Protein-coupled Signaling Research Articles

TRPV1-Expressing Sensory Neuron Stimulation-based Model of Inflammatory Injury Enhances the Excitability of Spinal Neurons Targeting the Periaqueductal Gray

Modeling inflammatory injury via high-intensity stimulation of peripheral afferents at low frequencies (LFS) potentiates excitatory input onto spinal neurons projecting (PNs) to the midbrain periaqueductal gray (PAG), a region critical to pain processing. Therefore, we sought to identify the underlying afferent population driving this potentiation and how this enhanced synaptic drive might affect spino-PAG PN output to supraspinal pain circuits. We used 3–6-week-old male and female mice expressing Cre recombinase driven by expression of the TRPV1 gene, which is expressed in the lineage of virtually all C fibers, and Cre-dependent channelrhodopsin to stimulate C fibers. Spinal PNs were retrogradely labeled from the PAG using diI. We performed whole-cell patch-clamp recordings in spino-PAG PNs using a semi-intact spinal cord preparation. Stimulation of TRPV1-expressing (TRPV1+) peripheral afferents induced burst firing in virtually all spino-PAG PNs sampled. Inflammation increases the firing rates of C fibers to 1-2 Hz; therefore, we used LFS (1 ms, 470 nm LED pulses at 2 Hz for 2 mins) to model inflammatory injury. LFS induced a persistent (≥20 min) increase in the number of action potentials (APs) within a C fiber-induced burst (n=10, p=0.029), rise in the frequency of synaptically evoked after discharge (n=10, p=0.0004), and decrease in intraburst afterhyperpolarization (n=10, p=0.037). Furthermore, LFS induced a transient membrane depolarization (Mean=6.796 mV, n=12, p<0.0001). Additional experiments suggested the enhanced synaptic excitability relied on postsynaptic G protein-coupled signaling, NMDA receptors, and TRPV1+ afferent input. Finally, LFS may increase the intrinsic excitability of spino-PAG PNs—by decreasing AP threshold (n=14, p=0.015) and increasing membrane resistance (n=14, p=0.0003). LFS had no effect on the membrane potential nor the firing frequency of spino-PAG PNs. In summary, this work suggests that LFS of TRPV1+ fibers persistently enhances spino-PAG projection neuron output, likely leading to a lasting increase in the activation of PAG neural circuits. Grant support from F32NS123008 NINDS; Brewer, C. B. T32DA035165 NIDA; Mackey, S. C. R01DA011289 NIDA; Kauer, J. A.

Protective Prognostic Biomarkers Negatively Correlated with Macrophage M2 Infiltration in Low-Grade Glioma.

Tumor-associated Macrophages (TAMs) play a vital role in the progression of glioma. Macrophage M2 has been confirmed to promote immunosuppression and proliferation of low-grade glioma (LGG). Here, we searched for genes negatively correlated with Macrophages M2 by bioinformatical methods and investigated their protective ability for prognosis. LGG and adjacent normal samples were screened out in TCGA and three GEO datasets. 326 overlapped differentially expressed genes were calculated, and their biological functions were investigated by Go and KEGG analyses. Macrophage M2 accounted for the highest proportion among all 22 immune cells by CIBERSORT deconvolution algorithm. The proportion of Macrophage M2 in LGG was also higher than that in normal tissue according to several deconvolution algorithms. 43 genes in the blue module negatively correlated with Macrophage M2 infiltration were identified by weighted gene coexpression network analysis (WGCNA). Through immune infiltration and correlation analysis, FGFBP3, VAX2, and SHD were selected and they were enriched in G protein-coupled receptors' signaling regulation and cytokine receptor interaction. They could prolong the overall and disease-free survival time. Univariate and multivariate Cox regression analyses were applied to evaluate prognosis prediction ability. Interestingly, FGFBP3 and AHD were independent prognostic predictors. A nomogram was drawn, and its 1-year, 3-year, and 5-year survival prognostic value was verified by ROC curves and calibration plots. In conclusion, FGFBP3, VAX2, and SHD were protective prognostic biomarkers against Macrophage M2 infiltration in low-grade glioma. The FGFBP3 and SHD were independent factors to effectively predict long-term survival probability.

