scholarly journals Maintenance of cellular levels of G-proteins: different efficiencies of αs and αo synthesis in GH3 cells

1996 ◽  
Vol 318 (3) ◽  
pp. 1071-1077 ◽  
Author(s):  
Ying LI ◽  
Ulrike MENDE ◽  
Carol LEWIS ◽  
Eva J NEER
Keyword(s):  
Protein Synthesis ◽  
G Protein ◽  
G Proteins ◽  
Gh3 Cells ◽  
Rate Limiting Step ◽  
Degradation Rates ◽  
Steady State Levels ◽  
Membrane Bound ◽  
External Signals ◽  
Rate Limiting

G-proteins couple membrane-bound receptors to intracellular effectors. Each cell has a characteristic complement of G-protein α, β and γ subunits that partly determines the cell's response to external signals. Very little is known about the mechanisms that set and maintain cellular levels of G-proteins or about potential points of regulation. We have assayed the steady-state levels of mRNA and protein for two types of G-protein subunits, αs and αo, in rat brain, heart and GH3 cells, and found that in all these cases, it takes 9- to 20-fold more mRNA to produce a given amount of αs protein than to produce the same amount of αo protein. Such a situation could arise from a relatively rapid rate of αs protein degradation, requiring rapid protein synthesis to compensate, or from relatively inefficient translation of αs mRNA compared with αo mRNA. The latter appears to be the case in GH3 cells. These cells contain 94 times more mRNA for αs than for αo, yet the rate of αs protein synthesis is only 9 times greater than αo protein synthesis. The degradation rates of the two proteins are similar (13 h for αs and 18 h for αo). To begin to define the mechanism that accounts for the fact that it takes more mRNA to synthesize a given amount of αs than αo, we asked whether there is a pool of αs mRNA that does not participate in protein synthesis. We found that virtually all αs and αo mRNA is associated with ribosomes. Therefore, all the mRNA is likely to be capable of directing protein synthesis. Since the rate-limiting step in protein synthesis is usually binding of the ribosome to mRNA at initiation, our results suggest that the relatively slow rate of αs protein synthesis is regulated by a mechanism that acts beyond initiation at peptide elongation and/or termination.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

Kinetic analysis of segmentation gene interactions in Drosophila embryos

Development ◽  
1999 ◽  
Vol 126 (7) ◽  
pp. 1515-1526 ◽  
Author(s):  
A. Nasiadka ◽  
H.M. Krause
Keyword(s):  
Target Genes ◽  
Vast Number ◽  
Rate Limiting Step ◽  
Degradation Rates ◽  
Time Required ◽  
Potential Interactions ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Synthesis And Degradation ◽  
Drosophila Embryos

A major challenge for developmental biologists in coming years will be to place the vast number of newly identified genes into precisely ordered genetic and molecular pathways. This will require efficient methods to determine which genes interact directly and indirectly. One of the most comprehensive pathways currently under study is the genetic hierarchy that controls Drosophila segmentation. Yet, many of the potential interactions within this pathway remain untested or unverified. Here, we look at one of the best-characterized components of this pathway, the homeodomain-containing transcription factor Fushi tarazu (Ftz), and analyze the response kinetics of known and putative target genes. This is achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, we find that both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (<10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes are analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy.

Material Properties for Degradation and Reduction of Perfluorinated Acid in Complex Pollution System by Photo-Induced Hydrated Electrons

2014 ◽  
Vol 540 ◽  
pp. 243-246
Author(s):  
Si Bo Li ◽  
Chao Jie Zhang ◽  
Yan Qu ◽  
Qi Zhou
Keyword(s):  
Reduction Rate ◽  
Carbon Chain ◽  
Perfluorinated Acids ◽  
Rate Limiting Step ◽  
Degradation Rates ◽  
Hydrated Electrons ◽  
Perfluorinated Acid ◽  
Rate Limiting ◽  
Linear Correlations ◽  
Complex Pollution

