Alpha Subunits Of Guanine Nucleotide-binding Proteins Research Articles

Effect of GNA13 on the alteration of tumor microenvironmental components through exosomes to promote the development of head and neck tumors.

e18035 Background: Guanine nucleotide-binding protein alpha subunit 13 (GNA13) is closely related to tumor development, but the effect of GNA13 on the biological behavior of head and neck tumors is unknown. The aim of this study was to investigate whether GNA13 can influence the alteration of tumor microenvironment components through exosomes, which in turn promotes the development of head and neck squamous carcinoma (HNSCC). Methods: Using the Gene Expression Omnibus database, the differentially expressed gene GNA13 was screened. Immunohistochemistry and western blotting were used to detect the expression of GNA13 in HNSCC tissues and control tissues. GNA13 expression was down-regulated in tumor cell lines using transfection techniques. The effect of GNA13 downregulation on tumor cell growth was examined by in vitro experiments. RT-PCR and dual luciferase reporter gene assays were used to validate GNA13-regulated miRNAs and their target genes. Clonogenesis assays were used to examine the effect of target gene expression on fibroblasts, and the HNSCC xenograft mouse model was used to verify the role of GNA13 in tumor growth in vivo. The peripheral blood from patients with different stages of HNSCC was collected to determine the expression level of GNA13 in serum exosomes and to analyze its relationship with metastasis and postoperative recurrence. Results: GNA13 expression was elevated in HNSCC tissues and cells. The growth capacity of HNSCC cells was significantly reduced after downregulation of GNA13, which induced fibroblast conversion to MAF via exosomes and promoted their proliferation and migration. Meanwhile, GNA13 was able to regulate miR-26a-5p/STRADB expression in fibroblasts via exosomes. The overexpression of STRADB promoted the proliferation and migration of fibroblasts and induced their conversion to MAF. More importantly, in the conditioned medium of STRADB overexpressing fibroblasts, the proliferation and migration of HNSCC cells were promoted. Furthermore, knockdown of GNA13 expression inhibited the tumor growth in nude mice. It appeared that high expression of GNA13 in serum exosomes of patients with HNSCC was associated with poor prognosis. Conclusions: GNA13 regulates the miR-26a-5p/STRADB axis in fibroblasts via exosomes to create a metastasis-friendly microenvironment for tumor cells, ultimately promoting the progression of HNSCC. Meanwhile, the expression of GNA13 was positively correlated with the pathological grade and clinical stage of HNSCC, which seems to be a reliable indicator of tumor recurrence. Targeting this signaling pathway would be a promising therapeutic strategy for HNSCC.

Circ_0000595 knockdown alleviates CoCl2-mediated effects in VSMCs by regulating the miR-582-3p/ADAM10 axis.

Thoracic aortic aneurysm (TAA) is a serious vascular disease causing the death of elder people. Accumulating studies have reported that circular RNAs (circRNAs) are implicated in the regulation of aortic aneurysms. However, the role of circ_0000595 in the progression of TAA is still unclear. Quantitative real-time PCR (qRT-PCR) and western blotting were implemented to assess circ_0000595, microRNA (miR)-582-3p, guanine nucleotide-binding protein alpha subunit (ADAM10), PCNA, Bax, and Bcl-2 expression. The proliferation of vascular smooth muscle cells was determined using cell counting kit 8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU). Cell apoptosis was measured using flow cytometry, and caspase-3 activity was analyzed using a commercial kit. After bioinformatics analysis, the interaction between miR-582-3p and circ_0000595 or ADAM10 was validated using a dual-luciferase reporter and RNA immunoprecipitation. As compared with controls, TAA tissues and CoCl2-induced VSMCs displayed high expression of circ_0000595 and ADAM10, and low expression of miR-582-3p. CoCl2 treatment evidently suppressed VSMC proliferation and promoted VSMCs apoptosis, and these impacts were reverted by circ_0000595 knockdown. Circ_0000595 acted as a molecular sponge for miR-582-3p, and circ_0000595 silencing-mediated influences in CoCl2-induced VSMCs were overturned by miR-582-3p inhibitor. ADAM10 was confirmed as a target gene of miR-582-3p, and miR-582-3p overexpression-induced influence was almost restored by overexpressed ADAM10 in CoCl2-induced VSMCs. Besides, circ_0000595 contributed to ADAM10 protein expression by sponging miR-582-3p. Our data verified that circ_0000595 silencing might attenuate CoCl2-mediated impacts in VSMCs by regulating the miR-582-3p/ADAM10 axis, providing new potential roads for treating TAA.