Defining a role of NADPH oxidase in myogenic tone development.

The myogenic response sets the foundation for blood flow control. Recent findings suggest a role for G protein-coupled receptors (GPCR) and signaling pathways tied to the generation of reactive oxygen species (ROS). In this regard, this study ascertained the impact of NADPH oxidase (Nox) on myogenic tone in rat cerebral resistance arteries. The study employed real-time qPCR (RT-qPCR), pressure myography, and immunohistochemistry. Gq blockade abolished myogenic tone in rat cerebral arteries, linking GPCR to mechanosensation. Subsequent work revealed that general (TEMPOL) and mitochondrial specific (MitoTEMPO) ROS scavengers had little impact on myogenic tone, whereas apocynin, a broad spectrum Nox inhibitor, initiated transient dilation. RT-qPCR revealed Nox1 and Nox2 mRNA expression in smooth muscle cells. Pressure myography defined Nox1 rather than Nox2 is facilitating myogenic tone. We rationalized that Nox1-generated ROS was initiating this response by impairing the ability of the CaV 3.2 channel to elicit negative feedback via BKCa . This hypothesis was confirmed in functional experiments. The proximity ligation assay further revealed that Nox1 and CaV 3.2 colocalize within 40nm of one another. Our data highlight that vascular pressurization augments Nox1 activity and ensuing ROS production facilitates myogenic tone by limiting Ca2+ influx via CaV 3.2.

G protein-coupled bile acid receptor TGR5 signaling regulates fibroblast growth factor 21

G protein-coupled bile acid receptor TGR5 signaling regulates fibroblast growth factor 21

Purification of Endogenous Drosophila Transient Receptor Potential Channels.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase Cβ/No receptor potential A (PLCβ/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Differential Actions of Muscarinic Receptor Subtypes in Gastric, Pancreatic, and Colon Cancer.

Cancers arising from gastrointestinal epithelial cells are common, aggressive, and difficult to treat. Progress in this area resulted from recognizing that the biological behavior of these cancers is highly dependent on bioactive molecules released by neurocrine, paracrine, and autocrine mechanisms within the tumor microenvironment. For many decades after its discovery as a neurotransmitter, acetylcholine was thought to be synthesized and released uniquely from neurons and considered the sole physiological ligand for muscarinic receptor subtypes, which were believed to have similar or redundant actions. In the intervening years, we learned this former dogma is not tenable. (1) Acetylcholine is not produced and released only by neurons. The cellular machinery required to synthesize and release acetylcholine is present in immune, cancer, and other cells, as well as in lower organisms (e.g., bacteria) that inhabit the gut. (2) Acetylcholine is not the sole physiological activator of muscarinic receptors. For example, selected bile acids can modulate muscarinic receptor function. (3) Muscarinic receptor subtypes anticipated to have overlapping functions based on similar G protein coupling and downstream signaling may have unexpectedly diverse actions. Here, we review the relevant research findings supporting these conclusions and discuss how the complexity of muscarinic receptor biology impacts health and disease, focusing on their role in the initiation and progression of gastric, pancreatic, and colon cancers.

Loss of β-Arrestins or six Gα proteins in HEK293 cells caused Warburg effect and prevented progesterone-induced rapid proteasomal degradation of progesterone receptor membrane component 1

Hormonal dysregulation plays a significant role in the metabolic switching during malignant transformation. Progesterone Receptor Membrane Component 1 (PGRMC1) is a single-pass transmembrane receptor activated by the binding of progesterone (P4), a sex hormone. In a previous study, P4 treatment caused rapid (within 30 min) induction of aerobic glycolysis in transformed HEK293 cells, a hallmark malignant phenotype known as the Warburg effect. This metabolic reprogramming was associated with the proteasomal degradation of a 70 kilodalton (kDa) PGRMC1. PGRMC1 interacts with a variety of proteins, including G protein-coupled receptors (GPCRs) and P4-PGRMC1 signaling modulates cyclic adenosine monophosphate (cAMP) production. Therefore, we hypothesized that the P4-induced Warburg effect and proteasomal degradation of PGRMC1 involve G proteins and β-Arrestins (ARRBs). In the present study, we investigated P4-induced aerobic glycolysis, proteasomal degradation of p70 PGRMC1, as well as abundance and subcellular translocation of PGRMC1 along with two key glycolytic enzymes Hexokinase 1 (HK1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in six Gα subunit (Gsix) proteins or ARRB1/2-deficient HEK293 cells. Loss of ARRB1/2 or Gsix proteins inhibited P4-induced p70 PGRMC1 degradation but failed to prevent the P4-induced Warburg effect. Also, deficiency of ARRB1/2 or Gsix proteins differentially affected the basal as well as P4-induced abundance and subcellular translocation of PGRMC1, HK1, and GAPDH proteins. Overall, the findings indicate that P4-PGRMC1-mediated metabolic reprogramming in HEK293 cells depends on β-Arrestins and Gα proteins suggesting the involvement of an underlying GPCR signal transduction pathway.

