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Enhancement of secretion by PMA and βγ-subunits. A suboptimal concentration (510-8 M) of βγ-subunits was applied together with PMA (100 nM) and the cells were allowed to run down for 10 min before stimulation. Boiled protein was used as control. The results of an analysis of variance (ANOVA), presented in the box, indicate that additional stimulation of secretion by βγ-subunits was highly significant both in the absence (6.9% secretion) and presence of PMA (6.4% secretion). Similar results were obtained on five separate occasions.
Source publication
We applied G protein-derived beta gamma-subunits to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and GTPgammaS over approximately 20-30 min. beta gamma-Subunits applied...
Contexts in source publication
Context 1
... have considered the possibility that the effect of βγ-subunits could be mediated by a phosphorylating enzyme, in particular PKC, due to the generation of diglyceride as a consequence of the activation of phospholipase C. We therefore investigated the effect of reagents known to cause stimulation or inhibition of PKC in the presence of either native or heat-inactivated βγ- subunits. The experiment illustrated in Figure 6 ('boiled') confirms that treatment of run-down cells with phorbol myristate acetate (PMA) enhances the extent of secretion. The amount of extra release due to PMA (100 nM), ~30% in the experiment shown, is comparable with that induced by an optimal concentration of βγ-subunits (see Figure 4). ...
Context 2
... this experiment, the βγ-subunits were applied at a suboptimal concentration (510 -8 M) which enhanced the extent of secretion by 6.9% (P 0.00019, one-way ANOVA). When the two reagents were applied together, their combined effect was greater (6.4%, P 0.0002) than that of PMA alone, and thus the enhancements were approximately additive (see Figure 6, 'native'). We conclude that although the enhancement of secretion by βγ-subunits may be due, in part, to activation of the PLC-PKC cascade, a significant component of the effect must have another origin. ...
Citations
... SCRN1 promotes exocytosis in cells and multiple proteins work methodically participating in exocytosis [10]. Several GTP binding proteins, for instance, rac, rho, and G alpha i3, participate in secretion [27,28]. These proteins are also involved in other biological functions, so SCRN1 might promote cell proliferation and clone formation through an indirect manner. ...
Emerging evidence shows that exocytosis plays a key role in tumor development and metastasis. Secernin-1 (SCRN1) is a novel regulator of exocytosis. Our previous work identified SCRN1 as a tumor-associated gene by bioinformatics analysis of transcriptomes. In this study, we demonstrated the aberrant overexpression of SCRN1 at mRNA and protein level in colon cancer. We also revealed that overexpression of SCRN1 was significantly associated with the tumor development and poor prognosis. Experiments in vitro validated that SCRN1 may promote cancer cell proliferation and secretion of matrix metalloproteinase-2/9 (MMP-2/9) proteins to accelerate tumor progression.
... The activity of exocytosis and endocytosis in AEC II can be regulated and influenced in different ways. It is believed that Ca 2+ signal, ATP, G protein beta gamma-subunits, physiologic stimulus and physical state of extracellular phosphatidylcholine etc. can regulate the activity of exocytosis and endocytosis [32][33][34][35]. So it is reasonable and valuable to do researches on the regulation mechanism of exocytosis and endocytosis for getting evidence to support this hypothesis. ...
Exocytosis and endocytosis are the way of macromolecules transmembrane transport. Pulmonary surfactant (PS), one of such macromolecules, is secreted via exocytosis of lamellar bodies and recycled via endocytosis by type II alveolar epithelial cells (AEC II). It maintains low alveolar surface tension and is therefore essential to normal lung function. PS deficiency causes respiratory distress syndrome in infants. Congenital diaphragmatic hernia is an abnormal condition in which low lung compliance is involved. This condition is multifactorial and a primary surfactant deficiency may be responsible for it. We hypothesize that surfactant deficiency is involved in CDH and depressed activity of exocytosis and endocytosis in AEC II is responsible for the surfactant deficiency in the lung of newborn with congenital diaphragmatic hernia.
... Indeed, although it is known that outwardly rectifying Cl − channels can be activated by compound 48 / 80, they have been shown not to be essential for it to cause exocytosis in mast cells (51) and that compound 48 / 80-induced exocytosis is largely independent of extracellular Ca 2+ influx (1). Taken together, the results from Ca 2+ -free and Cl − -free conditions further substantiate the view that activation of G-proteins in the presence of compound 48 / 80 may not require the absolute presence of extracellular Ca 2+ but, rather, involves the release of G-protein βγ-subunits (52,53). The lengthening of the latent periods and temporal measurements was probably due to the reduction of the electrochemical gradient of Ca 2+ as a direct result of an elevated V m , thus affecting the onset and duration of the HFP. ...
