Alpha O1 Research Articles

Gnao1 is a molecular switch that regulates the Rho signaling pathway in differentiating neurons

GNAO1 encodes G protein subunit alpha O1 (Gαo). Pathogenic variations in GNAO1 cause developmental delay, intractable seizures, and progressive involuntary movements from early infancy. Because the functional role of GNAO1 in the developing brain remains unclear, therapeutic strategies are still unestablished for patients presenting with GNAO1-associated encephalopathy. We herein report that siRNA-mediated depletion of Gnao1 perturbs the expression of transcripts associated with Rho GTPase signaling in Neuro2a cells. Consistently, siRNA treatment hampered neurite outgrowth and extension. Growth cone formation was markedly disrupted in monolayer neurons differentiated from iPSCs from a patient with a pathogenic variant of Gαo (p.G203R). This variant disabled neuro-spherical assembly, acquisition of the organized structure, and polarized signals of phospho-MLC2 in cortical organoids from the patient’s iPSCs. We confirmed that the Rho kinase inhibitor Y27632 restored these morphological phenotypes. Thus, Gαo determines the self-organizing process of the developing brain by regulating the Rho-associated pathway. These data suggest that Rho GTPase pathway might be an alternative target of therapy for patients with GNAO1-associated encephalopathy.

Both subthalamic and pallidal deep brain stimulation are effective for GNAO1-associated dystonia: three case reports and a literature review.

Background:Mutations in the G-protein subunit alpha o1 (GNAO1) gene have recently been shown to be involved in the pathogenesis of early infantile epileptic encephalopathy and movement disorders. The clinical manifestations of GNAO1-associated movement disorders are highly heterogeneous. However, the genotype–phenotype correlations in this disease remain unclear, and the treatments for GNAO1-associated movement disorders are still limited.Objective:The objective of this study was to explore diagnostic and therapeutic strategies for GNAO1-associated movement disorders.Methods:This study describes the cases of three Chinese patients who had shown severe and progressive dystonia in the absence of epilepsy since early childhood. We performed genetic analyses in these patients. Patients 1 and 2 underwent globus pallidus internus (GPi) deep brain stimulation (DBS) implantation, and Patient 3 underwent subthalamic nucleus (STN) DBS implantation. In addition, on the basis of a literature review, we summarized and discussed the clinical characteristics and outcomes after DBS surgery for all reported patients with GNAO1-associated movement disorders.Results:Whole-exome sequencing (WES) analysis revealed de novo variants in the GNAO1 gene for all three patients, including a splice-site variant (c.724–8G > A) in Patients 1 and 3 and a novel heterozygous missense variant (c.124G > A; p. Gly42Arg) in Patient 2. Both GPi and STN DBS were effective in improving the dystonia symptoms of all three patients.Conclusion:DBS is effective in ameliorating motor symptoms in patients with GNAO1-associated movement disorders, and both STN DBS and GPi DBS should be considered promptly for patients with sustained refractory GNAO1-associated dystonia.

GNAO1 as a Novel Predictive Biomarker for Late Relapse in Hepatocellular Carcinoma.

GNAO1, the alpha O1 subunit of G protein, was reported to be significantly downregulated in hepatocellular carcinoma (HCC), as well as being implicated in a variety of intracellular biological events; findings suggest that it may act as a tumor suppressor. Our goal was to further explore the expression of GNAO1 in HCC patients and its potential clinical significance. Oncomine and Kaplan–Meier plotter databases were used to assess the mRNA expression of GNAO1 in HCC tissues and patient survival time. Subsequently, immunohistochemistry (IHC) was used to measure GNAO1 protein level in tissue from 79 cases of HCC and paired adjacent tissues. The Kaplan–Meier survival analysis, Cox regression model, and prognostic nomogram were used to evaluate the prognostic role of GNAO1 in HCC. Results demonstrated that mRNA and protein expressions of GNAO1 were both lower in HCC tissues than in adjacent tissues (all p < 0.01). HCC patients with high expression of GNAO1 had better relapse-free survival (RFS) than those with low GNAO1 expression (all p < 0.05). A high expression of GNAO1, meanwhile, functioned as a good predictor of late relapse for HCC (p < 0.05). The nomogram consisting of GNAO1 expression and the tumor-node-metastasis (TNM) model presented good ability in predicting the 3-year relapse for HCC (C-index = 0.614). In conclusion, GNAO1 was a reliable biomarker of relapse prediction for HCC.