Involvement of GRK2 in modulating nalfurafine-induced reduction of excessive alcohol drinking in mice

Though it is well known that G protein-coupled receptor kinase 2 [GRK2] is involved in regulation of mu opioid receptor [MOR] desensitization and morphine-related behaviors, the potential role of GRK2 in regulation of kappa opioid receptor [KOR] functions in vivo has not been established yet. A couple of recent studies have found that GRK2 activity desensitizes KOR functions via decreasing G protein-coupled signaling with sensitizing arrestin-coupled signaling. Nalfurafine, a G protein-biased KOR full agonist, produces an inhibitory effect on alcohol intake in mice, with fewer side effects (sedation, aversion, or anxiety/depression-like behaviors). Using RNA sequencing (RNA-seq) analysis, we first identified that nuclear transcript level of grk2 [adrbk1] (but not other grks) was significantly up-regulated in mouse nucleus accumbens shell (NAcs) after chronic excessive alcohol drinking, suggesting alcohol specifically increased NAcs grk2 expression. We then tested whether selective GRK2/3 inhibitor CMPD101 could alter alcohol intake and found that CMPD101 alone had no effect on alcohol drinking. Therefore, we hypothesized that the grk2 increase in the NAcs could modulate the nalfurafine effect on alcohol intake via interacting with the G protein-mediated KOR signaling. Nalfurafine decreased alcohol drinking in a dose-related manner, and pretreatment with CMPD101 enhanced the reduction in alcohol intake induced by nalfurafine, indicating an involvement of GRK2/3 blockade in modulating G protein-biased KOR agonism of nalfurafine. Together, our study provides initial evidence relevant to the transcriptional change of grk2 gene in the NAc shell after excessive alcohol drinking. Pharmacological GRK2/3 blockade enhanced nalfurafine’s efficacy, suggesting a GRK2/3-mediated mechanism, probably through the G protein-mediated KOR signaling.

GABAB receptors in GtoPdb v.2021.2

Functional GABAB receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on GABAB receptors [11, 71]) are formed from the heterodimerization of two similar 7TM subunits termed GABAB1 and GABAB2 [11, 70, 28, 71, 87]. GABAB receptors are widespread in the CNS and regulate both pre- and postsynaptic activity. The GABAB1 subunit, when expressed alone, binds both antagonists and agonists, but the affinity of the latter is generally 10-100-fold less than for the native receptor. Co-expression of GABAB1 and GABAB2 subunits allows transport of GABAB1 to the cell surface and generates a functional receptor that can couple to signal transduction pathways such as high-voltage-activated Ca2+ channels (Cav2.1, Cav2.2), or inwardly rectifying potassium channels (Kir3) [12, 11, 5]. The GABAB1 subunit harbours the GABA (orthosteric)-binding site within an extracellular domain (ECD) venus flytrap module (VTM), whereas the GABAB2 subunit mediates G protein-coupled signalling [11, 70, 40, 39]. The cryo-electron microscopy structures of the human full-length GABAB1-GABAB2 heterodimer have been solved in the inactive apo state, two intermediate agonist-bound forms and an active state in which the heterodimer is bound to an agonist and a positive allosteric modulator [81]. The positive allosteric modulator binds to the transmembrane dimerization interface and stabilizes the active state. Recent evidence indicates that higher order assemblies of GABAB receptor comprising dimers of heterodimers occur in recombinant expression systems and in vivo and that such complexes exhibit negative functional cooperativity between heterodimers [69, 22]. Adding further complexity, KCTD (potassium channel tetramerization proteins) 8, 12, 12b and 16 associate as tetramers with the carboxy terminus of the GABAB2 subunit to impart altered signalling kinetics and agonist potency to the receptor complex [86, 3, 79] and are reviewed by [72]. The molecular complexity of GABAB receptors is further increased through association with trafficking and effector proteins [80] and reviewed by [68]. The predominant GABAB1a and GABAB1b isoforms, which are most prevalent in neonatal and adult brain tissue respectively, differ in their ECD sequences as a result of the use of alternative transcription initiation sites. GABAB1a-containing heterodimers localise to distal axons and mediate inhibition of glutamate release in the CA3-CA1 terminals, and GABA release onto the layer 5 pyramidal neurons, whereas GABAB1b-containing receptors occur within dendritic spines and mediate slow postsynaptic inhibition [74, 91]. Amyloid precursor protein (APP) and soluble APP (sAPP) bind to the N- terminal sushi domain of the GABAB1a isoform to regulate axonal trafficking of GABAB receptors and release of neurotransmitters [76].

