共查询到20条相似文献,搜索用时 31 毫秒
1.
S E Langille V Patki J K Klarlund J M Buxton J J Holik A Chawla S Corvera M P Czech 《The Journal of biological chemistry》1999,274(38):27099-27104
The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions. 相似文献
2.
Venkateswarlu K 《The Journal of biological chemistry》2003,278(44):43460-43469
The ADP-ribosylation factor 6 (ARF6) small GTPase functions as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganization and membrane ruffling. The formation of active ARF6GTP is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ARF6 activation. Using cytohesin 2 as bait in yeast two-hybrid screening, we have isolated a cDNA encoding a protein termed interaction protein for cytohesin exchange factors 1 (IPCEF1). Using yeast two-hybrid and glutathione S-transferase pull-down assays coupled with deletion mutational analysis, the specific domains required for the cytohesin 2-IPCEF1 interaction were mapped to the coiled-coil domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1. IPCEF1 also interacts with the other members of the cytohesin family of ARF GEFs, suggesting that the interaction with IPCEF1 is highly conserved among the cytohesin family of ARF GEFs. The interaction of cytohesin 2 and IPCEF1 in mammalian cells was demonstrated by immunoprecipitation. Immunofluorescence analysis revealed that IPCEF1 co-localizes with cytohesin 2 to the cytosol in unstimulated cells and translocates to the plasma membrane via binding to cytohesin 2 in epidermal growth factor-stimulated cells. However, a deletion mutant of IPCEF1 that lacks the cytohesin 2 binding site failed to co-migrate with cytohesin 2 to the membrane in stimulated cells. The functional significance of the IPCEF1-cytohesin 2 interaction is demonstrated by showing that IPCEF1 increases the in vitro and in vivo stimulation of ARFGTP formation by cytohesin 2. 相似文献
3.
Incubation of Swiss 3T3 cells with [2-3H]adenine, as in other cell types, reveals the ADP-ribosylation of GRP78 (the 78-kDa glucose-regulated protein, also known as BiP, the immunoglobulin heavy chain-binding protein), a resident endoplasmic reticulum protein that assists in the processing of proteins destined for secretion or cell surface expression. Here we show that Pasteurella multocida toxin, a potent growth factor for cultured fibroblasts, decreased the ADP-ribosylation of GRP78/BiP to 16 +/- 6% of the control value (n = 23). The action of the toxin occurred after a lag period, was blocked by lysosomotrophic agents, and potentiated by increased incubation time (ED50 4 ng/ml and 1 ng/ml in 4 and 8 h, respectively), thus indicating that the toxin enters the cells to act. Bombesin and platelet-derived growth factor (PDGF) similarly decreased the ADP-ribosylation of GRP78/BiP (ED50 0.5 nM and 2.5 ng/ml, respectively) but acted more rapidly than the toxin. Signaling pathways activated by the toxin, bombesin, and PDGF had effects on the ADP-ribosylation of GRP78/BiP. Thus, activation of protein kinase C alone by phorbol 12,13-dibutyrate was partially effective, and down-regulation of protein kinase C attenuated but did not block the action of the toxin, bombesin, and PDGF. Agents that mobilize Ca2+ from the endoplasmic reticulum (A23187, ionomycin, and thapsigargin) caused a decrease in the ADP-ribosylation of GRP78/BiP that was similar in magnitude to that achieved by the toxin, bombesin, and PDGF, implicating a role for inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization in the action of the mitogenic agents. The growth factor-induced decrease in the ADP-ribosylation of GRP78/BiP may represent its conversion from an inactive to an active state. 相似文献
4.
Starvation of mouse hepatoma cells for essential amino acids or glucose results in the ADP-ribosylation of the molecular chaperone BiP/GRP78. Addition of the missing nutrient to the medium reverses the reaction. The signal mediating the response to environmental nutrients involves the translational efficiency. An inhibitor of proteins synthesis, cycloheximide, or reduced temperature, both of which reduce translational efficiency, stimulate the ADP-ribosylation of BiP/GRP78. Inhibition of N-linked glycosylation of proteins results in the overproduction of BiP/GRP78. The over produced protein is not ADP-ribosylated suggesting that this is the functional form of BiP/GRP78. The over produced BiP/GRP78 can, however, be ADP-ribosylated if the cells are starved for an essential amino acid. BiP/GRP78 resides in the lumen of the endoplasmic reticulum where it participates in the assembly of secretory and integral membrane proteins. ADP-ribosylation of BiP/GRP78 during starvation is probably part of a nutritional stress response which conserves limited nutrients by slowing flow through the secretory pathway. 相似文献
5.
de Crom R van Haperen R Janssens R Visser P Willemsen R Grosveld F van der Kamp A 《Biochimica et biophysica acta》1999,1437(3):378-392
We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed. 相似文献
6.
