had been administered intravenously for about ten months before the onset of hyperthyroidism. TSAb became positive (591%) and technetium uptake was elevated (4.7%), which were similar to the typical observations in Grave's disease. Total thyroidectomy was needed to control the thyroid function in case 1. In case 2, no thyroid antibody was detected and technetium uptake was almost vanished, as in the destructive thyroiditis. The thyroid function eventually normalized without any intervention. In case 3, hyperthyroidism occurred during oral PGI2 analog selexipag administration, but no evidence of Grave's disease or destructive thyroiditis was observed. Hyperthyroidism was declined when the dose of selexipag was reduced. To investigate the molecular mechanism underlying each case, we added the drugs which were used in each case on a thyroid follicular cell line, FRTL5. The thyroid tissue which was resected in case 1 and FRTL5 cells expressed PGI2 receptors in immunohistochemistry and immunofluorescence study. Human thyrotropin alpha (TSH) and epoprostenol elevated the intracellular cyclic AMP (cAMP) in FRTL5 (1.33 fold and 1.20 fold each, P<0. 05, n=3), but the other PGI2 analogs and the other drugs for pulmonary hypertension didn't change the cAMP in FRTL5. The gene expression level of Na/I symporter was up-regulated only by TSH (2.39 fold, P=0. 02, n=3), but not by PGI2 analogs in qPCR study. It is suggested that PGI2 increases the intracellular cAMP in thyroid follicular cells, but does not cause the same genetic changes as TSH. In conclusion, PGI2 analog may not directly affect the thyroid hormone synthesis in follicular cells, but further analysis is needed to elucidate the molecular mechanism underlying hyperthyroidism during PGI2 analog treatment. Reference: (1) Satoh et al.,Endocrine Journal 2017,64(12),1173-1180 Presentation: No date and time listed

Cushing syndrome as a failed cardiac screen in a patient with McCune–Albright syndrome: a case report

BackgroundMcCune–Albright syndrome is a complex disorder encompassing multiple endocrinopathies. These manifestations are secondary to a mutation in the stimulatory G-protein alpha subunit. Cushing syndrome is due to autonomous secretory function of the adrenal gland and is present in 7.1% of patients with McCune–Albright syndrome. Cardiac newborn screenings assist in the identification of critical congenital heart disease. These screenings have become part of routine postnatal care nationwide.Case reportA 6-week-old Caucasian male presented to a cardiologist at the University of Tennessee Health Science Center with left ventricular hypertrophy and poor feeding after a failed cardiac newborn screen. He had been previously seen at 2 weeks by a cardiologist on follow-up for abnormal critical congenital heart disease screening. Electrocardiogram and echocardiographic studies identified hypertrophic cardiomyopathy. Other examination findings revealed multiple characteristic café-au-lait lesions along with hypotonia and rounded facies. Given his cardiac disease, he was admitted to the hospital, where an evaluation was done for Cushing syndrome, showing elevated cortisol by immunoassay of 38 μg/dL (1.7–14.0 μg/dL, Vitros 5600) after a dexamethasone suppression test and urinary cortisol elevated to 35 μg/dL/24 hours (reference range 3–9 μg/dL/24 hours) (Esoterix; Calabasas, CA). He was started on metyrapone therapy to block synthesis of cortisol. His cortisol improved and was suppressed less than 2 μg/dL. His hypertension and clinical features of Cushing syndrome improved.ConclusionsThis case demonstrates a unique presentation of Cushing syndrome in a young infant. This is the first case to our knowledge showing significant left ventricular hypertrophy resulting from Cushing syndrome identified following a failure on a critical congenital heart disease screen. It highlights the importance of considering of McCune–Albright syndrome in patients with Cushing syndrome, especially if other clinical features are present. Medical therapy can be used to treat Cushing syndrome and can result in improvement in the cardiovascular pathology.

Novel Insights into Understanding the Molecular Dialogues between Bipolaroxin and the Gα and Gβ Subunits of the Wheat Heterotrimeric G-Protein during Host-Pathogen Interaction.

Spot blotch disease of wheat, caused by the fungus Bipolaris sorokiniana (Sacc.) Shoem., produces several toxins which interact with the plants and thereby increase the blightening of the wheat leaves, and Bipolaroxin is one of them. There is an urgent need to decipher the molecular interaction between wheat and the toxin Bipolaroxin for in-depth understanding of host–pathogen interactions. In the present study, we have developed the three-dimensional structure of G-protein alpha subunit from Triticum aestivum. Molecular docking studies were performed using the active site of the modeled G-protein alpha and cryo-EM structure of beta subunit from T. aestivum and ‘Bipolaroxin’. The study of protein–ligand interactions revealed that six H-bonds are mainly formed by Glu29, Ser30, Lys32, and Ala177 of G-alpha with Bipolaroxin. In the beta subunit, the residues of the core beta strand domain participate in the ligand interaction where Lys256, Phe306, and Leu352 formed seven H-bonds with the ligand Bipolaroxin. All-atoms molecular dynamics (MD) simulation studies were conducted for G-alpha and -beta subunit and Bipolaroxin complexes to explore the stability, conformational flexibility, and dynamic behavior of the complex system. In planta studies clearly indicated that application of Bipolaroxin significantly impacted the physio-biochemical pathways in wheat and led to the blightening of leaves in susceptible cultivars as compared to resistant ones. Further, it interacted with the Gα and Gβ subunits of G-protein, phenylpropanoid, and MAPK pathways, which is clearly supported by the qPCR results. This study gives deeper insights into understanding the molecular dialogues between Bipolaroxin and the Gα and Gβ subunits of the wheat heterotrimeric G-protein during host–pathogen interaction.