Discovery of an Orally Efficacious Positive Allosteric Modulator of the Glucagon-like Peptide-1 Receptor.

The identification of LSN3318839, a positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), is described. LSN3318839 increases the potency and efficacy of the weak metabolite GLP-1(9-36)NH2 to become a full agonist at the GLP-1R and modestly potentiates the activity of the highly potent full-length ligand, GLP-1(7-36)NH2. LSN3318839 preferentially enhances G protein-coupled signaling by the GLP-1R over β-arrestin recruitment. Ex vivo experiments show that the combination of GLP-1(9-36)NH2 and LSN3318839 produces glucose-dependent insulin secretion similar to that of GLP-1(7-36)NH2. Under nutrient-stimulated conditions that release GLP-1, LSN3318839 demonstrates robust glucose lowering in animal models alone or in treatment combination with sitagliptin. From a therapeutic perspective, the biological properties of LSN3318839 support the concept that GLP-1R potentiation is sufficient for reducing hyperglycemia.

Optineurin deletion disrupts metabotropic glutamate receptor 5-mediated regulation of ERK1/2, GSK3β/ZBTB16, mTOR/ULK1 signaling in autophagy

Optineurin (OPTN) is a multifunctional protein that mediates a network of cellular processes regulating membrane trafficking, inflammatory responses and autophagy. The OPTN-rich interactome includes Group I metabotropic glutamate receptors (mGluR1 and 5), members of the Gαq/11 protein receptor family. Recent evidence has shown that mGluR5, in addition to its canonical Gαq/11 protein-coupled signaling, regulates autophagic machinery via mTOR/ULK1 and GSK3β/ZBTB16 pathways in both Alzheimer’s and Huntington’s disease mouse models. Despite its potential involvement, the role of OPTN in mediating mGluR5 downstream signaling cascades remains largely unknown. Here, we employed a CRISPR/Cas9 OPTN-deficient STHdhQ7/Q7 striatal cell line and global OPTN knockout mice to investigate whether Optn gene deletion alters both mGluR5 canonical and noncanonical signaling. We find that OPTN is required for mGluR5-activated Ca2+ flux and ERK1/2 signaling following receptor activation in STHdhQ7/Q7 cells and acute hippocampal slices. Deletion of OPTN impairs both GSK3β/ZBTB16 and mTOR/ULK1 autophagic signaling in STHdhQ7/Q7 cells. Furthermore, mGluR5-dependent regulation of GSK3β/ZBTB16 and mTOR/ULK1 autophagic signaling is impaired in hippocampal slices of OPTN knockout mice. Overall, we show that the crosstalk between OPTN and mGluR5 can have major implication on receptor signaling and therefore potentially contribute to the pathophysiology of neurodegenerative diseases.