Identify Cross Talk Between Circadian Rhythm and Coronary Heart Disease by Multiple Correlation Analysis.

Disorder in circadian rhythm has been revealed as a risk factor for coronary heart disease. Several studies in molecular biology established a gene interaction network using coronary heart susceptibility genes and the circadian rhythm pathway. However, cross talk between genes was mostly discovered in single gene pairs. There might be combination sets of genes intergraded as a unit to regulate the network. To resolve multiple variables in coronary heart susceptibility genes controlling circadian rhythm pathways, a multiple correlation analysis was applied to the transcriptome. Nine genes, including CUGBP, Elav-like family member (CELF); sodium leak channel, nonselective (NALCN); protein phosphatase 2 regulatory subunit B gamma (PPP2R2C); tubulin alpha 1c (TUBA1C); microtubule-associated protein 4 (MAP4); cofilin 1 (CFL1); myosin heavy chain 7 (MYH7); QKI, KH domain containing RNA binding (QKI); and maternal embryonic leucine zipper kinase (MELK), from coronary heart susceptibility were identified to predict the outcome of a linear combination of circadian rhythm pathway genes with R factor more than 0.7. G protein subunit alpha o1 (GNAO1), protein kinase C gamma (PRKCG), RBX, and G protein subunit beta 1 (GNB1) in the circadian rhythm pathway are characterized as combination variables to coexpress with coronary heart susceptibility genes.

Designing a model to detect the brain connections abnormalities in children with autism using 3D-cellular neural networks.

In neuropsychological disorders, the significant abnormalities in the brain connections in some regions are observed. This paper presents a novel model to demonstrate the connections between different regions in children with autism. The proposed model first conducts the wavelet decomposition of electroencephalography signals by wavelet transform then the features are extracted, such as relative energy and entropy. These features are fed to the 3D-cellular neural network model as inputs to indicate the brain connections. The results showed that there are significant differences and abnormalities in the left hemisphere, (p<0.05) at the electrodes AF3, F3, P7, T7 and O1 in alpha band, AF3, F7, T7 and O1 in beta band, T7 and P7 in gamma band for children with autism compared with the control children. Also, the evaluation of the obtained connections values between brain regions indicated that there are more abnormalities in the connectivity of frontal and parietal lobes and the relations of the neighboring regions in all three bands especially in gamma band for autistic children. Evaluation of the analysis demonstrated that alpha frequency band had the best distinction level of 96.6% based on the obtained values of the cellular neural network using support vector machine method.

Electroencephalographic changes after one nigth of sleep deprivation

Total or partial sleep deprivation (SD) causes degrading effects on different cognitive and psychomotor functions that might be related to electrophysiological changes frequently observed. In the present study, we investigated the effects of one night of sleep deprivation on waking EEG. Experimental protocol consisted of recording electroencephalographic data from eleven healthy young subjects before (baseline) and after (time 2) one night of sleep deprivation. A natural log transformation was carried out and showed a significant increase in theta T6 (p=0.041), O2 (p=0.018) and OZ (p=0.028); and delta T6 (p=0.043) relative power; and a decrease in alpha Fp1 (p=0.040), F3 (p=0.013), Fp2 (p=0.033), T4 (p=0.050), T6 (p=0.018), O2 (p=0.011) and Oz (p=0.025) and beta (p=0.022) absolute power. These outcomes show that the EEG power spectra, after sleep deprivation, exhibit site-specific differences in particular frequency bands and corroborate for the premise of local aspects of brain adaptation after sleep deprivation, rather than global.

Levels of IL-1 beta control stimulatory/inhibitory growth of cancer cells

Different cellular signaling pathways operate in response to varying levels of IL-1 beta leading to genotoxic damage, cell apoptosis or cell growth. At high levels of IL-1 beta, cells receiving genotoxic insults engage apoptotic pathways. The IL-1 beta over expressing stable MCF7 cell secreting high level of IL-1 beta peptides undergo cell apoptosis. Cotreatment with an inhibitor of IL-1 beta and TNF-alpha synthesis prevented stilbene estrogen-induced lesions. In addition to direct effect of 17 beta-estradiol (E2) on mitochondria and redox cycling of catechol estrogens, E2-induced overexpression of IL-1 beta can produce an increase in the level of ROS. Our recent data showed that MCF7 cell growth and cyclin D1 expression are suppressed by antioxidants and mitochondrial blockers. Stably IL-1 beta transfected cells secreting moderate level of IL-1 beta peptides stimulated the clonal expansion of MCF7 cells. These studies support that in addition to ovarian estrogens, mitogenic signals may also come from TNF-alpha and IL-1 beta-generated O2*- and hydrogen peroxide. Further validation of this concept that the concentrations of the peptide interleukin-1 beta within the cells determine its stimulatory or inhibitory signals regulating the growth of estrogen-dependent tumors might result in novel preventive strategies.