Pharmacological Interventions Targeting Pain in Fibrous Dysplasia/McCune-Albright Syndrome.

Fibrous dysplasia (FD) is a rare, non-inherited bone disease occurring following a somatic gain-of-function R201 missense mutation of the guanine-nucleotide binding protein alpha subunit stimulating activity polypeptide 1 (GNAS) gene. The spectrum of the disease ranges from a single FD lesion to a combination with extraskeletal features; an amalgamation with café-au-lait skin hyperpigmentation, precocious puberty, and other endocrinopathies defines McCune-Albright Syndrome (MAS). Pain in FD/MAS represents one of the most prominent aspects of the disease and one of the most challenging to treat-an outcome driven by (i) the heterogeneous nature of FD/MAS, (ii) the variable presentation of pain phenotypes (i.e., craniofacial vs. musculoskeletal pain), (iii) a lack of studies probing pain mechanisms, and (iv) a lack of rigorously validated analgesic strategies in FD/MAS. At present, a range of pharmacotherapies are prescribed to patients with FD/MAS to mitigate skeletal disease activity, as well as pain. We analyze evidence guiding the current use of bisphosphonates, denosumab, and other therapies in FD/MAS, and also discuss the potential underlying pharmacological mechanisms by which pain relief may be achieved. Furthermore, we highlight the range of presentation of pain in individual cases of FD/MAS to further describe the difficulties associated with employing effective pain treatment in FD/MAS. Potential next steps toward identifying and validating effective pain treatments in FD/MAS are discussed, such as employing randomized control trials and probing new pain pathways in this rare bone disease.

Circ_0004491 stimulates guanine nucleotide-binding protein alpha subunit to inhibit the malignant progression of oral squamous cell carcinoma by sponging miR-2278

Background/purposeCircular RNA origin recognition complex subunit 4 (circORC4; ID: hsa_circ_0004491) have been confirmed to be a novel potential biomarker of oral squamous cell carcinoma (OSCC). This study aimed to explore the molecular mechanism of circ_0004491 in OSCC progression. Materials and methodsLevels of circ_0004491, microRNA (miR)-2278, guanine nucleotide-binding protein alpha subunit (GNAS), Bax, Bcl-2, E-cadherin and ki-67 were detected by quantitative real-time PCR, western blotting and immunohistochemistry. The proliferation of OSCC cells was measured using colony formation assay and EdU staining. Cell apoptosis and motility were detected by flow cytometry and transwell assays respectively. Interaction between miR-2278 and circ_0004491 or GNAS was predicted by bioinformatics analysis and confirmed via luciferase reporter assay and RNA immunoprecipitation assay. Xenograft tumor model was used to analyze the role of circ_0004491 in tumor growth in vivo. ResultsCirc_0004491 was downregulated in OSCC tissues and cell lines. Circ_0004491 overexpression suppressed the proliferation, migration and invasion whereas facilitated the apoptosis of OSCC cells. Circ_0004491 acted as a molecular sponge for miR-2278, and circ_0004491 overexpression-mediated effect was partly reversed by miR-2278 mimic in OSCC cells. MiR-2278 interacted with the 3’UTR of GNAS. Circ_0004491 contributed to GNAS level by sponging miR-2278 in OSCC cells. GNAS knockdown restored miR-2278 inhibitor-mediated effect in OSCC cells. Circ_0004491 overexpression repressed xenograft tumor growth in vivo. ConclusionCirc_0004491 can repress OSCC progression by regulation of miR-2278/GNAS axis, providing a possible circRNA-targeted therapy for OSCC.