F, the most electronegative element, is easy to catch the electrons in the bonds to form the stabilized C-F. Perfluorinated acids have high chemical stability which can tolerate acid, alkali, etc. Inhibition effect was observed in the complex pollution system in which 7 kinds of perfluorinated acids existed simultaneously. The formation of hydrated electrons was the rate-limiting step in complex pollution system, and the inhibition was increased as the carbon chain increasing. Compared the reduction rate of perfluorinated acids by photo-induced hydrated electrons in the complex pollution system with the single rate, the results showed that there were positive linear correlations between the degradation rate and photo-induced hydrated electrons. The degradation multiples of perfluorinated acids were studied, it concluded that, because of the competition among the 7 kinds of perfluorinated acids, the degradation rates were influenced.

scholarly journals Heterotrimeric G-Protein Signalers and RGSs in Aspergillus fumigatus

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 902
Author(s):  
Hee-Soo Park ◽  
Min-Ju Kim ◽  
Jae-Hyuk Yu ◽  
Kwang-Soo Shin
Keyword(s):  
Aspergillus Fumigatus ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
G Protein ◽  
G Proteins ◽  
Pathogenic Fungus ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Heterotrimeric G Protein ◽  
External Signals

The heterotrimeric G-protein (G-protein) signaling pathway is one of the most important signaling pathways that transmit external signals into the inside of the cell, triggering appropriate biological responses. The external signals are sensed by various G-protein-coupled receptors (GPCRs) and transmitted into G-proteins consisting of the α, β, and γ subunits. Regulators of G-protein signaling (RGSs) are the key controllers of G-protein signaling pathways. GPCRs, G-proteins, and RGSs are the primary upstream components of the G-protein signaling pathway, and they are highly conserved in most filamentous fungi, playing diverse roles in biological processes. Recent studies characterized the G-protein signaling components in the opportunistic pathogenic fungus Aspergillus fumigatus. In this review, we have summarized the characteristics and functions of GPCRs, G-proteins, and RGSs, and their regulatory roles in governing fungal growth, asexual development, germination, stress tolerance, and virulence in A. fumigatus.

Selective inactivation of guanine-nucleotide-binding regulatory protein (G-protein) α and βγ subunits by urea

2001 ◽  
Vol 354 (2) ◽  
pp. 337-344 ◽  
Author(s):  
William K. LIM ◽  
Richard R. NEUBIG
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Regulatory Protein ◽  
Significant Loss ◽  
Membrane Extraction ◽  
Α Subunit ◽  
Mammalian Cell Lines ◽  
Membrane Bound ◽  
Guanine Nucleotide Binding ◽  
G Protein Coupled

G-protein-coupled receptors activate signal-transducing G-proteins, which consist of an α subunit and a βγ dimer. Membrane extraction with 5–7M urea has been used to uncouple receptors from endogenous G-proteins to permit reconstitution with purified G-proteins. We show that αi subunits are inactivated with 5M urea whereas the βγ dimer requires at least 7M urea for its inactivation. There is no significant loss of receptors. Surprisingly, Western-blot analysis indicates that the urea-denatured αi subunit remains mostly membrane-bound and that β is only partially removed. After 7M urea treatment, both αi1 and βγ subunits are required to restore high-affinity agonist binding and receptor-catalysed guanosine 5′-[γ-thio]triphosphate binding. We demonstrate the generality of this approach for four Gi-coupled receptors (α2A-adrenergic, adenosine A1, 5-hydroxytryptamine1A and µ-opioid) expressed in insect cells and two mammalian cell lines. Thus a selectivity of urea for G-protein α versus βγ subunits is established in both concentration and mechanism.

scholarly journals Phospholipase C-β1 is regulated by a pertussis toxin-insensitive G-protein

1991 ◽  
Vol 280 (3) ◽  
pp. 753-760 ◽  
Author(s):  
T F J Martin ◽  
J E Lewis ◽  
J A Kowalchyk
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Gh3 Cells ◽  
Phospholipid Vesicles ◽  
Protein Tyrosine Phosphorylation ◽  
Gtp Binding ◽  
Gamma Subunits ◽  
Gh3 Cell

Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.