Type I interferon upregulation and deregulation of genes involved in monopoiesis in chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is characterized by myelomonocytic bias and monocytic proliferation. Whether cell-intrinsic innate immune or inflammatory upregulation mediate disease pathogenesis and phenotype or whether the degree of aberrant monocytic differentiation influences outcomes remains unclear. We compared the transcriptomic features of bone marrow CD34+ cells from 19 patients with CMML and compared to healthy individuals. A total of 1495 genes had significantly differential expression in CMML (q<0.05, fold change>2), including 1271 genes that were significantly upregulated and 224 that were significantly downregulated in CMML. Top upregulated genes were associated with interferon (IFN) alpha and beta signaling, chemokine receptors, IFN gamma, G protein-coupled receptor ligand signaling, and genes involved in immunomodulatory interactions between lymphoid and non-lymphoid cells. Additionally, 6 gene sets were differentially upregulated and 139 were significantly downregulated in patients with myeloproliferative compared to myelodysplastic CMML. A total of 23 genes involved in regulation of monopoiesis were upregulated in CMML compared to healthy controls. We developed a prediction model using Cox regression including 3 of these genes, which differentiated patients into two prognostic subsets with distinct survival outcomes. This data warrants further evaluation of the roles and therapeutic potential of type I IFN signaling and monopoiesis in CMML.

Post-Translational Modifications of G Protein-Coupled Receptors Control Cellular Signaling Dynamics in Space and Time.

G protein-coupled receptors (GPCRs) are a large family comprising >800 signaling receptors that regulate numerous cellular and physiologic responses. GPCRs have been implicated in numerous diseases and represent the largest class of drug targets. Although advances in GPCR structure and pharmacology have improved drug discovery, the regulation of GPCR function by diverse post-translational modifications (PTMs) has received minimal attention. Over 200 PTMs are known to exist in mammalian cells, yet only a few have been reported for GPCRs. Early studies revealed phosphorylation as a major regulator of GPCR signaling, whereas later reports implicated a function for ubiquitination, glycosylation, and palmitoylation in GPCR biology. Although our knowledge of GPCR phosphorylation is extensive, our knowledge of the modifying enzymes, regulation, and function of other GPCR PTMs is limited. In this review we provide a comprehensive overview of GPCR post-translational modifications with a greater focus on new discoveries. We discuss the subcellular location and regulatory mechanisms that control post-translational modifications of GPCRs. The functional implications of newly discovered GPCR PTMs on receptor folding, biosynthesis, endocytic trafficking, dimerization, compartmentalized signaling, and biased signaling are also provided. Methods to detect and study GPCR PTMs as well as PTM crosstalk are further highlighted. Finally, we conclude with a discussion of the implications of GPCR PTMs in human disease and their importance for drug discovery. SIGNIFICANCE STATEMENT: Post-translational modification of G protein-coupled receptors (GPCRs) controls all aspects of receptor function; however, the detection and study of diverse types of GPCR modifications are limited. A thorough understanding of the role and mechanisms by which diverse post-translational modifications regulate GPCR signaling and trafficking is essential for understanding dysregulated mechanisms in disease and for improving and refining drug development for GPCRs.

Tctex-1 augments G protein-coupled receptor-mediated Gs signaling by activating adenylyl cyclase

Proteins interacting with G protein-coupled receptors (GPCRs) can modulate signal transduction of these receptors. However, the regulatory mechanisms of the interacting proteins are diverse and largely unknown. We have previously shown that Tctex-1 (or DYNLT1) can interact with the parathyroid hormone receptor (PTHR). In the present study, we investigated the role of Tctex-1 in the PTHR signaling and found that Tctex-1 augmented the PTHR-mediated Gs/adenylyl cyclase (AC) pathway by activating AC regardless of the binding to PTHR. Furthermore, Tctex-1 directly bound to AC type 6. These data demonstrate a novel mechanism underlying GPCR/Gs signaling regulated by Tctex-1.

Functional Selectivity and Antinociceptive Effects of a Novel KOPr Agonist.

