Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.
Apoptosis/*radiation effects ; Cell Line, Tumor ; Cyclic AMP Response Element-Binding Protein/metabolism ; GTP-Binding Protein alpha Subunits, Gs/*metabolism ; *Gamma Rays ; Heterotrimeric GTP-Binding Proteins/metabolism ; Humans ; Lung/*cytology/physiology/radiation effects ; Lung Neoplasms ; Transcription Factor AP-1/metabolism ; *Up-Regulation ; bcl-2 Homologous Antagonist-Killer Protein/*metabolism

Heterotrimeric G proteins are key intracellular coordinators that receive signals from cells through activation of cognate G protein-coupled receptors (GPCRs). The details of their atomic interactions and structural mechanisms have been described by many biochemical and biophysical studies. Specifically, a framework for understanding conformational changes in the receptor upon ligand binding and associated G protein activation was provided by description of the crystal structure of the β2-adrenoceptor-Gs complex in 2011. This review focused on recent findings in the conformational dynamics of G proteins and GPCRs during activation processes.
GTP-Binding Proteins* ; Heterotrimeric GTP-Binding Proteins

G protein-coupled receptors (GPCRs) are membrane receptors; approximately 40% of drugs on the market target GPCRs. A precise understanding of the activation mechanism of GPCRs would facilitate the development of more effective and less toxic drugs. Heterotrimeric G proteins are important molecular switches in GPCR-mediated signal transduction. An agonist-activated receptor interacts with specific sites on G proteins and promotes the release of GDP from the Galpha subunit. Because of the important biological role of the GPCR-G protein coupling, conformational changes in the G protein upon receptor coupling have been of great interest. One of the most important questions was the interface between the GPCR and G proteins and the structural mechanism of GPCR-induced G protein activation. A number of biochemical and biophysical studies have been performed since the late 80s to address these questions; there was a significant breakthrough in 2011 when the crystal structure of a GPCR-G protein complex was solved. This review discusses the structural aspects of GPCR-G protein coupling by comparing the results of previous biochemical and biophysical studies to the GPCR-G protein crystal structure.
GTP-Binding Proteins ; Guanosine Diphosphate ; Heterotrimeric GTP-Binding Proteins ; Membranes ; Signal Transduction

The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.
Anti-Infective Agents ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; genetics ; Cyanogen Bromide ; pharmacology ; Escherichia coli ; genetics ; metabolism ; GTP-Binding Protein alpha Subunits, G12-G13 ; biosynthesis ; genetics ; Inclusion Bodies ; metabolism ; Protein Structure, Tertiary ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thioredoxins ; genetics ; Transfection

Heterotrimeric G proteins mediate signals generated by neurotransmitters and hormones. Among G proteins, Go is found in a large quantity in brain and growth cone membranes of neurons. In spite of its abundance in neurons, the role of Go is not fully understood. In the previous study, we showed that transient expression of the alpha subunit of Go (alpha o) modulated neurite outgrowth in F11 cells. It is possible that transient transfection may cause transient accumulation of the protein, which itself may alter differentiation process in non-specific manner. In this study, we determined that modulation of neurite outgrowth by alpha o was specific by evaluating the effect of alpha o in stably transformed F11 cells. F11 cells stably expressing the wild type alpha o (alphao(wt)) and a constitutively active form of alpha o (alpha oQ205L) were established. In normal F11 cells and alpha o-stable cell lines, the neurite length was measured in the presence of dibutyryl cAMP. In normal F11 cells, the average length of neurites was 57.9+/-7.0 microgram. In alpha o(wt)- and alpha o(Q205L)-expressing cells, the average length were 34.4+/-5.1 microgram 30.5+/-3.6 microgram, respectively. Thus, stable expression of alpha o(wt) and alpha o(Q205L) caused a decrease in neurite outgrowth by 40.6%, 47.3% respectively. This result indicates that modulation of neurite by alpha o was specific to the function of alpha o but not due to accumulation of exogenous proteins.
Brain ; Cell Line ; Growth Cones ; GTP-Binding Proteins ; Heterotrimeric GTP-Binding Proteins ; Membranes ; Neurites* ; Neurons ; Neurotransmitter Agents ; Transfection

The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling-Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surface receptor capping. A remarkably large family of 91 Rab GTPases has multiple roles in a complex amebic vesicular trafficking system required for phagocytosis and pinocytosis and secretion of known virulence factors, such as amebapores and cysteine proteases. Although much remains to be discovered, recent studies of G protein signaling in E. histolytica have enhanced our understanding of parasitic pathogenesis and have also highlighted possible targets for pharmacological manipulation.
Animals ; Entamoeba histolytica/*metabolism ; Entamoebiasis/parasitology ; GTP-Binding Proteins/*metabolism ; Heterotrimeric GTP-Binding Proteins/metabolism ; Humans ; *Signal Transduction ; ras Proteins/metabolism

