Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.
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ABSTRACT:
SUBMITTER: Waldo GL
PROVIDER: S-EPMC1148293 | biostudies-other | 1987 Sep
REPOSITORIES: biostudies-other
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Project description:In human adipocyte plasma membranes, pertussis toxin catalysed the ADP-ribosylation of an apparently single 40 kDa protein. The same protein was also observed in Western blots by using an antibody which identifies the C-terminal decapeptide of Gi alpha (alpha subunit of Gi). In analogous experiments, cholera toxin and an antibody raised against the C-terminal decapeptide of Gs alpha (alpha subunit of Gs) were used to identify two proteins of 42 and 45 kDa, the former of which was more prominent. A method was developed to estimate the relative amounts of Gi and Gs in crude adipocyte plasma membranes in a single immunoblot by using the two antisera. In animal models, changes in the amounts of G-proteins have been suggested to explain alterations in hormone-responsiveness in hypothyroidism and obesity. However, the amounts of Gi and Gs were unaltered in thyroidectomized papillary-carcinoma patients who had been without hormone substitution for 4 weeks. In adipocyte plasma membranes prepared from markedly obese subjects, the amounts of both Gi alpha and Gs alpha as calculated per mg of protein were decreased, but the Gi/Gs ratio remained unaltered in comparison with control subjects.
| S-EPMC1138753 | biostudies-other
Project description:Incubation of the neuroblastoma x glioma hybrid cell line NG108-15 in tissue culture with dibutyryl cyclic AMP (1 mM) for up to 8 days produced a morphological differentiation of the cells, during which they extended neurite-like processes. Pertussis-toxin-catalysed ADP-ribosylation indicated that amounts of guanine-nucleotide-binding proteins (G-proteins), which are substrates for this toxin, were approximately doubled in membranes from the 'differentiated' cells in comparison with the control cells. Immunoblotting of membranes derived from either untreated or dibutyryl cyclic AMP-treated cells with anti-peptide antisera specific for the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi and Go demonstrated that amounts of these G-proteins were reciprocally modulated during the differentiation process. In comparison with the untreated cells, the amount of Gi in the 'differentiated' cells was decreased, whereas the amount of Go was substantially increased. Stimulation of high-affinity GTPase activity in response to opioid peptides, which in this cell line interact with an opioid receptor of the delta subclass, was much decreased, and inhibition of adenylate cyclase activity was almost entirely attenuated in the 'differentiated'-cell membranes in comparison with membranes of untreated cells. Opioid receptor number was also decreased in membranes of the dibutyryl cyclic AMP-treated cells in comparison with the control cells. These data demonstrate that relatively small changes in the observed pattern of pertussis-toxin-catalysed ADP-ribosylation of membranes can mask more dramatic alterations in amounts of the individual pertussis-toxin-sensitive G-proteins, and further demonstrate the importance of methodologies able to discriminate between the different gene products.
| S-EPMC1135458 | biostudies-other
Project description:A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5' and 3' rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (approximately 208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a approximately 7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.
| S-EPMC24240 | biostudies-literature
Project description:Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named 'alpha sL', with an apparent Mr of 42,000-45,000, and 'alpha sH' with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.
| S-EPMC1147565 | biostudies-other
Project description:ObjectiveThe sympathetic nervous system (SNS) is a key regulator of the metabolic and endocrine functions of adipose tissue. Increased SNS outflow promotes fat mobilization, stimulates non-shivering thermogenesis, promotes browning, and inhibits leptin production. Most of these effects are attributed to norepinephrine activation of the Gs-coupled beta adrenergic receptors located on the surface of the adipocytes. Evidence suggests that other adrenergic receptor subtypes, including the Gi-coupled alpha 2 adrenergic receptors might also mediate the SNS effects on adipose tissue. However, the impact of acute stimulation of adipocyte Gs and Gi has never been reported.MethodsWe harness the power of chemogenetics to develop unique mouse models allowing the specific and spatiotemporal stimulation of adipose tissue Gi and Gs signaling. We evaluated the impact of chemogenetic stimulation of these pathways on glucose homeostasis, lipolysis, leptin production, and gene expression.ResultsStimulation of Gs signaling in adipocytes induced rapid and sustained hypoglycemia. These hypoglycemic effects were secondary to increased insulin release, likely consequent to increased lipolysis. Notably, we also observed differences in gene regulation and ex vivo lipolysis in different adipose depots. In contrast, acute stimulation of Gi signaling in adipose tissue did not affect glucose metabolism or lipolysis, but regulated leptin production.ConclusionOur data highlight the significance of adipose Gs signaling in regulating systemic glucose homeostasis. We also found previously unappreciated heterogeneity across adipose depots following acute stimulation. Together, these results highlight the complex interactions of GPCR signaling in adipose tissue and demonstrate the usefulness of chemogenetic technology to better understand adipocyte function.