Kim DW 《Biochemical and biophysical research communications》2003,303(1):370-378
A high copy suppressor screen with sec34-2, a temperature-sensitive mutant defective in the late stages of ER to Golgi transport, has resulted in the identification of a novel gene called GRP1 (also called RUD3). GRP1 encodes a hydrophilic yeast protein related to the mammalian Golgi matrix protein golgin-160. A large portion of the protein is predicted to form a coiled-coil structure. Although GRP1 is not essential for growth, the loss of Grp1p results in a growth defect at high temperature. GRP1 genetically interacts with several genes involved in vesicle targeting/fusion stages of ER to Golgi transport. Despite these interactions, pulse chase analysis using Grp1p-depleted cells did not reveal a significant delay in the transit of the vacuolar protease carboxypeptidase Y. Grp1p-depleted cells efficiently secreted invertase which was underglycosylated, suggesting some disturbance of Golgi function. Grp1p-GFP predominantly colocalizes with the cis-Golgi marker Och1p. Despite lacking a signal peptide and a significant stretch of hydrophobic amino acids, Grp1p pellets with membranes. It is extracted with 1M NaCl or 0.1M Na(2)CO(3) (pH 11.0), but is surprisingly insoluble in 1% Triton X-100. Grp1p does not recycle to the ER when forward transport is blocked and a cis-Golgi marker (Och1p-HA), but not a trans-Golgi marker (Chs5p-HA), became dispersed in grp1 Delta cells after 1.5h incubation at 38.5 degrees C. Together, these data suggest that Grp1p is a novel matrix protein that is involved in the structural organization of the cis-Golgi. 相似文献
7.
Wang D De Deken X Milenkovic M Song Y Pirson I Dumont JE Miot F 《The Journal of biological chemistry》2005,280(4):3096-3103
H(2)O(2) is a crucial substrate of thyroproxidase (TPO) to iodinate thyroglobulin and synthesize thyroid hormones in thyroid. ThOX proteins (thyroid oxidase) also called Duox are believed to be responsible for H(2)O(2) generation. Duoxs expressed in transfected cells do not generate an active system, nor permit their membrane localization suggesting that other proteins are required to fulfill these functions. In this study, we demonstrate interactions of Duoxs with TPO and with p22(phox) without any effect on Duox activity. By yeast two-hybrid method using EF-hand fragment of dog Duox1 as the bait we have isolated EFP1 (EF-hand binding protein 1), one partner of Duoxs that belongs to the thioredoxin-related protein family. EFP1 shares moderate similarities with other members of thioredoxin-related proteins, but the characteristic active site and the folding structures are well conserved. EFP1 can be co-immunoprecipitated with Duoxs in transfected COS cells as well as in primary cultured human thyrocytes. It interacts also with TPO but not thyroglobulin. Immunofluorescence studies show that EFP1 and Duox proteins are co-localized inside the transfected cells, suggesting that EFP1 is not sufficient to induce either the expression of Duox at the plasma membrane or to permit H(2)O(2) production. EFP1 and Duox mRNA share similar distribution in nine different tissues. These results suggest that EFP1 could be one of the partners in the assembly of the multiprotein complex constituting the thyroid H(2)O(2) generating system but is certainly not sufficient to permit H(2)O(2) generation. 相似文献
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Maclean LM O'Toole PJ Stark M Marrison J Seelenmeyer C Nickel W Smith DF 《Cellular microbiology》2012,14(5):740-761
Proteins of the Leishmania hydrophilic acylated surface protein B (HASPB) family are only expressed in infective parasites (both extra- and intracellular stages) and, together with the peripheral membrane protein SHERP (small hydrophilic endoplasmic reticulum-associated protein), are essential for parasite differentiation (metacyclogenesis) in the sand fly vector. HASPB is a 'non-classically' secreted protein, requiring N-terminal acylation for trafficking to and exposure on the plasma membrane. Here, we use live cell imaging methods to further explore this pathway to the membrane and flagellum. Unlike HASPB trafficking in transfected mammalian cells, we find no evidence for a phosphorylation-regulated recycling pathway in metacyclic parasites. Once at the plasma membrane, HASPB18-GFP (green fluorescent protein) can undergo bidirectional movement within the inner leaflet of the membrane and on the flagellum. Transfer of fluorescent protein between the flagellum and the plasma membrane is compromised, however, suggesting the presence of a diffusion barrier at the base of the Leishmania flagellum. Full-length HASPB is released from the metacyclic parasite surface on to macrophages during phagocytosis but while expression is maintained in intracellular amastigotes, HASPB cannot be detected on the external surface in these cells. Thus HASPB may be a dual function protein that is shed by the infective metacyclic but retained internally once Leishmania are taken up by macrophages. 相似文献
11.
