Recent Progress in Understanding the Conformational Mechanism of Heterotrimeric G Protein Activation

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.

Influence of G-protein β-Polypeptide 3 C825T Polymorphism on Antihypertensive Response to Telmisartan and Amlodipine in Chinese Patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>G-protein β-polypeptide 3 (GNB3) is a β subunit isoform of G-protein that plays important role in signal transduction of membrane G-protein coupled receptors (GPCRs). The GNB3 splice variant C825T (rs5443) is associated with risk for essential hypertension (EH) and efficacy of therapeutic drugs targeting GPCRs. It is unknown whether the polymorphism is associated with blood pressure (BP) response to telmisartan or amlodipine, two widely prescribed antihypertensive drugs.</p><p><b>METHODS</b>A total of 93 subjects initially diagnosed as EH were recruited and underwent a 4-week treatment with telmisartan (42 patients) or amlodipine (51 patients) monotherapy. Both baseline and after-treatment BP were measured. GNB3 C825T polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism.</p><p><b>RESULTS</b>Baseline systolic BP (SBP) and diastolic BP (DBP) were comparable among C825T genotypes in both telmisartan and amlodipine treatment groups. Patients with the CT or TT genotypes showed significantly lower body mass index (BMI) as compared with CC homozygotes in both groups (P < 0.05, respectively). GNB3 825TT homozygotes showed significantly higher after-treatment DBP and mean arterial pressure (MAP) than those carrying at least one 825C allele (P < 0.01) in the telmisartan treatment group. No difference in after-treatment SBP, DBP, and MAP levels among C825T genotypes was observed in the amlodipine treatment group. No significant difference in absolute changes in BP levels was observed among the genotypes in either treatment group.</p><p><b>CONCLUSION</b>The GNB3 C825T splice variant is associated with the DBP-lowering effect of telmisartan but not amlodipine in Chinese EH patients.</p>

C825T polymorphism of G protein beta3 subunit gene and Uygur Hilit type of essential hypertension: a correlation study / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the association between C825T polymorphism of G protein beta3 subunit (GNB3) gene and different Hilit types of essential hypertension (EH) in the Uygur nationality of Xinjiang.</p><p><b>METHODS</b>According to Uygur medical theories, EH patients (as the EH group) and non-EH patients (as the control group) were assigned to four Hilit groups. The C825T polymorphism of GNB3 was detected in 161 EH patients and 379 non-EH subjects of different Hilit types by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to explore the difference of the genotypes and allelic frequencies and hypertension.</p><p><b>RESULTS</b>(1) In Xinjiang Uygur population, the distribution frequencies of GNB3 C825T polymorphism were in accordance with Hardy-Weinberg (chi2 = 0.871, P = 0.647). (2) There was no statistical difference in the distribution frequencies of three genotypes and two alleles of GNB3 between the EH group and the control group (P > 0.05). (3) There was statistical difference in distribution frequencies of three genotypes between the abnormal Sapra and non-abnormal Sapra group (the sum of abnormal Sewda, abnormal Kan, and abnormal Balhem) (chi2 = 6.905, P = 0.032), especially between the abnormal Sapra and abnormal Balhem groups (chi2 = 10.404, P = 0.006), but there was no statistical difference in distribution frequencies of alleles between the two groups (P > 0.05). (4) In 161 EH patients, there was statistical difference in the distribution frequencies of three genotypes and two alleles between the abnormal Sapra and non-abnormal Sapra group (chi2 = 9.034, P = 0.011; chi2 = 4.701, P = 0.03).</p><p><b>CONCLUSIONS</b>Both TT genotype and T allele of GNB3 C825T polymorphism might not be associated with EH patients in Xinjiang Uygur populations. However, they were correlated with hypertension patients of non-abnormal Sapra, indicating the pathogeneses of EH with different Hilit types might be different.</p>

G Protein beta3 Subunit Polymorphism and Long-Term Prognosis of Functional Dyspepsia