Kappa opioid receptor (KOPr) agonists represent alternative analgesics for their low abuse potential, although relevant adverse effects have limited their clinical use. Functionally selective KOPr agonists may activate, in a pathway-specific manner, G protein-mediated signaling, that produces antinociception, over β-arrestin 2-dependent induction of p38MAPK, which preferentially contributes to adverse effects. Thus, functionally selective KOPr agonists biased toward G protein-coupled intracellular signaling over β-arrestin-2-mediated pathways may be considered candidate therapeutics possibly devoid of many of the typical adverse effects elicited by classic KOPr agonists. Nonetheless, the potential utility of functionally selective agonists at opioid receptors is still highly debated; therefore, further studies are necessary to fully understand whether it will be possible to develop more effective and safer analgesics by exploiting functional selectivity at KOPr. In the present study we investigated in vitro functional selectivity and in vivo antinociceptive effects of LOR17, a novel KOPr selective peptidic agonist that we synthesized. LOR17-mediated effects on adenylyl cyclase inhibition, ERK1/2, p38MAPK phosphorylation, and astrocyte cell proliferation were studied in HEK-293 cells expressing hKOPr, U87-MG glioblastoma cells, and primary human astrocytes; biased agonism was investigated via cAMP ELISA and β-arrestin 2 recruitment assays. Antinociception and antihypersensitivity were assessed in mice via warm-water tail-withdrawal test, intraperitoneal acid-induced writhing, and a model of oxaliplatin-induced neuropathic cold hypersensitivity. Effects of LOR17 on locomotor activity, exploratory activity, and forced-swim behavior were also assayed. We found that LOR17 is a selective, G protein biased KOPr agonist that inhibits adenylyl cyclase and activates early-phase ERK1/2 phosphorylation. Conversely to classic KOPr agonists as U50,488, LOR17 neither induces p38MAPK phosphorylation nor increases KOPr-dependent, p38MAPK-mediated cell proliferation in astrocytes. Moreover, LOR17 counteracts, in a concentration-dependent manner, U50,488-induced p38MAPK phosphorylation and astrocyte cell proliferation. Both U50,488 and LOR17 display potent antinociception in models of acute nociception, whereas LOR17 counteracts oxaliplatin-induced thermal hypersensitivity better than U50,488, and it is effective after single or repeated s.c. administration. LOR17 administered at a dose that fully alleviated oxaliplatin-induced thermal hypersensitivity did not alter motor coordination, locomotor and exploratory activities nor induced pro-depressant-like behavior. LOR17, therefore, may emerge as a novel KOPr agonist displaying functional selectivity toward G protein signaling and eliciting antinociceptive/antihypersensitivity effects in different animal models, including oxaliplatin-induced neuropathy.

Crosstalk between heterotrimeric G protein-coupled signaling pathways and WRKY transcription factors modulating plant responses to suboptimal micronutrient conditions.

Nutrient stresses induce foliar chlorosis and growth defects. Here we propose heterotrimeric G proteins as signaling mediators of various nutrient stresses, through meta-analyses of >20 transcriptomic data sets associated with nutrient stresses or G protein mutations. Systematic comparison of transcriptomic data yielded 104 genes regulated by G protein subunits under common nutrient stresses: 69 genes under Gβ subunit (AGB1) control and 35 genes under Gα subunit (GPA1) control. Quantitative real-time PCR experiments validate that several transcription factors and metal transporters changed in expression level under suboptimal iron, zinc, and/or copper concentrations, while being misregulated in the Arabidopsis Gβ-null (agb1) mutant. The agb1 mutant had altered metal ion profiles and exhibited severe growth arrest under zinc stress, and aberrant root waving under iron and zinc stresses, while the Gα-null mutation attenuated leaf chlorosis under iron deficiency in both Arabidopsis and rice. Our transcriptional network analysis inferred computationally that WRKY-family transcription factors mediate the AGB1-dependent nutrient responses. As corroborating evidence of our inference, ectopic expression of WRKY25 or WRKY33 rescued the zinc stress phenotypes and the expression of zinc transporters in the agb1-2 background. These results, together with Gene Ontology analyses, suggest two contrasting roles for G protein-coupled signaling pathways in micronutrient stress responses: one enhancing general stress tolerance and the other modulating ion homeostasis through WRKY transcriptional regulatory networks. In addition, tolerance to iron stress in the rice Gα mutant provides an inroad to improve nutrient stress tolerance of agricultural crops by manipulating G protein signaling.

Higher burden of rare frameshift indels in genes related to synaptic transmission separate familial hemiplegic migraine from common types of migraine

BackgroundFamilial hemiplegic migraine (FHM) is a rare form of migraine with aura that often has an autosomal dominant mode of inheritance. Rare mutations in the CACNA1A, ATP1A2 and SCN1A genes...