BACKGROUND/AIMS: Guanine nucleotide binding protein (G-protein) beta polypeptide 3 (GNB3) C825T polymorphism alters intracellular signal transduction, which may lead to motor or sensory abnormalities of the gastrointestinal tract. The aim of the present study was to evaluate the association of the GNB3 C825T polymorphism with susceptibility to overlap syndrome of functional dyspepsia (FD) and irritable bowel syndrome (IBS) in a Korean population. METHODS: One hundred sixty-seven patients with FD alone, 60 patients with IBS alone, 85 patients with the overlap of FD and IBS, and 434 asymptomatic healthy subjects participated in the study. Genotyping for GNB3 C825T polymorphism was performed using their blood samples. RESULTS: No association of GNB3 genotypes in patients with FD alone, IBS alone or overlap phenotype, when compared to genotypes in controls, was detected. The frequency of CT and TT genotypes relative to the CC genotype for the phenotypes of FD alone, IBS alone and the coexistence of FD and IBS did not significantly differ. Comparison of the TT genotype with the CC/CT genotype showed no significant association for each phenotype group. CONCLUSIONS: There is no apparent association of the GNB3 C825T polymorphism with the susceptibility to FD, IBS or the overlap of FD and IBS. Larger-scale studies and further investigation on other candidate genes are required.
Carrier Proteins ; Dyspepsia ; Gastrointestinal Tract ; Genotype ; GTP-Binding Proteins ; Guanine ; Heterotrimeric GTP-Binding Proteins ; Humans ; Irritable Bowel Syndrome ; Phenotype ; Signal Transduction

BACKGROUND/AIMS: Many candidate gene studies have revealed that polymorphisms of the 5'-flanking controlled SERT gene linked polymorphic region (5HTT-LPR) gene and G-protein beta3 C825T gene might be associated with functional dyspepsia (FD) and irritable bowel syndrome (IBS). This study was performed to investigate polymorphisms of the 5HTT-LPR gene and G-protein beta3 C825T gene in FD and IBS in Korean children. METHODS: In total, 102 patients with FD, 72 patients with IBS based on the Rome III criteria and 148 healthy controls without gastrointestinal symptoms were included in the study to analyze 5HTT-LPR and G-protein beta3 C825T polymorphisms. RESULTS: 5HTT-LPR genotype analysis revealed no signifi cant differences in FD and IBS patients compared with controls. The GNbeta3 C825T genotype distribution for CC, CT, and TT was 23.6%, 53.4%, and 23.0% in controls, 36.3%, 38.2%, and 25.5% in FD and 37.5%, 38.9%, and 23.6% in IBS, respectively. The CC genotype was more common in FD and IBS patients than controls (p<0.05). When the IBS patients were grouped according to IBS subtypes, CC genotype GNbeta3 C825T was common in diarrhea-dominant IBS, and the TT genotype was common in constipation-dominant IBS (p<0.05). CONCLUSIONS: The CC genotype of G-protein beta3 C825T may be associated with FD and diarrhea-predominant IBS. The TT genotype may be associated with constipation-predominant IBS.
Child ; Dyspepsia ; Genotype ; GTP-Binding Proteins ; Heterotrimeric GTP-Binding Proteins ; Humans ; Irritable Bowel Syndrome ; Rome ; Serotonin ; Serotonin Plasma Membrane Transport Proteins

OBJECTIVE: Weight gain is a possible adverse effect of the use of antipsychotics, and is an important factor for long-term health and treatment compliance. Olanzapine is an atypical antipsychotic known to cause considerable weight gain. A relationship between weight gain and the G protein beta3 subunit gene (GNB3) 825C/T polymorphism has been reported. We therefore examined this possible association in a Korean schizophrenic patient group receiving olanzapine treatment. METHODS: Weight and height measurements were obtained prior to starting olanzapine and measured again after long-term treatment. Genotyping for the 825C/T polymorphism was performed using a PCR-based method. RESULTS: We found that long-term treatment with olanzapine resulted in mean gains in weight and body mass index (BMI) of 5.2 kg and 1.93 kg/m2, respectively. There was a no significant difference in the mean body weight change from baseline to the endpoint after olanzapine treatment between the genotype groups (p=0.796). There were also no significant differences in genotype or allele frequencies between the severe weight-gain (more than 10%) and minimal weight-gain (less than 10%) groups (chi-square=0.037, p=0.98; chi-square=0.020, p=0.89). CONCLUSION: The finding from this study thus does not support a relationship between the GNB3 825C/T polymorphism and weight gain in Korean schizophrenic patients receiving olanzapine treatment.
Antipsychotic Agents ; Benzodiazepines ; Body Mass Index ; Body Weight Changes ; Compliance ; Gene Frequency ; Genotype ; GTP-Binding Proteins ; Heterotrimeric GTP-Binding Proteins ; Humans ; Schizophrenia ; Weight Gain

A variety of ligands differ in their capacity to bind the receptor, elicit gene expression, and modulate physiological responses. Such receptors include Toll-like receptors (TLRs), which recognize various patterns of pathogens and lead to primary innate immune activation against invaders, and G-protein coupled receptors (GPCRs), whose interaction with their cognate ligands activates heterotrimeric G proteins and regulates specific downstream effectors, including immuno-stimulating molecules. Once TLRs are activated, they lead to the expression of hundreds of genes together and bridge the arm of innate and adaptive immune responses. We characterized the gene expression profile of Toll-like receptor 4 (TLR4) in RAW 264.7 cells when it bound with its ligand, 2-keto-3-deoxyoctonate (KDO), the active part of lipopolysaccharide. In addition, to determine the network communications among the TLR, Janus kinase (JAK)/signal transducer and activator of transcription (STAT), and GPCR, we tested RAW 264.7 cells with KDO, interferon-beta, or cAMP analog 8-Br. The ligands were also administered as a pair of double and triple combinations.
Arm ; Gene Expression ; GTP-Binding Proteins ; Heterotrimeric GTP-Binding Proteins ; Immunity, Innate ; Interferon-beta ; Ligands ; Macrophages ; Phosphotransferases ; Sugar Acids ; Toll-Like Receptor 4 ; Toll-Like Receptors ; Transcriptome ; Transducers