Characterization and expression of the Pirin gene family in Triticum aestivum

Pirins are nuclear bicupin proteins, encoded by genes that are one of several gene families that comprise the cupin superfamily in plants. Pirin genes have been implicated in stress response pathways studied in Arabidopsis and At-Pirin1 has been shown to interact with the heterotrimeric G-protein alpha subunit (GPA1). The aim of this study was to identify the members of the Pirin gene family in Triticum aestivum, to correct their annotations in the whole genome, and gain an insight into their tissue-specific expression as well as their response to abiotic and biotic stresses. The Pirin gene family in T. aestivum is comprised of 18 genes that represent six paralogous gene copies, each having an A, B, and D homeolog. Expression analysis of the Pirin genes in T. aestivum Illumina RNA-seq libraries, which included sampling from differing tissue types as well as abiotic and biotic stresses, indicates that the members of the Pirin gene family have specialized expression and play a role in stress responses. Pirin gene families are also identified in other monocots including Aegilops tauschii, Hordeum vulgare, Brachypodium distachyon, Oryza sativa, Zea mays, Sorghum bicolor, and the dicot Arabidopsis thaliana.

Receptor-proximal effectors mediating GnRH actions in the goldfish pituitary: Involvement of G protein subunits and GRKs

In goldfish (Carassius auratus), two endogenous isoforms of gonadotropin-releasing hormone (GnRH) stimulate luteinizing hormone (LH) and growth hormone (GH) secretion. These isoforms, GnRH2 and GnRH3, act on a shared population of cell-surface GnRH receptors (GnRHRs) expressed on both gonadotrophs and somatotrophs, and can signal through unique, yet partially overlapping, suites of intracellular effectors, in a phenomenon known as functional selectivity or biased signalling. In this study, G-protein alpha (Gα) subunits were targeted with two inhibitors, YM-254890 and BIM-46187, to ascertain the contribution of specific G-protein subunits in GnRH signalling. Results with the Gαq/11-specific inhibitor YM-254890 on primary cultures of goldfish pituitary cells revealed the use of these subunits in GnRH control of both LH and GH release, as well as GnRH-induced elevations in phospho-ERK levels. Results with the pan-Gα inhibitor BIM-46187 matched those using YM-254890 in LH release but GH responses differed, indicating additional, non-Gαq/11 subunits may be involved in somatotrophs. BIM-46187 also elevated unstimulated LH and GH release suggesting that Gα subunits regulate basal hormone secretion. Furthermore, G-protein-coupled receptor kinase (GRK2/3) inhibition reduced LH responses to GnRH2 and GnRH3, and selectively enhanced GnRH2-stimulated GH release, indicating differential use of GRK2/3 in GnRH actions on gonadotrophs and somatotrophs. These findings in a primary untransformed system provide the first direct evidence to establish Gαq/11 as an obligate driver of GnRH signalling in goldfish pituitary cells, and additionally describe the differential agonist- and cell type-selective involvement of GRK2/3 in this system.

Gs\u03b1 deficiency facilitates cardiac remodeling via CREB/ Bmp10-mediated signaling

The stimulatory G-protein alpha subunit (Gsα), a ubiquitously expressed protein, mediates G-protein receptor-stimulated signal transduction. To investigate the functions of Gsα in cardiomyocytes. We developed transverse aortic constriction (TAC)-induced heart failure mouse models and tamoxifen-inducible transgenic mice with cardiac-specific Gsα disruption. We detected alterations in Gsα expression in TAC-induced heart failure mice. Moreover, we examined cardiac function and structure in mice with genetic Gsα deletion and investigated the underlying molecular mechanisms of Gsα function. We found that Gsα expression increased during the compensated cardiac hypertrophy period and decreased during the heart failure period. Moreover, cardiac-specific Gsα disruption deteriorated cardiac function and induced severe cardiac remodeling. Mechanistically, Gsα disruption decreased CREB1 expression and inhibited the Bmp10-mediated signaling pathway. In addition, we found that Gsα regulates Bmp10 expression through the binding of CREB1 to the Bmp10 promoter. Our results suggest that fluctuations in Gsα levels may play a vital role in the development of heart failure and that loss of Gsα function facilitates cardiac remodeling.