BACKGROUND/AIMS: A link between G protein beta3 (GNB3) polymorphism and functional dyspepsia (FD) has been suggested. The aim of this study was to determine the role of GNB3 polymorphism in the long-term prognosis of FD in Koreans. METHODS: FD patients and normal healthy controls were recruited from patients who visited our center between December 2006 and June 2007. All of the subjects completed Rome III questionnaires before undergoing upper gastrointestinal endoscopy and colonoscopy. Genomic DNA was extracted for GNB3 genotyping. After 5 years, the subjects were reevaluated using the same questionnaires. RESULTS: GNB3 825T carrier status was significantly related to FD in Koreans (p=0.04). After 5 years, 61.0% of the initial FD patients and 12.2% of the initial normal subjects were diagnosed with FD (odds ratio [OR], 11.7; 95% confidence interval [CI], 4.3 to 31.1; p<0.001). Regardless of the GNB3 genotype (p=0.798), female sex was strongly correlated with FD after 5 years (OR, 3.3; 95% CI, 1.2 to 9.1; p=0.017). CONCLUSIONS: The T allele of GNB3 is linked to FD in Koreans but does not predict long-term prognosis. Female sex is related to a higher prevalence of FD after 5 years.

Structural Aspects of GPCR-G Protein Coupling

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.

G protein signaling in the parasite Entamoeba histolytica

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.

Association between polymorphism of the G-protein β3 subunit C825T and essential hypertension: an updated meta-analysis involving 36,802 subjects

Purpose: The G-protein β3-subunit gene C825T polymorphism (GNB3-C825T) has been reported to be associated with essential hypertension (EH), but results from previous studies are conflicting. The present study aimed at investigating the association between this polymorphism and risk of EH using a meta-analysis on the published studies. Materials and Methods: PubMed, Embase, CBM (China Biological Medicine Database), Wanfang and VIP databases were searched to identify eligible studies published in English and Chinese before March 2013. Data were extracted using standardized methods. The association was assessed by the odds ratio (OR) with 95% confidence intervals (CI). Begg's test was used to measure publication bias. Results: A total of 40 case-control studies containing 16,518 EH patients and 20,284 controls were involved in this meta-analysis. Overall, a significant association was found between GNB3 C825T polymorphism and risk of EH when all studies were pooled with a random-effects model for T versus C (OR=1.09, 95% CI: 1.04-1.19). In the subgroup analysis, the same association was found in overall Caucasian (T versus C, OR=1.16, 95% CI 1.08-1.24) and Chinese populations (TT versus CC, OR=1.23, 95% CI 1.06-1.57). No associations were detected between GNB3-C825T and the risk of EH overall in Asian and Japanese people. Conclusions: Meta-analysis results suggest that the GNB3-C825T polymorphism is associated with risk of EH in the overall population, the Caucasians and the Chinese. The effect of the variants on the expression levels and the possible functional role of the variants in EH should be addressed in further studies.

Systems Biological Approaches Reveal Non-additive Responses and Multiple Crosstalk Mechanisms between TLR and GPCR Signaling

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.

G-Protein Beta3 Subunit C825T Polymorphism in Patients With Overlap Syndrome of Functional Dyspepsia and Irritable Bowel Syndrome

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.

Polymorphisms of the Serotonin Transporter Gene and G-Protein beta3 Subunit Gene in Korean Children with Irritable Bowel Syndrome and Functional Dyspepsia

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.

Tumor suppressor genes on frequently deleted chromosome 3p in nasopharyngeal carcinoma / 癌症

Nasopharyngeal carcinoma (NPC) is among the most common malignancies in southern China. Deletion of genomic DNA, which occurs during the complex pathogenesis process for NPC, represents a pivotal mechanism in the inactivation of tumor suppressor genes (TSGs). In many circumstances, loss of TSGs can be detected as diagnostic and prognostic markers in cancer. The short arm of chromosome 3 (3p) is a frequently deleted chromosomal region in NPC, with 3p21.1-21.2 and 3p25.2-26.1 being the most frequently deleted minimal regions. In recent years, our research group and others have focused on the identification and characterization of novel target TSGs at 3p, such as RASSF1A, BLU, RBMS3, and CHL1, in the development and progression of NPC. In this review, we summarize recent findings of TSGs at 3p and discuss some of these genes in detail. A better understanding of TSGs at 3p will significantly improve our understanding of NPC pathogenesis, diagnosis, and treatment.