Palmitoylation Regulates Regulator of G-protein Signaling (RGS) 16 Function: II. PALMITOYLATION OF A CYSTEINE RESIDUE IN THE RGS BOX IS CRITICAL FOR RGS16 GTPase ACCELERATING ACTIVITY AND REGULATION OF Gi-COUPLED SIGNALING

Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301-19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/G alpha o1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/G alpha o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein alpha subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/G alpha o1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells.

Light Response of Retinal ON Bipolar Cells Requires a Specific Splice Variant of Gαo

Glutamate released onto retinal ON bipolar neurons binds to a metabotropic receptor to activate a heterotrimeric G-protein (G(o)) that ultimately closes a nonspecific cation channel. Signaling requires the alpha subunit (Galpha(o)), but its effector is unknown. Because Galpha(o) is transcribed into two splice variants (alpha(o1) and alpha(o2)) that differ in the key GTPase domain, the next step in elucidating this pathway was to determine which splice variant carries the signal. Here we show by reverse transcription-PCR and Western blots that retina expresses both splice variants. Furthermore, in situ hybridization and immunostaining on mouse retina deficient in one splice variant or the other show that both alpha(o1) and alpha(o2) are expressed by ON bipolar cells but that alpha(o1) is much more abundant. Finally, electroretinography performed on mice deficient for one splice variant or the other shows that the positive b-wave (response of ON bipolar cells to rod and cone input) requires alpha(o1) but not alpha(o2). Thus, the light response of the ON bipolar cell is probably carried by its strongly expressed splice variant, Galpha(o1).

Localization of G protein alpha-subunits in the human fetal adrenal gland.

The aim of the present study was to investigate the presence and localization of the main G protein alpha-subunits in the human fetal adrenal gland during the second trimester of gestation. Immunofluorescence studies conducted on sections from frozen glands obtained immediately after therapeutic abortion indicated that the alpha s subunit of the heterotrimeric Gs protein was detected in all adrenal cell types, except for endothelial cells. The other alpha-subunits had a more specific pattern of distribution. Indeed, the alpha il-2 protein was restricted to the definitive zone, whereas alpha i3 labeling was mainly expressed in the fetal zone. The alpha q protein subunit was localized in vascular endothelial cells at the periphery of the adrenal gland and in fetal cells at the center. Finally, chromaffin cells expressed alpha s, alpha q, and alpha o1, but not alpha o2 nor alpha i. Altogether, these results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of G protein alpha-subunits. Such cell specific distribution in glands from midgestational fetuses may account for the absence or the different responses to stimuli, when compared with the adult adrenal gland.

Alkyl-Substituted Amino Acid Amides and Analogous Di- and Triamines: New Non-Peptide G Protein Activators

Synthesis and pharmacological properties of new potent direct activators of heterotrimeric G proteins are described. Compounds were synthesized from protected amino acids with alkylamines using coupling reagents (CDI, DCC, and EDC). Alkyl-substituted amino acid amides and their corresponding di- and triamines were subjected to structure-activity analysis. All compounds activated membrane-bound HL-60 GTPases in a pertussis toxin-sensitive fashion. This suggests a specific effect of compounds on the carboxy terminus of a defined subclass of heterotrimeric G proteins, i.e., members of the G alpha i subfamily. Elongation of the alkyl chain and increasing the number of amino groups enhanced the potency of compounds on HL-60 membrane-bound GTPase. N-(2,5-Diaminopentyl)dodecylamine (21) was selected to study its mode of action employing purified pertussis toxin-sensitive G proteins. It stimulated G alpha subunits by inducing the release of bound GDP. In contrast to receptors G beta gamma complexes were not required for 21-mediated activation of G alpha. Moderate isoform selectivity of its action was observed within a group of highly homologous members of the Gi subfamily with G alpha o1 being activated at lowest concentrations, whereas higher concentrations were necessary for the stimulation of G alpha i1 or transducin. We conclude that these compounds represent important tools for studying G protein-dependent cellular functions.

G(o)2 and Gi3 proteins mediate the action of somatostatin on membrane Ca2+ and K+ currents in ovine pituitary somatotrophs.