We performed this study to demonstrate the applicability of the microelectrode array (MEA) to study electrophysiological changes of rat peritoneal mast cells in the presence of compound 48/80 under normal, Ca(2+)-free, Ca(2+)-free with EDTA, and Cl(-)-free conditions. The use of high extracellular K(+) (KCl, 150 mM), charybdotoxin (ChTX, 100 nM), and Cl(-)-free containing ChTX buffers verified that the hyperpolarizing signal was due to the activation of mainly K(+) and, to a lesser extent, Cl(-) channels. Compound 48/80 concentration-dependently shortened the latent periods (the onset of response) and increased both the spatial (the K(+) and Cl(-) hyperpolarizing field potentials, HFP) and temporal measurements (the duration of response). Ca(2+)-free buffer had no effect on the latent period of compound 48/80 but increased the HFP at high concentrations. The latent period increased while the HFP diminished when cells were equilibrated in Ca(2+)-free buffer containing EDTA. Durations of the HFP were generally longer when cells were in either Ca(2+)-free or Ca(2+)-free containing EDTA buffers than when cells were in normal buffer. The EC(50) values confirmed that effects were only affected in Ca(2+)-free buffer containing EDTA but not in Ca(2+)-free or Cl(-)-free buffers, further reinforcing the hypothesis that the presence of Ca(2+) is not essential to the action of compound 48/80. The present study is the first application of MEA to study rat peritoneal mast cells, and our results indicate that it could be of value in future pharmacological research on other non-excitable cells.
... SCRN1 might cause the recruitment of secretory granules to the site of exocytosis or increase granule swelling, core expulsion, or breakdown. Notably, a number of GTP binding proteins such as G alpha i3, (36) beta gamma subunits, (37) rac, (38) rho, (39) and cdc42 (40) play a role in secretion. These molecules are involved in other physiological functions, so SCRN1 might also play different roles other than exocytosis in the cells. ...
Despite the discovery of multiple TAAs, only a limited number is available for clinical application, particularly against epithelial malignancies. In this study we searched for novel TAAs using expression profiles of gastric cancer examined with cDNA microarray, and identified the SCRN1 gene as a candidate. SCRN1 was confirmed to be expressed in five out of seven gastric cancers with semiquantitative RT-PCR. With Northern blot analysis, it was detected abundantly in the testis and ovary, but it was barely detectable in 14 other normal human adult organs. Colony formation assay revealed that its augmented expression is associated with promoted cell growth. As these expression profiles and functional features of SCRN1 appeared to be compatible with the characteristics of the hypothesized ideal TAAs, we examined whether SCRN1 protein contains antigenic epitope peptides restricted to HLA-A*0201. We synthesized the candidate peptides derived from SCRN1, and tried to induce CTLs with each peptide. The CTL clones were successfully induced with a peptide SCRN1-196 (KMDAEHPEL), and they lyzed not only the peptide-pulsed targets but also the tumor cells expressing both SCRN1 and HLA-A*0201 endogenously. These results strongly suggest that SCRN1-196 is an epitope peptide restricted to HLA-A*0201. Furthermore, we synthesized an anchor-modified peptide SCRN1-9 V (KMDAEHPEV), in which leucine at position 9 was substituted for valine to increase the binding affinity to the HLA-A*0201 molecules. The CTL clones induced by SCRN1-9 V also recognized tumor cells expressing its natural SCRN1 protein endogenously. These results strongly suggest that SCRN1 is a novel TAA and these peptides, both native and modified, may be applicable for cancer vaccines to treat gastric cancer.
... This model was attractive because a direct interaction between bg subunit and the exocytotic machinery has also been suggested to inhibit presynaptic release downstream of Ca 2þ entry (64). However, it was subsequently found that the heterotrimetric G proteins are unable to induce exocytosis by themselves (21). It follows that they operate in conjunction with other monomeric GTPases. ...
Many cells utilize a GTP-dependent pathway to trigger exocytosis in addition to Ca(2+)-triggered exocytosis. However, little is known about the mechanism by which GTP triggers exocytosis independent of Ca(2+). We used dual-color evanescent field microscopy to compare the motion and fusion of large dense core vesicles stimulated by either mastoparan (Mas) in Ca(2+)-free conditions or high K(+) in the presence of Ca(2+). We demonstrate that Mas is hardly effective in triggering the fusion of the predocked vesicles but predominantly mobilizes cytosolic vesicles. In contrast, Ca(2+)-dependent exocytosis is largely due to predocked vesicles. Fusion kinetics analysis and carbon-fiber amperometry reveal that Mas induces a brief 'kiss-and-run' fusion and releases only a small amount of the cargo, whereas Ca(2+) stimulates a more persistent opening of the fusion pore and larger release of the contents. Furthermore, we show that Mas-released vesicles require a much shorter time to reach fusion competence once they approach the plasma membrane. Our data suggest the involvement of different mechanisms not only in triggering and fusion but also in the docking and priming process for Ca(2+)- and GTP-dependent exocytosis.