MAGED2 controls vasopressin-induced aquaporin-2 expression in collecting duct cells

Mutations in the Melanoma-Associated Antigen D2 (MAGED2) cause antenatal Bartter syndrome type 5 (BARTS5). This rare disease is characterized by perinatal loss of urinary concentration capability and large urine volumes. The underlying molecular mechanisms of this disease are largely unclear. Here, we study the effect of MAGED2 knockdown on kidney cell cultures using proteomic and phosphoproteomic analyses. In HEK293T cells, MAGED2 knockdown induces prominent changes in protein phosphorylation rather than changes in protein abundance. MAGED2 is expressed in mouse embryonic kidneys and its expression declines during development. MAGED2 interacts with G-protein alpha subunit (GNAS), suggesting a role in G-protein coupled receptors (GPCR) signalling. In kidney collecting duct cell lines, Maged2 knockdown subtly modulated vasopressin type 2 receptor (V2R)-induced cAMP-generation kinetics, rewired phosphorylation-dependent signalling, and phosphorylation of CREB. Maged2 knockdown resulted in a large increase in aquaporin-2 abundance during long-term V2R activation. The increase in aquaporin-2 protein was mediated transcriptionally. Taken together, we link MAGED2 function to cellular signalling as a desensitizer of V2R-induced aquaporin-2 expression. SignificanceIn most forms of Bartter Syndrome, the underlying cause of the disease is well understood. In contrast, the role of MAGED2 mutations in a newly discovered form of Bartter Syndrome (BARTS5) is unknown. In our manuscript we could show that MAGED2 modulates vasopressin-induced protein and phosphorylation patterns in kidney cells, providing a broad basis for further studies of MAGED2 function in development and disease.

Obesity-Associated GNAS Mutations and the Melanocortin Pathway

BackgroundGNAS encodes the Gαs (stimulatory G-protein alpha subunit) protein, which mediates G protein–coupled receptor (GPCR) signaling. GNAS mutations cause developmental delay, short stature, and skeletal abnormalities in a syndrome called Albright’s hereditary osteodystrophy. Because of imprinting, mutations on the maternal allele also cause obesity and hormone resistance (pseudohypoparathyroidism).MethodsWe performed exome sequencing and targeted resequencing in 2548 children who presented with severe obesity, and we unexpectedly identified 22 GNAS mutation carriers. We investigated whether the effect of GNAS mutations on melanocortin 4 receptor (MC4R) signaling explains the obesity and whether the variable clinical spectrum in patients might be explained by the results of molecular assays.ResultsAlmost all GNAS mutations impaired MC4R signaling. A total of 6 of 11 patients who were 12 to 18 years of age had reduced growth. In these patients, mutations disrupted growth hormone–releasing hormone receptor signaling, but growth was unaffected in carriers of mutations that did not affect this signaling pathway (mean standard-deviation score for height, −0.90 vs. 0.75, respectively; P=0.02). Only 1 of 10 patients who reached final height before or during the study had short stature. GNAS mutations that impaired thyrotropin receptor signaling were associated with developmental delay and with higher thyrotropin levels (mean [±SD], 8.4±4.7 mIU per liter) than those in 340 severely obese children who did not have GNAS mutations (3.9±2.6 mIU per liter; P=0.004).ConclusionsBecause pathogenic mutations may manifest with obesity alone, screening of children with severe obesity for GNAS deficiency may allow early diagnosis, improving clinical outcomes, and melanocortin agonists may aid in weight loss. GNAS mutations that are identified by means of unbiased genetic testing differentially affect GPCR signaling pathways that contribute to clinical heterogeneity. Monogenic diseases are clinically more variable than their classic descriptions suggest. (Funded by Wellcome and others.)

The G-protein alpha subunit CgGa1 mediates growth, sporulation, penetration and pathogenicity in Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is the main pathogen causing rubber anthracnose, which brings huge economic loss to the natural rubber industry. Heterotrimeric G proteins play a vital role in signal transduction in filamentous fungi, and G alpha subunits are the major component of G proteins. In this study, we characterize a group I Gα subunit CgGa1 in C. gloeosporioides as a homolog of MagB in Pyricularia oryzae. CgGa1 encodes a 353-amino acid protein and has a G_alpha domain. Deletion of CgGa1 results in reduced vegetative growth and conidia yield, and the mutant cannot produce a fruiting body. The CgGa1 deletion mutant also exhibits decreased conidial germination and appressorium formation significantly. Moreover, the mutant has an obvious deficiency in penetration and loses its virulence completely. Transcriptome analysis showed that CgGa1 could affect the expression of many genes related to carbohydrate metabolism, amino acid metabolism and signal transduction, etc. In conclusion, CgGa1 regulates growth, asexual and sexual sporulation, appressorium formation, penetration and pathogenicity of C. gloeosporioides.