Organelle tethering by a homotypic PDZ interaction underlies formation of the Golgi membrane network
Debrup Sengupta Steven Truschel Collin Bachert Adam D. Linstedt 《The Journal of cell biology》2009,186(1):41-55
Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells. 相似文献
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Molecular cloning and characterization of a novel chloride intracellular channel-related protein, parchorin, expressed in water-secreting cells 总被引:2,自引:0,他引:2
Nishizawa T Nagao T Iwatsubo T Forte JG Urushidani T 《The Journal of biological chemistry》2000,275(15):11164-11173
We previously reported a 120-kDa phosphoprotein that translocated from cytosol to the apical membrane of gastric parietal cells in association with stimulation of HCl secretion. To determine the molecular identity of the protein, we performed molecular cloning and expression of the protein. Immunoblot analysis showed that this protein was highly enriched in tissues that secrete water, such as parietal cell, choroid plexus, salivary duct, lacrimal gland, kidney, airway epithelia, and chorioretinal epithelia. We named this protein "parchorin" based on its highest enrichment in parietal cells and choroid plexus. We obtained cDNA for parchorin from rabbit choroid plexus coding a protein consisting of 637 amino acids with a predicted molecular mass of 65 kDa. The discrepancy in size on 6% SDS-polyacrylamide gel electrophoresis is considered to be due to its highly acidic nature (pI = 4.18), because COS-7 cells transfected with parchorin cDNA produced a protein with apparent molecular mass of 120 kDa on 6% SDS-polyacrylamide gel electrophoresis. Parchorin is a novel protein that has significant homology to the family of chloride intracellular channels (CLIC), especially the chloride channel from bovine kidney, p64, in the C-terminal 235 amino acids. When expressed as a fusion protein with green fluorescent protein (GFP) in the LLC-PK1 kidney cell line, GFP-parchorin, unlike other CLIC family members, existed mainly in the cytosol. Furthermore, when Cl(-) efflux from the cell was elicited, GFP-parchorin translocated to the plasma membrane. These results suggest that parchorin generally plays a critical role in water-secreting cells, possibly through the regulation of chloride ion transport. 相似文献
14.
J K Klarlund J Holik A Chawla J G Park J Buxton M P Czech 《The Journal of biological chemistry》2001,276(43):40065-40070
GRP1 is a member of a family of proteins that contain a coiled-coil region, a Sec7 homology domain with guanosine nucleotide exchange activity for the ARF GTP-binding proteins, and a pleckstrin homology domain at the C terminus. The pleckstrin homology domain of GRP1 binds phosphatidylinositol (3,4,5) trisphosphate and mediates the translocation of GRP1 to the plasma membrane upon agonist stimulation of PI 3-kinase activity. Using a (32)P-labeled GRP1 probe to screen a mouse brain cDNA expression library, we isolated a cDNA clone encoding a GRP1-binding partner (GRSP1) that exists as two different splice variants in brain and lung. The GRSP1 protein contains a FERM protein interaction domain as well as two coiled coil domains and may therefore function as a scaffolding protein. Mapping experiments revealed that the interaction of GRP1 and GRSP1 occurs through the coiled coil domains in the two proteins. Immunodepletion experiments indicate that virtually all of the endogenous GRSP1 protein exists as a complex with GRP1 in lung. When co-expressed in Chinese hamster ovary cells expressing the human insulin receptor, both proteins display a diffuse, cytoplasmic localization. Acute translocation and co-localization of GRSP1 and GRP1 to ruffles in the plasma membrane was evident after insulin stimulation. These results identify GRSP1 as a novel member of GRP1 signaling complexes that are acutely recruited to plasma membrane ruffles in response to insulin receptor signaling. 相似文献
15.
Fiscella M Perry JW Teng B Bloom M Zhang C Leung K Pukac L Florence K Concepcion A Liu B Meng Y Chen C Elgin EC Kanakaraj P Kaufmann TE Porter J Cibotti R Mei Y Zhou J Chen G Roschke V Komatsoulis G Mansfield B Ruben S Sanyal I Migone TS 《Nature biotechnology》2003,21(3):302-307
A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins. 相似文献
16.