Association between the polymorphism of GNB3C825T gene and vasovagal syncope in children / 中华儿科杂志

<p><b>OBJECTIVE</b>Investigate the association between GNB3C825T gene polymorphism and pediatric vasovagal syncope.</p><p><b>METHOD</b>Syncope group consisted of 54 cases of unexplained syncope in children, including 18 males and 36 females, at the age of 11.8 years; control group consisted of 54 healthy children over the same period, of whom 20 were male and 34 female, at the age of 11.2 years. The patients underwent head-up tilt test (HUTT). According to HUTT test results, HUTT-positive group and HUTT-negative group were further classified. For cases in HUTT-positive group, based on the changes in blood pressure and in heart rate during HUTT, vasodepressor, mixed and cardioinhibitory patterns were studied. DNA was extracted from peripheral blood in all the patients. A pair of primers was designed flanking 825 polymorphic loci. Products were recovered by using polymerase chain reaction (PCR). GNB3C825T polymorphism was detected by using gene-side GNB3C825T sequencing. Allele distribution between the various groups were studied.</p><p><b>RESULT</b>Among fifty-four children with syncope, HUTT was positive in 30 cases, including vasodepressor pattern in 15 cases (50.0%), mixed pattern in 9 cases (30.0%) and cardioinhibitory pattern in 6 cases (20.0%). Whereas the subjects in control group had negative HUTT response. GNB3C825T allele C in the control and syncope groups was 81.5% and 65.7%, respectively. GNB3C825T allele T in the control and syncope groups was 18.5% and 34.3%, respectively (χ(2) = 6.888, P < 0.05). GNB3C825T allele C in HUTT-positive and negative groups was 61.7% and 81.3%, respectively. And GNB3C825T allele T in HUTT-positive and negative groups was 38.3% and 18.7%, respectively (χ(2) = 4.905, P < 0.05). GNB3C825T allele frequency did not show statistically significant difference among the 3 hemodynamic patterns of VVS (χ(2) = 0.658, P > 0.05).</p><p><b>CONCLUSION</b>Study on GNB3C825T allele frequency in children with vasovagal syncope is of significant value for a better understanding of the pathophysiology of VVS and provide a molecular biologic basis for its mechanisms.</p>

G-protein beta3 Subunit Gene 825C/T Polymorphism Is Not Associated with Olanzapine-Induced Weight Gain in Korean Schizophrenic Patients

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.

Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells

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.

Analysis of the GNB3 gene 825C/T polymorphism in non-obese and obese Chinese / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the G-protein beta 3 subunit (GNB3) gene 825C/T polymorphism and its relationship to obesity in Chinese population.</p><p><b>METHODS</b>Three hundred and ninety nine subjects (270 non-obese and 129 obese individuals) from a population of Chinese Han nationality in Chengdu area were studied using PCR-restriction fragment length polymorphisms (PCR-RFLPs). Serum lipids were measured by enzymatic kits and apolipoproteins A I, A II, B100, C II, C III and E were measured by RID kits.</p><p><b>RESULTS</b>The frequencies of C and T alleles at the 825C/T site in obese and non-obese groups were 0.531 and 0.469, and 0.528 and 0.472, respectively. It showed no significant difference in both genotypes and allele frequencies between the non-obese and obese groups (P> 0.05). The frequency of T allele at 825C/T site in GNB3 gene in the population (0.471) was significantly higher than that of German white (0.319), lower than that of African black (0.788), and similar to that of Japanese (0.487). In the non-obese group, subjects with genotype TT had higher serum triglyceride(TG) concentrations than those with genotype CT (P< 0.05). In the obese group, subjects with genotype CC had lower serum high-density lipoprotein cholesterol(HDL-C) levels than those with CT genotype (P< 0.05). Similar results were only observed in non-obese male and obese female subgroups, respectively, when male and female subgroups were further separated in the two groups. In addition, non-obese males with genotype TT and obese females with genotype CC had lower HDL-C and higher apoA I levels than those with genotype CT, respectively. Obese males with genotype TT had higher apoA I levels than those with genotype CC.</p><p><b>CONCLUSION</b>The 825C/T polymorphism in the GNB3 gene was not associated with obesity in Chinese Han population of Chengdu area. However, it may be associated with serum triglyceride, HDL-C and apoAI levels, with some gender-specific effect, in this population.</p>

Papel de las proteínas G heterotriméricas sensibles a mastoparán en la activación muscarínica del músculo liso de vías aéreas / Paper of heterotrimetric G-proteins sensitive to mastoparan in the muscarinic activation of tracheal smooth muscle