| S-EPMC6717754 | biostudies-literature
Project description:The protein Lgl has key roles in the regulation of cell polarity. We have shown that Lgl is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl is phosphorylated by atypical protein kinase C in a complex with Par6 and either activated Cdc42 or activated Rac. Here we have investigated the role of specific Rac guanine nucleotide exchange factors in Lgl hyperphosphorylation in glioblastoma. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor that is overexpressed in glioblastoma. Knockout of PREX1 in patient-derived glioblastoma cells resulted in a reduction in Lgl phosphorylation and this could be reversed by re-expressing PREX1. PREX1 knockout cells showed reduced motility and altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses of these cells identified sets of PREX1-regulated genes with roles in promoting cell motility and repressing neuronal differentiation. Knockout of PREX1 in glioblastoma cells derived from a second patient did not affect Lgl phosphorylation. These cells overexpressed a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in PREX1-knockout cells from this patient reduced Lgl phosphorylation. These data show that PREX1 links aberrant PI 3-kinase to Lgl phosphorylation in glioblastoma, but that TIAM1 can also promote Lgl phosphorylation in a subset of patients. While this shows redundant mechanisms for Lgl phosphorylation, PREX1 appears to have a non-redundant role in glioblastoma cell motility, as this was impaired in PREX1 knockout cells from both patients.
2021-08-23 | GSE162783 | GEO
Project description:More than two decades ago, the activation mechanism for the membrane-bound photoreceptor and prototypical G protein-coupled receptor (GPCR) rhodopsin was uncovered. Upon light-induced changes in ligand-receptor interaction, movement of specific transmembrane helices within the receptor opens a crevice at the cytoplasmic surface, allowing for coupling of heterotrimeric guanine nucleotide-binding proteins (G proteins). The general features of this activation mechanism are conserved across the GPCR superfamily. Nevertheless, GPCRs have selectivity for distinct G-protein family members, but the mechanism of selectivity remains elusive. Structures of GPCRs in complex with the stimulatory G protein, Gs, and an accessory nanobody to stabilize the complex have been reported, providing information on the intermolecular interactions. However, to reveal the structural selectivity filters, it will be necessary to determine GPCR-G protein structures involving other G-protein subtypes. In addition, it is important to obtain structures in the absence of a nanobody that may influence the structure. Here, we present a model for a rhodopsin-G protein complex derived from intermolecular distance constraints between the activated receptor and the inhibitory G protein, Gi, using electron paramagnetic resonance spectroscopy and spin-labeling methodologies. Molecular dynamics simulations demonstrated the overall stability of the modeled complex. In the rhodopsin-Gi complex, Gi engages rhodopsin in a manner distinct from previous GPCR-Gs structures, providing insight into specificity determinants.
| S-EPMC5877964 | biostudies-literature
Project description:The protein Lgl1 is a key regulator of cell polarity. We previously showed that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN tumour suppressor loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C that has been activated by binding to a complex of the scaffolding protein Par6 and active, GTP-bound Rac. The specific Rac guanine nucleotide exchange factors that generate active Rac to promote Lgl1 hyperphosphorylation in glioblastoma are unknown. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation, which was reversed by re-expressing PREX1. They also had reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. This was due to overexpression of a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in these PREX1 knockout cells reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 is also to fill this role in a subset of patients. This redundancy between PREX1 and TIAM1 is only partial, as motility was impaired in PREX1 knockout cells from both patients.
| S-EPMC8551657 | biostudies-literature
Project description:BACKGROUND:Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. As such, opsins have potential applications in both biomedical research and therapy. Selecting the opsin with the best balance of activity and selectivity for a given application requires knowing their ability to couple to a full range of relevant G? subunits. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels, measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration. RESULTS:Of the opsins tested, wild-type human rod opsin had the highest activity for chimeric Gs proxies for Gi and Gt (Gsi and Gst) and was matched in Go proxy (Gso) activity only by a human rod opsin/scallop opsin chimera. Rod opsin drove roughly equivalent responses via Gsi, Gso, and Gst, while cone opsins showed much lower activities with Gso than Gsi or Gst, and a human rod opsin/amphioxus opsin chimera demonstrated higher activity with Gso than with Gsi or Gst. We failed to detect activity for opsin chimeras bearing three intracellular fragments of mGluR6, and observed unexpectedly complex response profiles for scallop and amphioxus opsins thought to be specialized for Go. CONCLUSIONS:These results identify rod opsin as the most potent non-selective Gi/o/t-coupled opsin, long-wave sensitive cone opsin as the best for selectively activating Gi/t over Go, and a rod opsin/amphioxus opsin chimera as the best choice for selectively activating Go over Gi/t.
| S-EPMC5771134 | biostudies-literature
Project description:BackgroundBacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology.Methodology/findingsThe strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database.ConclusionsWe have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.
| S-EPMC3293901 | biostudies-literature