... Adenosine in turn binds to and activates G-protein coupled adenosine A 3 receptors [22, 23] , whose G i bc subunits activate PI3K p110c. Supporting this scheme, it has been observed that bc subunits will activate permeabilised mast cells, and the secretagogues mastoparan and compound 48/80 [24, 25] trigger histamine release via G i3 proteins [26, 27]. Why does this systematic PI generation exist? ...
The release of pre-formed mediators such as histamine from mast cells and basophils is an integral part of the normal immune response to infection by parasites. This exocytosis is also characteristic response in a number of disease states including asthma, which, due to their prevalence in western society are becoming of increasing clinical importance. In an effort to tackle this growing problem much work has gone into unlocking the mechanisms through which mast cells function in health and disease. To date we have learned a lot about the various proteins that regulate degranulation. However, our knowledge on the contribution of lipids to this process is less clear. This review will discuss the role of phospholipids, particularly the phosphoinositides (PIs) in the processes that regulate mast cell exocytosis.
... The search for intracellular proteins other than a cell surface receptor that regulated G-protein function was initiated in part following observations of variations in the efficiency and/or specificity of receptor-heterotrimeric G-protein coupling in different cell backgrounds, as well as differences between the kinetic properties of the G-protein activation/deactivation cycle for selected signaling pathways within the cell and those observed with purified proteins (Duzic and Lanier 1992;Herzog et al. 1992;Perez et al. 1993;Sato et al. 1995Sato et al. , 1996Marjamaki et al. 1997 and references therein). Additional studies demonstrating the localization of heterotrimeric G-protein subunits in intracellular compartments, suggesting a role in controlling such basic cellular functions as vesicular trafficking and exocytosis, lent further momentum to this search (Toutant et al. 1987;Ngsee et al. 1990;Stow et al. 1991;Aronin and DiFiglia 1992;Bomsel and Mostov 1992;Ahnert-Hilger et al. 1994;Helms 1995;Maier et al. 1995;Denker et al. 1996;Nurnberg and Ahnert-Hilger 1996;Ogier-Denis et al. 1996;Crouch and Simson 1997;Pinxteren et al. 1998;Martin et al. 1999;Crouch et al. 2000;Yamaguchi et al. 2000;Fisher 2000a,2000b;Blackmer et al. 2001;Blumer et al. 2002). From this body of work, it was hypothesized that there were cell-specific accessory proteins or signal regulators that influence the transfer of signal from receptor to G-protein or that regulate the activation state and/or subcellular localization of heterotrimeric G-proteins (Fig. 1). ...
During protein translation, a variety of quality control checks ensure that the resulting polypeptides deviate minimally from their genetic encoding template. Translational fidelity is central in order to preserve the function and integrity of each cell. Correct termination is an important aspect of translational fidelity, and a multitude of mechanisms and players participate in this exquisitely regulated process. This review explores our current understanding of eukaryotic termination by highlighting the roles of the different ribosomal components as well as termination factors and ribosome-associated proteins, such as chaperones.
... In mast cells, G protein hg-subunits are implicated in controlling exocytosis while they are not sufficient by themselves to trigger exocytosis. Instead, Gomberts et al. suggest that activation of Rho-related GTPases is likely necessary for induction of exocytosis [39]. This mechanism is further postulated in the action of both compound 48/80 and mastoparan activation of mast cells. ...
We investigated G protein-stimulated release of ATP from human umbilical vein endothelial cells (HUVECs) using the G protein stimulant compound 48/80. Application of compound 48/80 resulted in dose-dependent ATP evolution from cultured HUVECs. This release was not cytotoxic as demonstrated by a lactate dehydrogenase assay and the ability of the cells to load and retain the viability dye calcein following stimulation. Mastoparan also stimulated release of ATP, further suggesting the process was G-protein initiated. This G protein was insensitive to pertussis toxin and appeared to be of the Gq-subtype. The ATP efflux was completely abolished in the presence of EGTA and thapsigargin signifying a strict Ca2+ dependence. Furthermore, compound 48/80-induced release was significantly decreased in cells pretreated with the phospholipase C inhibitor U73122. Thus, the release pathway appears to proceed through an increase in intracellular Ca2+ via PLC activation. Additionally, the G protein-initiated release was attenuated by pretreatment of the cells with either phorbol ester or indolactam V, both activators of protein kinase C. Finally, ATP release was not affected by treating HUVECs with nitric oxide synthase (NOS) inhibitors or glybenclamide.