A novel GNAS mutation in pseudohypoparathyroidism type 1a in a Chinese man presented with recurrent seizure: a case report

BackgroundPseudohypoparathyroidism is a rare genetic disease characterized by hypocalcaemia and hyperphosphataemia due to the defect to the guanine nucleotide-binding protein alpha subunit (GNAS) gene. Patients with pseudoparathyroidism type 1a and 1c could manifest Albright’s hereditary osteodystrophy and multiple hormone resistance including gonadotropin and thyroid stimulating hormone.Case presentationHere we report a Chinese man who presented with fatigue, recurrent seizure and Albright’s hereditary osteodystrophy. His genetic study revealed a heterozygote mutation in the GNAS gene [NM_000516.4(GNAS): c2787_2788del (p.Val930AspfsTer12)]. After calcium and calcitriol supplement, his seizures achieved partially remission.ConclusionsWe report a case of PHP1a or 1c with a novel frameshift mutation in GNAS gene in a patient presenting with AHO, as well as TSH and partial gonadotropin resistance. This mutation in this case has not been reported in literature and adds to the spectrum of genetic mutations related to PHP.

Molecular Markers of Fibro-Osseous Lesions and Osteosarcomas of the Craniofacial Complex—Current Situation and Recent Advances

Fibro-osseous lesions share clinical, radiological, and pathological features, particularly in the craniofacial complex, a location where diagnosing these lesions is challenging. Some molecular markers involved in the pathogenesis of these lesions offer significant diagnostic support. Guanine nucleotide-binding protein/alpha subunit (GNAS) mutations are specific to fibrous dysplasias and are not encountered in ossifying fibromas (including juvenile variants), or in low-grade and high-grade osteosarcomas of the jaw. Low-grade and dedifferentiated osteosarcomas show murine double-minute 2 (MDM2) amplification, with overexpression of MDM2, abnormalities not encountered in fibrous dysplasia, or in ossifying fibroma. No recurrent cytogenetic or molecular abnormality has been reported to date in conventional ossifying fibromas. The juvenile variants of ossifying fibroma exhibit MDM2/RAS protein activator like-1 (RASAL1) co-amplification, without MDM2 overexpression, abnormalities also observed in certain aggressive osteosarcomas of the jaw, which may derive from juvenile ossifying fibroma. MDM2/RASAL1 co-amplification may constitute an early molecular marker of aggressive juvenile ossifying fibroma and may indicate a need for closer follow-up and more radical treatment.

Molecular Changes Leading to Gastric Cancer: A Suggestion From Rare-Type Gastric Tumors With GNAS Mutations

Molecular Changes Leading to Gastric Cancer: A Suggestion From Rare-Type Gastric Tumors With GNAS Mutations

Interaction of α1-syntrophin with multiple isoforms of heterotrimeric G protein α subunits

Syntrophins are components of the dystrophin-glycoprotein complex of the plasma membrane in muscular and neuronal cells, and recruit signaling proteins such as neuronal nitric oxide synthase via their multiple protein-protein interaction motifs. In this study, we found that alpha1-syntrophin binds to various subtypes of guanine nucleotide-binding protein alpha subunits (Galpha). A pull-down analysis using full-length recombinant alpha1-syntrophin and MS analysis showed that alpha1-syntrophin was coprecipitated with several isoforms of Galpha proteins in addition to known binding partners such as dystrobrevin and neuronal nitric oxide synthase. Further analysis using recombinant Galpha isoforms showed that alpha1-syntrophin associates with at least Galphai, Galphao, Galphas and Galphaq subtypes. The region of alpha1-syntrophin required for its interaction with Galphas was determined as the N-terminal half of the first pleckstrin homology domain. In addition, the syntrophin unique domain of alpha1-syntrophin was suggested to contribute to this interaction. In COS-7 cells, downregulation of alpha1-syntrophin by RNAi resulted in enhanced cAMP production and cAMP response element-binding protein phosphorylation induced by isoproterenol treatment. These results suggest that alpha1-syntrophin provides a scaffold for the Galpha family of heterotrimeric G proteins in the brain to regulate the efficiency of signal transduction evoked by G-protein-coupled receptors.