1. Growth hormone (GH) secretion from the anterior pituitary gland is mainly regulated by hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIF). Somatostatin reduces both spontaneous and GHRH-stimulated GH secretion. 2. Exocytosis of GH is mainly determined by the intracellular free Ca2+ concentration ([Ca2+]i), which is regulated by the influx of Ca2+ via membrane Ca2+ channels. Somatostatin reduces the influx of Ca2+ through two separate mechanisms, namely a direct action on Ca2+ channels and an indirect action on membrane potentials through the activation of K+ channels. 3. In the present experiments, somatotroph-enriched cells were obtained from the ovine pituitary gland by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the effect of SRIF (10 nmol/L) on Ca2+ or K+ currents. 4. A significant reduction in Ca2+ currents and an increase in K+ currents was obtained in response to local application of SRIF (10 nmol/L), but vehicle application had no effect. The responses of Ca2+ and K+ currents to SRIF were reversible after removal of SRIF. 5. Dialysis of GTP-gamma-s (200 mumol/L) abolished the recovery phase of K+ current response to SRIF after its removal, whereas GDP-beta-s (200 mumol/L) totally blocked the response. Pretreatment of the cells with pertussis toxin (100 nmol/L) overnight abolished the Ca2+ current response to SRIF. 6. Intracellular dialysis of antibodies to alpha o, alpha i1-3, alpha i1-2 and alpha i3 subunits of the G-proteins into cells via whole-cell patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. 7. Dialysis of anti-alpha i1-3 or anti-alpha i3 antibodies significantly attenuated the increase in the K+ current in response to 10 nmol/L SRIF, whereas neither anti-alpha o nor anti-alpha i1-2 antibodies diminished the effect of SRIF on the K+ current. 8. Dialysis of anti-alpha o antibodies significantly attenuated the reduction in the Ca2+ current that was obtained upon application of 10 nmol/L SRIF. Neither anti-alpha i1-2 nor anti-alpha i3 antibody dialysis diminished the effect of SRIF on the Ca2+ current. 9. Dialysis of the alpha o common antisense oligonucleotides (ASm) but not the alpha i3 AS significantly diminished the inhibitory effect of SRIF on the Ca2+ current. This effect of alpha o ASm dialysis occurred at 12 h incubation after dialysis, reaching a maximal level at 48 h and partially recovering at 72 h incubation. Antisense oligonucleotides specific for alpha o1 (alpha o1 AS) or alpha o2 (alpha o2 AS) were dialysed into somatotrophs and only alpha o2 AS significantly attenuated the inhibition of SRIF on the Ca2+ current. 10. It is concluded that the Gi3 protein mediates the effect of SRIF on the K+ current and that the G(o)2 protein mediates the effect of SRIF on the Ca2+ current in primary cultured ovine somatotrophs.

Receptors couple to L-type calcium channels via distinct Go proteins in rat neuroendocrine cell lines.

1. The present study examines the hypothesis of G protein subtype selectivity in receptor-induced inhibition of calcium channel currents (ICa) in the insulin-secreting RINm5F and pituitary GH3 rat cell lines. Specificity of receptor coupling to G proteins was studied by infusion of purified G alpha isoforms into cells via a patch pipette. 2. In RINm5F cells, the neuropeptide galanin inhibited dihydropyridine (DHP)- and omega-conotoxin-sensitive components of ICa and slowed down their activation kinetics. In GH3 cells, DHP-sensitive ICa was inhibited by galanin, as well as by somatostatin and carbachol. Agonist-induced ICa inhibition was suppressed by pertussis toxin (PTX) pretreatment of the cells. In PTX-pretreated cells of either cell line, the response to galanin was restored only by the G alpha o1 subunit. Following PTX treatment of GH3 cells, only the G alpha o1 subunit restored carbachol-induced inhibition of ICa, whereas only the G alpha o2 subunit restored somatostatin-induced inhibition of ICa. G(i) subtypes had no effect on ICa inhibition. 3. Both cell lines expressed two distinct immunoreactive Go proteins. Whereas in RINm5F cell membranes Go1 was found to be the predominant isoform, we detected more Go2 than Go1 in GH3 cell membranes. Nevertheless, all agonists stimulated incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into both G(o) isoforms. 4. The results indicate that the same Go subtype, i.e. Go1, mediates galanin-induced inhibition of ICa in both cell lines and that the Go subtype specificity of receptor-G protein coupling is confined to intact cells.