La activación muscarínica del músculo liso traqueal de bovino (MLTB) genera de dos señales de GMPc (20 y 60s), la primera atribuida a la Guanililciclasa soluble (sGC) y la segunda a la Guanililciclasa particulada (NPR-GC). Existen evidencias que la activación muscarínica es mediada. En este sentido determinamos el efecto del tetradecapéptido desacoplante de las proteínas G Mastoparán sobre dicha activación, evaluando los niveles de GMPc y la actividad contráctil del MLTB. Mastoparán inhibió en un 63 por ciento la primera señal de GMPc y desapareció la segunda. Posteriormente evaluamos el efecto de Mastoparán sobre las sGC y NPR-GC. La enzima sGG se evalúo utilizando el Nitroprusiato de Sodio (SNP), el cual indujo un patrón bifásico de acumulación GMPc, las NPR-GC, fueron evaluadas utilizando los péptidos natriuréticos ANP y CNP-53 los cuales indujeron patrones monotónico y bifásicos respectivamente. Observando en los tres casos que la preincubación con Mastoparán, disminuyó los niveles de GMPc de manera significativa (p<0.05). Dicho efecto fue atribuido a proteínas Gi ya que al pre-tratar el MLTB con PTX desaparecía el efecto de Mastoparán. También evaluamos la actividad contráctil del MLTB, encontrándose que Mastoparán se comportan como un "inhibidor no competitivo" de la contracción muscarínica, sin comprometer la integridad funcional de la maquinaria contráctil ya que las aminas biogenas histamina y serotonina produjeron potentes contracciones. Estos hallazgos nos sugieren que la inhibición selectiva de las señales de GMPc esta relacionada con el bloqueo de la contracción muscarínica.

Cloning and molecular characterization of the Schistosoma mansoni genes RbAp48 and histone H4

The human nuclear protein RbAp48 is a member of the tryptophan/aspartate (WD) repeat family, which binds to the retinoblastoma (Rb) protein. It also corresponds to the smallest subunit of the chromatin assembly factor and is able to bind to the helix 1 of histone H4, taking it to the DNA in replication. A cDNA homologous to the human gene RbAp48 was isolated from a Schistosoma mansoni adult worm library and named SmRbAp48. The full length sequence of SmRbAp48 cDNA is 1036 bp long, encoding a protein of 308 amino acids. The transcript of SmRbAp48 was detected in egg, cercariae and schistosomulum stages. The protein shows 84% similarity with the human RbAp48, possessing four WD repeats on its C-terminus. A hypothetical tridimensional structure for the SmRbAp48 C-terminal domain was constructed by computational molecular modeling using the b-subunit of the G protein as a model. To further verify a possible interaction between SmRbAp48 and S. mansoni histone H4, the histone H4 gene was amplified from adult worm genomic DNA using degenerated primers. The gene fragment of SmH4 is 294 bp long, encoding a protein of 98 amino acids which is 100% identical to histone H4 from Drosophila melanogaster

Inhibition of Neurite Outgrowth by Stably Expressed Go alpha in F11 Cells / 대한해부학회지

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.

Adaptation of cAMP signaling system in SH-SY5Y neuroblastoma cells following expression of a constitutively active stimulatory G protein alpha, Q227L Gsalpha

Heterotrimeric GTP-binding proteins (G protein) are known to participate in the transduction of signals from ligand activated receptors to effector molecules to elicit cellular responses. Sustained activation of cAMP-G protein signaling system by agonist results in desensitization of the pathway at receptor levels, however it is not clear whether such receptor responses induce other changes in post-receptor signaling path that are associated with maintenance of AMP levels, i.e. cAMP-forming adenylate cyclase (AC), cAMP-degrading cyclic nucleotide phosphodiesterase (PDE) and cAMP-dependent protein kinase (PKA). Experiments were performed to determine the expression of AC, PDE, and PKA isoforms in SH-SY5Y neuroblastoma cells, in which cAMP system was activated by expressing a constitutively activated mutant of stimulatory G protein (Q227L Gsalpha). Expression of ACI mRNA was increased, but levels of ACVIII and ACIX mRNA were decreased. All of the 4 expressed isoforms of PDE (PDE1C, PDE2, PDE 4A, and PDE4B) were increased in mRNA expression; the levels of PKA RIalpha, RIbeta, and RIIbeta were increased moderately, however, those of RIIalpha and Calpha were increased remarkably. The activities of AC, PDE and PKA were also increased in the SH-SY5Y cells expressing Q227L Gsalpha. The similar changes in expression and activity of AC, PDE and PKA were observed in the SH-SY5Y cells treated with dbcAMP for 6 days. Consequently, it is concluded that the cAMP system adapts at the post-receptor level to a sustained activation of the system by differential expression of the isoforms of AC, PDE, and PKA in SH-SY5Y neuroblastoma. We also showed that an increase in cellular cAMP concentration might mediate the observed changes in the cAMP system.