Morinaga N Kaihou Y Vitale N Moss J Noda M 《The Journal of biological chemistry》2001,276(25):22838-22843
ADP-ribosylation factor 1 (ARF1) was originally found as a cofactor in CT-catalyzed ADP-ribosylation of Galpha(s) but is now known to participate in vesicle trafficking. We asked whether ARF1 function in vesicular trafficking is necessary for CT-induced morphological changes in Chinese hamster ovary (CHO) cells, which result from increased intracellular cAMP. Brefeldin A treatment of cells suppressed CT action, confirming a requirement for Golgi integrity. Overexpression of a GFP-ARF1 fusion protein did not affect the morphological changes induced by CT, but changes were reduced in cells overexpressing guanine nucleotide exchange-defective ARF1(T31N) or GTP hydrolysis-deficient ARF1(Q71L) mutants. In cells expressing these mutants, 8-bromo-cAMP induced changes similar to those seen in cells transfected with ARF1 or vector. Inhibition of CT action was specific for mutants of ARF1 and not reproduced by analogous mutants of ARF5 or ARF6. ARF1(Q71L) was mostly colocalized with betaCOP, but ARF5(Q71L) less so. ARF6(Q67L) did not colocalize with betaCOP and was partially associated with the plasma membrane. These data are consistent with the conclusion that ARF1 influenced CT action in cells by its specific function in the vesicular transport pathway used by CT to travel from plasma membrane to Golgi to ER. 相似文献
17.
Danming Tang Xiaoyan Zhang Shijiao Huang Hebao Yuan Jie Li Yanzhuang Wang 《Molecular biology of the cell》2016,27(1):137-152
In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking. 相似文献
18.
Perestenko PV Pooler AM Noorbakhshnia M Gray A Bauccio C Jeffrey McIlhinney RA 《The FEBS journal》2010,277(24):5174-5189
The copines are a family of C2- and von Willebrand factor A-domain-containing proteins that have been proposed to respond to increases in intracellular calcium by translocating to the plasma membrane. The copines have been reported to interact with a range of cell signalling and cytoskeletal proteins, which may therefore be targeted to the membrane following increases in cellular calcium. However, neither the function of the copines, nor their actual movement to the plasma membrane, has been fully established in mammalian cells. Here, we show that copines-1, -2, -3, -6 and -7 respond differently to a methacholine-evoked intracellular increase in calcium in human embryonic kidney cell line-293 cells, and that their membrane association requires different levels of intracellular calcium. We demonstrate that two of these copines associate with different intracellular vesicles following calcium entry into cells, and identify a novel conserved amino acid sequence that is required for their membrane translocation in living cells. Our data show that the von Willebrand factor A-domain of the copines modulates their calcium sensitivity and intracellular targeting. Together, these findings suggest a different set of roles for the members of this protein family in mediating calcium-dependent processes in mammalian cells. 相似文献
19.
Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells. 总被引:12,自引:5,他引:7
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S H Low S J Chapin T Weimbs L G Kmüves M K Bennett K E Mostov 《Molecular biology of the cell》1996,7(12):2007-2018
Syntaxins, integral membrane proteins that are part of the ubiquitous membrane fusion machinery, are thought to act as target membrane receptors during the process of vesicle docking and fusion. Several isoforms of the syntaxin family have been previously identified in mammalian cells, some of which are localized to the plasma membrane. We investigated the subcellular localization of these putative plasma membrane syntaxins in polarized epithelial cells, which are characterized by the presence of distinct apical and basolateral plasma membrane domains. Syntaxins 2, 3, and 4 were found to be endogenously present in Madin-Darby canine kidney cells. The localization of syntaxins 1A, 1B, 2, 3, and 4 in stably transfected Madin-Darby canine kidney cell lines was studied with confocal immunofluorescence microscopy. Each syntaxin isoform was found to have a unique pattern of localization. Syntaxins 1A and 1B were present only in intracellular structures, with little or no apparent plasma membrane staining. In contrast, syntaxin 2 was found on both the apical and basolateral surface, whereas the plasma membrane localization of syntaxins 3 and 4 were restricted to the apical or basolateral domains, respectively. Syntaxins are therefore the first known components of the plasma membrane fusion machinery that are differentially localized in polarized cells, suggesting that they may play a central role in targeting specificity. 相似文献
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