High lipid content enhances the rate of oxygen diffusion through fish skeletal muscle

The diffusion coefficient for O2 (Do2) and the solubility constant for O2 (alpha O2) were measured at 15 degrees C in oxidative muscle from striped bass (Morone saxatilis) that had been acclimated to 5 degrees and 25 degrees C. This design allowed us to test the hypothesis that changes in composition of the tissue that are known to occur during thermal acclimation may affect O2 movement. Our measurements permitted calculation of the diffusion constant for O2 (Ko2) through the tissue, which is a primary determinant of capacity for O2 flux. Under isothermal conditions, alpha O2 was 3.59 +/- 0.20 x 10(-2) and 6.64 +/- 0.27 x 10(-2) ml O2.cm-3.atm-1 in tissues from 25 degrees- and 5 degrees C-acclimated animals, respectively. Because O2 is more soluble in lipid than aqueous phase, higher alpha O2 in tissues from cold-acclimated animals can be accounted for by the 13-fold increase in lipid content that is known to occur in oxidative muscle of striped bass during acclimation from 25 degrees to 5 degrees C. When measured under similar isothermal conditions, Do2 showed no significant difference between animals acclimated to warm or cold temperature; Do2 through tissues from 25 degrees- and 5 degrees C-acclimated animals was 2.50 +/- 0.18 and 2.57 +/- 0.40 cm2/s, respectively. Because alpha O2 increases, the calculated KO2 (DO2. alpha O2) is greater in tissue from cold- than from warm-acclimated fish. At physiological temperature, elevated lipid content in oxidative muscle of cold-acclimated striped bass should result in enhanced intracellular movement of O2 and at least partially offset the expected decrease in DO2 at cold temperature.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

ADP-ribosylation of G alpha i and G alpha o in pituitary cells enhances their recognition by antibodies directed against their carboxyl termini.

Using antibodies raised against synthetic peptides of heterotrimeric GTP binding proteins, we demonstrate the presence of G alpha s, G alpha i1,2, G alpha i3, G alpha o2, and G beta subunits in pituitary cells. Pretreatment of pituitary cells with cholera toxin diminished the immunoreactivity of G alpha s and this decrease was kinetically coupled to the rate of G alpha s ADP-ribosylation. ADP-ribosylation by islet activating protein (IAP or Bordetella pertussis toxin) of G alpha i and G alpha o enhanced their immunoreactivities to antibodies raised against synthetic decapeptides that correspond to the G alpha carboxyl termini. Such enhancement was not observed when antibodies directed against the NH2-termini were used. These findings are consistent with the fact that ADP-ribosylation by IAP occurs on the cysteine located in the carboxyl terminal part of G alpha i and G alpha o. These observations mean that the kinetics and extent of Gi and Go ADP-ribosylation by IAP in whole pituitary cells and membrane preparations can be followed. It could be that ADP-ribosylation causes conformational changes in G alpha i and G alpha o. Indeed, we observed that ADP-ribosylated G alpha i was more sensitive to trypsin proteolysis and that the ADP-ribosylation rates of G alpha i and G alpha o in whole cells were comparable to the rate of loss of coupling between inhibitory neurohormone receptors and adenylyl cyclase.

Selective Inhibition of the Expression of Signal Transduction Proteins by Lithium in Nerve Growth Factor‐Differentiated PC12 Cells

This investigation examined if lithium, the primary therapeutic treatment for bipolar affective disorder, modulated the levels of selected signal transduction proteins in PC12 cells. Nerve growth factor (NGF) induced differentiation of PC12 cells, and after 12 days of NGF treatment there were large increases in the levels of the heterotrimeric G protein subunits alpha o1, alpha i1, beta, and alpha s, small increases in those of alpha i2 and alpha q, and a slight decrease in that of alpha o2. Lithium (1 mM, equivalent to the therapeutic concentration) selectively reduced NGF-induced increases in levels of G protein subunits, generally having the greatest inhibition on those that were increased the most by NGF. Lithium at 5 mM had greater inhibitory effects than 1 mM lithium on NGF-induced increases in levels of G proteins, but neither concentration of lithium affected the induction of the cytoskeletal protein beta-tubulin. Examination of other proteins involved in signal transduction revealed that 12 days of NGF treatment increased the level of protein kinase C-alpha, but not those of the beta, epsilon, or zeta subtypes, and did not alter the levels of beta, gamma, or delta phospholipase C. Pretreatment with lithium inhibited the increase in content of protein kinase C-alpha induced by NGF but had little effect on the proteins not responsive to NGF except for decreasing the levels of protein kinase C-epsilon. The inhibitory effect of lithium was found not to be due to inhibition of NGF-induced tyrosine phosphorylation, which was unaffected by 5 mM lithium, or to inositol depletion. In summary, use of the dynamic system of NGF-induced PC12 cell differentiation provided a sensitive model in which to identify signal transduction proteins that were influenced by lithium treatment. The large changes caused by a therapeutically equivalent concentration of lithium lend support to the proposal that the selective inhibitory effects of lithium on subtypes of G proteins and protein kinase C may be important therapeutic targets.