Disruption of 17β-estradiol secretion by persistent organic pollutants present in human follicular fluid is dependent on the potential of ovarian granulosa tumor cell lines to metabolize estrogen

Endocrine-disrupting chemicals (EDCs), such as perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, hexachlorobenzene, and polychlorinated biphenyl 153 are persistent pollutants that are found in human follicular fluid (FF). These compounds may affect endocrine function, disrupt steroid secretion by granulosa cells, and play a role in granulosa cell tumor (GCT) development. GCTs demonstrate endocrine activity, expressing aromatase and secreting 17β-estradiol (E2). We aimed to determine the effects of a mixture of EDCs, similar to that found in human FF, on human granulosa tumor cell lines representing the juvenile (JGCT) and adult (AGCT) forms (COV434 and KGN cells, respectively). We found that all the individual compounds and mixtures tested altered granulosa tumor cell function by disrupting E2 secretion. In KGN cells, which possess significantly higher basal aromatase gene expression, and therefore secrete more E2 than JGCT cells, EDC mixtures activated estrogen receptors (ERs) and G protein-coupled receptor-30 signaling, thereby stimulating E2 secretion, without affecting aromatase expression. By contrast, in COV434 cells, which demonstrate higher CYP1A1 expression, a key mediator of estrogen metabolism, than KGN cells, EDC mixtures reduced E2 secretion in parallel with increases in the 2-hydroxyestrogen 1/E2 ratio and CYP1A1 expression, implying an upregulation of E2 metabolism. These results indicate that the EDC mixture present in FF disrupts E2 secretion in JGCT and AGCT cells according to the estrogen metabolic potential of the cell type, involving both classical and non-classical ER pathways.

Computational design of G Protein-Coupled Receptor allosteric signal transductions.

Membrane receptors sense and transduce extracellular stimuli into intracellular signaling responses but the molecular underpinnings remain poorly understood. We report a computational approach for designing protein allosteric signaling functions. By combining molecular dynamics simulations and design calculations, the method engineers amino-acid 'microswitches' at allosteric sites that modulate receptor stability or long-range coupling, to reprogram specific signaling properties. We designed 36 dopamine D2 receptor variants, whose constitutive and ligand-induced signaling agreed well with our predictions, repurposed the D2 receptor into a serotonin biosensor and predicted the signaling effects of more than 100 known G-protein-coupled receptor (GPCR) mutations. Our results reveal the existence of distinct classes of allosteric microswitches and pathways that define an unforeseen molecular mechanism of regulation and evolution of GPCR signaling. Our approach enables the rational design of allosteric receptors with enhanced stability and function to facilitate structural characterization, and reprogram cellular signaling in synthetic biology and cell engineering applications.

An Overview on G Protein-coupled Receptor-induced Signal Transduction in Acute Myeloid Leukemia.

Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by uncontrolled proliferation of precursor myeloid-lineage cells in the bone marrow. AML is also characterized by patients with poor long-term survival outcomes due to relapse. Many efforts have been made to understand the biological heterogeneity of AML and the challenges to develop new therapies are therefore enormous. G Protein-coupled Receptors (GPCRs) are a large attractive drug-targeted family of transmembrane proteins, and aberrant GPCR expression and GPCR-mediated signaling have been implicated in leukemogenesis of AML. This review aims to identify the molecular players of GPCR signaling, focusing on the hematopoietic system, which are involved in AML to help developing novel drug targets and therapeutic strategies. We undertook an exhaustive and structured search of bibliographic databases for research focusing on GPCR, GPCR signaling and expression in AML. Many scientific reports were found with compelling evidence for the involvement of aberrant GPCR expression and perturbed GPCR-mediated signaling in the development of AML. The comprehensive analysis of GPCR in AML provides potential clinical biomarkers for prognostication, disease monitoring and therapeutic guidance. It will also help to provide marker panels for monitoring in AML. We conclude that GPCR-mediated signaling is contributing to leukemogenesis of AML, and postulate that mass spectrometrybased protein profiling of primary AML cells will accelerate the discovery of potential GPCR related biomarkers for AML.