scholarly journals GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling

2007 ◽  
Vol 104 (44) ◽  
pp. 17317-17322 ◽  
Author(s):  
C. A. Johnston ◽  
J. P. Taylor ◽  
Y. Gao ◽  
A. J. Kimple ◽  
J. C. Grigston ◽  
...  
Keyword(s):  
G Protein ◽  
Sugar Signaling ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
G Protein Coupled

Regulation of Protein Synthesis by eIF4E in the Brain

2020 ◽  
Author(s):  
Kleanthi Chalkiadaki ◽  
Stella Kouloulia ◽  
Clive R. Bramham ◽  
Christos G. Gkogkas
Keyword(s):  
Protein Synthesis ◽  
Brain Function ◽  
Regulation Of Gene Expression ◽  
Mrna Translation ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Eukaryotic Mrnas ◽  
Rate Limiting ◽  
The Brain ◽  
Key Pathways

Regulation of gene expression at the level of mRNA translation is crucial for all the functions our brains carry out. eIF4E binds to the 5′-end of eukaryotic mRNAs and dictates the rate-limiting step of cap-dependent initiation. This chapter reviews the key pathways regulating eIF4E function, but also the less studied and novel mechanisms of eIF4E modulation, linked to synaptic plasticity, learning and memory, and nervous system disorders. Understanding how regulation of protein synthesis by eIF4E affects different aspects of brain function is yet elusive.

Activation of BK Channels in GH3 Cells by a c-PLA2-Dependent G-Protein Signaling Pathway

2005 ◽  
Vol 93 (6) ◽  
pp. 3146-3156 ◽  
Author(s):  
D. D. Denson ◽  
J. Li ◽  
X. Wang ◽  
D. C. Eaton
Keyword(s):  
Arachidonic Acid ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Adrenergic Receptor ◽  
Bk Channels ◽  
Bk Channel ◽  
Gh3 Cells ◽  
Channel Activity ◽  
Excised Patches

BK-channels in GH3 cells are activated by arachidonic acid produced by c-PLA2. β-adrenergic agonists also activate BK channels and were presumed to do so via production of cAMP. We, however, show for the first time in GH3 cells that a β-adrenergic agonist activates a pertussis-toxin-sensitive G protein that activates c-PLA2. The arachidonic acid produced by c-PLA2 then activates BK channels. We examined BK channels in cell-attached patches and in excised patches from untreated GH3 cells and from GH3 cells exposed to c-PLA2 antisense oligonucleotides. For the cell-attached patch experiments, physiologic pipette and bath solutions were used. For the excised patches, 150 mM KCl was used in both the pipette and bath solutions, and the cytosolic surface contained 1 μM free Ca2+ (buffered with 5 mM K2EGTA). Treatment of GH3 cells with the G protein activator, fluoroaluminate, (AlF4−) produced an increase in the Po of BK channels of 177 ± 41% (mean ± SD) in cell-attached patches. Because G proteins are membrane associated, we also added an activator of G proteins, 100 μM GTP-γ-S, to the cytosolic surface of excised patches. This treatment leads to an increase in Po of 50 ± 9%. Similar treatment of excised patches with GDP-β-S had no effect on Po. Isoproterenol (1 μM), an activator of β-adrenergic receptors and, consequently, some G proteins, increased BK channel activity 229 ± 37% in cell-attached patches from cultured GH3 cells. Western blot analysis showed that GH3 cells have β-adrenergic receptor protein and that isoproterenol acts through these receptors because the β-adrenergic receptor antagonist, propanolol, blocks the action of isoproterenol. To test whether G protein activation of BK channels involves c-PLA2, we studied the effects of GTP-γ-S on excised patches and isoproterenol on cell attached patches from GH3 cells previously treated with c-PLA2 antisense oligonucleotides or pharmacological inhibitors of c-PLA2. Neither isoproterenol nor GTP-γ-S had any effect on Po in these patches. Similarly, neither isoproterenol nor GTP-γ-S had any effect on Po in cultured GH3 cells pretreated with pertussis toxin. Isoproterenol also significantly increased the rate of arachidonic production in GH3 cells. These results show that some receptor-linked, pertussis-toxin-sensitive G protein in GH3 cells can activate c-PLA2 to increase the amount of arachidonic acid present and ultimately increase BK-channel activity.

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