G alpha o1 decapeptide modulates the hippocampal 5-HT1A potassium current

1. The serotonin1A (5-HT1A) receptor is coupled to an inwardly rectifying potassium current (IKir) via a G protein. The identity of the G-protein subtype was investigated with 2 10-amino acid peptides derived from the carboxyl (C) terminus of the alpha-subunits of the Go1 and Gi2 proteins (G alpha o1 and G alpha i2). The synthetic decapeptides were applied by intracellular perfusion during whole cell recording from dentate granule cells in the hippocampal slice preparation. 2. Bath application of 5-HT produced an IKir, which was blocked by the selective 5-HT1A receptor antagonist, pindobind5-HT1A. The G alpha o1 peptide inhibited the 5-HT1A IKir by 60 +/- 7% (mean +/- SE; t = 30 min), whereas the G alpha i2 peptide had no effect. The G alpha o1 peptide produced a slowly developing outward current that was not observed in the absence of peptide or in the presence of the G alpha i2 peptide. 3. The results indicate that G alpha o1 and not G alpha i2 modulates the 5-HT1A IKir in hippocampal granule cells. They also suggest that G alpha o1 occludes the 5-HT1A response by direct activation of the IKir. The intracellular perfusion of synthetic G alpha peptides provides a new approach to identify the G-protein subtype(s) in a receptor-mediated electrophysiological response.

Involvement of G-protein alpha il subunits in activation of G-protein gated inward rectifying K+ channels (GIRK1) by human NPY1 receptors.

This study investigated the type of G-protein alpha subunit(s) that human neuropeptide Y (NPY)1 receptors preferentially utilize when activating G-protein gated K+ currents. Two electrode voltage-clamp recordings were made from Xenopus oocytes that had been injected with cDNAs encoding either human NPY1 or D2(short) dopamine receptors, and GIRK1 a cloned rat brain K+ channel. These receptors were also co-injected with G-protein alpha i1, alpha i2, alpha i3 and alpha o1 subunits to determine which subunit(s) modulate the efficiency of signal transduction. In NPY1 receptor injected cells neuropeptide Y (100 nM) caused a 53 +/- 10 nA inward current (n = 14; EC50 = 3 nM) and this effect was blocked by pertussis toxin (500 ng ml-1 24 h). Activation of GIRK1 currents by neuropeptide Y was selectively potentiated by alpha i1 subunit cDNA whereas coupling dopamine of D2 receptors to this channel was not.

Effect of thyroid deficiency on Go alpha-subunit isoforms in developing rat cerebral cortex.

Postnatal development of G alpha o isoforms in rat cerebral cortex was studied by SDS-PAGE and immunoblotting. When rat cerebral cortical membranes were resolved on separating gels containing 9% acrylamide and 8 M urea, three electrophoretically distinct G alpha o-immunoreactive proteins were evident. Comparison of their electrophoretic mobilities and partial tryptic digest pattern with recombinant G alpha o1 or G alpha o1-specific antibody revealed that the slowest and intermediate-migrating bands represent unmodified and fatty acylated forms of G alpha o1 protein, respectively. The fastest-migrating band corresponds to G alpha o2. While the fatty acylated form of G alpha o1 is the predominant species, its appearance paralleled that observed for G alpha o2 in developing rat cortex. Perinatal hypothyroidism induced by methimazole treatment did not significantly alter the appearance of cerebral cortical G alpha o1 and G alpha o2 between days 1 and 22 postpartum. Our findings support the earlier idea that heterogeneity of G alpha o proteins in mammalian brain is likely the result of different co- or post-translational processings of each splice variant of G alpha o. While the appearance of G alpha o isoforms is developmentally regulated, they likely do not play an obligatory role in neonatal brain development. Alternatively, the expression of G alpha o isoforms in developing rat cortex may be controlled by an intrinsic signal(s) that is independent of the thyroid status.