... Fractions were immediately frozen and stored at −70 • C prior to protein purification or gel analysis. Heterotrimeric GTP-binding proteins were purified from total membrane fractions by adaptation of established methods for bovine brain G␣␥ purification [33] [34]. G␣␥ purification was monitored with a GTP␥S binding assay as described in [33] and used mammalian G␣o and G␣i1 as positive controls. ...
... Heterotrimeric GTP-binding proteins were purified from total membrane fractions by adaptation of established methods for bovine brain G␣␥ purification [33] [34]. G␣␥ purification was monitored with a GTP␥S binding assay as described in [33] and used mammalian G␣o and G␣i1 as positive controls. Membranes were extracted with 0.4% Genapol-C100 (Calbiochem) in TEDP buffer (20 mM Tris, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, pH 8.0). ...
We have cloned a single copy gene from the human parasite Trichomonas vaginalis that encodes a putative protein of 402 amino acids with approximately 35% sequence identity to known alpha subunits of heterotrimeric G-proteins. It contains the characteristic GTP binding domains G-1 to G-5 with the key residues conserved. The new sequence has an unusual N-terminal extension of approximately 70 residues that cannot be aligned to reference G-proteins and which is characterised by proline-rich repeats. To investigate the expression and cellular localisation of the protein we produced specific antisera against a recombinant fusion protein. The antisera recognised a protein of an apparent molecular mass of 51 kDa in protein extracts from T. vaginalis and immunofluorescent microscopy established that the protein is localised to discrete endomembranes. Using a protocol designed to purify mammalian heterotrimeric G-proteins incorporating a GTPgammaS binding assay, we isolated two proteins from Trichomonas that are recognised by an heterologous GA/1 antisera raised to a peptide of the conserved G-1 domain of G-protein alpha subunits. These two proteins have an apparent molecular mass of 61 and 48 kDa, respectively, larger and smaller than the translation product of the cloned gene. Consistent with these results, the GA/1 antisera did not cross-react with the fusion protein produced from the gene we have cloned. These data suggest T. vaginalis possesses more than one heterotrimeric G-protein alpha subunit. Based on the sequence features of the cloned gene and the biochemical properties of the purified proteins, we suggest that these alpha subunits are likely to be part of classic heterotrimeric G-protein complexes.
... Incubation of BAPTA-AM-treated platelets with SFLLRN increased the association of FITC-QR-LFQVKGRR with platelets following permeabilization with SL-O (Fig. 6A). Neomycin binds PtdIns(4,5)P 2 strongly (46) and has been used to study the role of PtdIns(4,5)P 2 in secretory processes (47). In these experiments, neomycin abolished SFLLRN-induced FITC-QRLFQVKGRR binding to BAPTA-AM-treated, permeabilized platelets, consistent with the supposition that the FITC-labeled PtdIns(4,5)P 2 -binding domain of gelsolin associates with platelet PtdIns(4,5)P 2 (Fig. 6A). ...
To better understand the molecular mechanisms of platelet granule secretion, we have evaluated the role of type II phosphatidylinositol (PtdIns) 5-phosphate 4-kinase in agonist-induced platelet alpha-granule secretion. SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets was inhibited by either antibodies directed at type II PtdIns 5-phosphate 4-kinase or by a kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase. In contrast, recombinant type IIbeta PtdIns 5-phosphate 4-kinase augmented SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets. SFLLRN-stimulated alpha-granule secretion was inhibited by a protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. Phorbol 12-myristate 13-acetate-stimulated alpha-granule secretion was inhibited by anti-type II PtdIns 5-phosphate 4-kinase antibodies or the kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase and augmented by recombinant type IIbeta PtdIns 5-phosphate 4-kinase. Immunoblot analysis demonstrated that type II PtdIns 5-phosphate 4-kinase remained associated with SL-O-permeabilized platelets when incubated in the presence, but not the absence, of SFLLRN. This SFLLRN-induced translocation of type II PtdIns 5-phosphate 4-kinase was blocked by either the protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. In addition to stimulating alpha-granule secretion, both SFLLRN and PMA enhanced the association of a fluorescein isothiocyanate-labeled peptide derived from the PtdIns (4,5)P(2)-binding domain of gelsolin to permeabilized platelets. Agonist-induced recruitment of the PtdIns (4,5)P(2)-binding domain was inhibited by neomycin, bisindolylmaleimide I, and anti-type II PtdIns 5-phosphate 4-kinase antibody. These results suggest a mechanism whereby protein kinase C-mediated translocation of type II PtdIns 5-phosphate 4-kinase leads to the recruitment of PtdIns (4,5)P(2)-binding proteins.