Acute amphetamine down‐regulates RGS4 mRNA and protein expression in rat forebrain: distinct roles of D1 and D2 dopamine receptors

Administration of psychostimulants modulates mRNA of several regulators of guanine nucleotide-binding protein signaling (RGSs) proteins in the brain. In the present study, the regulation of amphetamine-induced decrease of RGS4 expression in the rat forebrain was evaluated. RGS4 mRNA was reduced by amphetamine in an inverse, dose-dependent manner. The lowest dose (2.5 mg/kg) decreased RGS4 mRNA in caudate putamen for up to 6 h after injection whereas the decrease in several frontal cortical areas was detected at 3 h only. Analysis of RGS4 immunoreactivity by western blotting revealed a decrease 3 h after amphetamine solely in the caudate putamen. Systemic administration of D(1) (SCH23390) or D(2) (eticlopride) receptor antagonists blocked amphetamine-induced locomotion but amphetamine augmented both the SCH23390-induced increase and the eticlopride-induced decrease in RGS4 mRNA in the caudate putamen. Further, the down-regulation of RGS4 immunoreactivity by eticlopride was robust whereas the effect of SCH23390 was blunted as compared with its effect on mRNA. These data suggest that, by decreasing RGS4 expression in the caudate putamen via D(1) receptors, acute amphetamine could disinhibit RGS4-sensitive guanine nucleotide-binding protein alpha-subunit i- and/or q-coupled signaling pathways and favor mechanisms that counterbalance D(1) receptor stimulation.

Biochemical characteristics of guanine nucleotide binding protein alpha-subunit recombinant protein and three mutants: investigation of a domain motion involved in GDP-GTP exchange.

Our previous studies using molecular dynamics have shown a hinge bending motion between the helical and the GTPase domains of GalphaT (Mello et al., 1998). The hypothesis that this motion is allowed by residues Gly56 and Gly179 and that this motion may affect the ligand exchange was tested in this work. Mutations of Gly 56 were carried out and the mutant proteins were expressed in Sf9 cells using the Baculovirus expression system. The recombinant proteins were purified using Ni-NTA affinity chromatography. The results for the (GDP/GTP) exchange assays showed that G56S and double mutants (D55G/G56S) proteins differ significantly from the wild type and D55G mutant forms. The Kd values for GTPgammaS binding of those mutants have decreased by approximately 10-fold. No difference in the GTPase activity was detected for the mutants. Thus, the biochemical results obtained support the conclusions of the computational studies.

Local changes in GTP-binding protein immunoreactivities in human epileptogenic neocortex.

The relative levels of guanine nucleotide-binding protein alpha-subunits Gi1alpha, Gi2alpha, Gi3alpha, Go(alpha), Gs(alpha), and Gx/z(alpha) were measured in neocortex removed at surgery from patients with intractable temporal lobe epilepsy. Immunoreactivity was quantified using specific polyclonal antisera against the Galpha-subunits according to the Laurell "rocket" immunoelectrophoresis technique. We compared the G protein contents of spiking (active) and nonspiking (nonactive) cortical regions, based on intraoperative electrocorticography, within the same and different patients. There were no clear trends for lower or higher levels of G-protein subtypes to be found in the samples of protein extracts from nonspiking regions as compared to spiking regions. However, comparison of paired samples of spiking and nonspiking cortex within the same patient demonstrated that levels of certain G-protein subtypes were either increased or decreased in all patients. This indicates that cortical regions with enhanced neuronal activity may produce microzonal alterations in the levels of G proteins. Moreover, our results suggest that high levels of Gi1alpha and low levels of the other G-protein subtypes appear to be associated with a greater susceptibility to maintaining spiking activity.

Chronic Exposure of NG 108‐15 Cells to Opiate Agonists Does Not Alter the Amount of the Guanine Nucleotide‐Binding Proteins Gi and GO

We have characterized the pertussis toxin substrate in NG 108-15 cell membranes using site-specific antisera and ADP-ribosylation. Cell membranes contain two pertussis toxin-sensitive guanine nucleotide-binding protein alpha-subunits (G alpha) whose Rf values in gel electrophoresis coincide with those of G alpha o and G alpha i2. The total quantity of Gi and Go immunoreactivity amounted to 24.3 +/- 2.8 pmol/mg, whereas only 1.5 +/- 0.2 pmol/mg are capable of undergoing ADP-ribosylation catalyzed by pertussis toxin. Pretreatment of cells with the agonist [D-Ala2,D-Leu2]-enkephalin (DADLE) for 24 h and DADLE or morphine for 72 h did not alter the incorporation of ADP-ribose or the immunoreactive amount of Gi and Go subunits. However, pretreatment for 72 h with naloxone increased the incorporation of ADP-ribose without an apparent change in affinity or in the immunochemically determined protein levels of Gi and Go. This indicates that the process of down-regulation and desensitization of the delta-opioid receptor neither requires quantitative alterations in the levels of Gi and Go nor changes in the degree of coupling among their subunits. In contrast, chronic exposure to antagonists seems to alter the degree of precoupling between alpha- and beta-subunits of Gi and/or Go.

Tissue- and species-specific expression of inhibitory guanine nucleotide-binding proteins. Cloning of a full-length complementary DNA from canine heart.

Three different isoforms of the inhibitory guanine nucleotide-binding protein alpha-subunit (Gi alpha) have been cloned. However, little information on the cardiac-specific expression of these isoforms is available. The deduced amino acid sequence of a full-length Gi alpha 2 complementary DNA from a canine ventricular library displays several unique residues compared with those from other species. In contrast to previously described complementary DNA clones for Gi alpha 2, this complementary DNA has a complete 3'-untranslated region with the expected consensus sequence for polyadenylation. Gi alpha 2 is the predominant isoform in the heart, and its message level of 14.7 +/- 4.9 pg/10 micrograms total ventricular RNA is about fourfold lower than that of the stimulatory guanine nucleotide-binding protein.

Angiotensin II-induced stimulation of voltage-dependent Ca2+ currents in an adrenal cortical cell line.

Biochemical studies suggest that stimulation of aldosterone secretion by angiotensin II involves activation of voltage-dependent Ca2+ channels. We used an adrenocortical cell line (Y1) to study the effect of angiotensin II on transmembranous currents. The hormone (1 nM to 1 microM) caused depolarization of the plasma membrane (from -35 to 10 mV) and elicited repetitive action potentials. Using the whole-cell clamp technique, we identified two types of voltage-dependent Ca2+ currents which differed with respect to their threshold potential and time course of inactivation. Angiotensin II (1 nM to 1 microM) stimulated a slowly inactivating Ca2+ current on average up to 1.7-fold whereas a fast inactivating Ca2+ current remained almost unaffected by the hormone. Ca2+ currents were not influenced by forskolin (1 microM) or intracellularly applied cAMP (50 microM). Pretreatment of cells with pertussis toxin abolished the hormonal stimulation of the slowly inactivating Ca2+ current but was without effect on control currents. The toxin ADP-ribosylated a single membranous peptide of 40 kd Mr. An antiserum raised against a synthetic peptide corresponding to a region common to all sequenced alpha-subunits of guanine nucleotide-binding proteins (G-proteins) and an antiserum raised against a peptide corresponding to a region of alpha-subunits of Gi-like G-proteins reacted with membranous 40 kd peptides, whereas an antiserum raised against a synthetic peptide corresponding to a region specific for the alpha-subunit of the G-protein, G0, failed to recognize a peptide in the 39 to 40 kd region.(ABSTRACT TRUNCATED AT 250 WORDS)