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Peter J Cullen - One of the best experts on this subject based on the ideXlab platform.
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tissue specific expression and endogenous subcellular distribution of the inositol 1 3 4 5 tetrakisphosphate binding proteins gap1 ip4bp and gap1 m
Biochemical and Biophysical Research Communications, 1999Co-Authors: Peter J Lockyer, Jon S Reynolds, Sara Vanlingen, Tracy J Mcnulty, Robin F Irvine, Jan B Parys, Peter J CullenAbstract:Abstract GAP1 IP4BP and GAP1 m belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP 4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1 IP4BP is primarily localised to the plasma membrane, whereas GAP1 m appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1 m and several Ca 2+ store markers, consistent with the lack of co-localisation between GAP1 m and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1 m does not reside at a site where it could regulate the ability of InsP 4 to release intracellular Ca 2+ . As GAP1 m is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane.
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structural and functional analysis of the putative inositol 1 3 4 5 tetrakisphosphate receptors gap1ip4bpand gap1m
Biochemical and Biophysical Research Communications, 1998Co-Authors: Joanna R Bottomley, Jon S Reynolds, Peter J Lockyer, Peter J CullenAbstract:Abstract Previously we have purified and cloned a high affinity isomerically specific inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 )-binding protein which, because it is clearly a member of the GAP1 family of Ras GTPase-activating proteins (GAP), we have termed GAP1 IP4BP . Here we show that expressed full-length GAP1 IP4BP binds Ins(1,3,4,5)P 4 with an affinity and specificity similar to that of the originally purified protein, a binding activity which is dependent on a functional PH/Btk domain. Furthermore, we highlight a fundamental distinction between GAP1 IP4BP and its homologue GAP1 m , namely that both proteins function as Ras GAPs but only GAP1 IP4BP displays Rap GAP activity.
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distinct subcellular localisations of the putative inositol 1 3 4 5 tetrakisphosphate receptors gap1ip4bp and gap1m result from the gap1ip4bp ph domain directing plasma membrane targeting
Current Biology, 1997Co-Authors: Peter J Lockyer, Joanna R Bottomley, Jon S Reynolds, Tracy J Mcnulty, K Venkateswarlu, Barry V L Potter, Christopher E Dempsey, Peter J CullenAbstract:Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.
Paul Melançon - One of the best experts on this subject based on the ideXlab platform.
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bioid performed on golgi enriched fractions identify c10orf76 as a gbf1 binding protein essential for golgi maintenance and secretion
Molecular & Cellular Proteomics, 2019Co-Authors: Calvin J Chan, Kaylan Burns, Khadra Ahmed, Etienne Coyaud, Estelle M N Laurent, Brian Raught, Paul MelançonAbstract:The Golgi-specific Brefeldin-A resistance factor 1 (GBF1) is the only large GEF that regulates Arf activation at the cis-Golgi and is actively recruited to membranes on an increase in Arf-GDP. Recent studies have revealed that GBF1 recruitment requires one or more heat-labile and protease-sensitive protein factor(s) (Quilty et al., 2018, J. Cell Science, 132). Proximity-dependent biotinylation (BioID) and mass spectrometry from enriched Golgi fractions identified GBF1 proximal proteins that may regulate its recruitment. Knockdown studies revealed C10orf76 to be involved in Golgi maintenance. We find that C10orf76 interacts with GBF1 and rapidly cycles on and off GBF1-positive Golgi structures. More importantly, its depletion causes Golgi fragmentation, alters GBF1 recruitment, and impairs secretion. Homologs were identified in most species, suggesting its presence in the last eukaryotic common ancestor.
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the arf gdp regulated recruitment of gbf1 to golgi membranes requires domains hds1 and hds2 and a golgi localized protein receptor
Journal of Cell Science, 2019Co-Authors: Douglas Quilty, Calvin J Chan, Katherine Yurkiw, Alexandra Bain, Ghazal Babolmorad, Paul MelançonAbstract:We previously proposed a novel mechanism by which the enzyme Golgi-specific Brefeldin A resistance factor 1 (GBF1) is recruited to the membranes of the cis-Golgi, based on in vivo experiments. Here, we extended our in vivo analysis on the production of regulatory Arf-GDP and observed that ArfGAP2 and ArfGAP3 do not play a role in GBF1 recruitment. We confirm that Arf-GDP localization is critical, as a TGN-localized Arf-GDP mutant protein fails to promote GBF1 recruitment. We also reported the establishment of an in vitro GBF1 recruitment assay that supports the regulation of GBF1 recruitment by Arf-GDP. This in vitro assay yielded further evidence for the requirement of a Golgi-localized protein because heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. Finally, combined in vivo and in vitro measurements indicated that the recruitment to Golgi membranes via a putative receptor requires only the HDS1 and HDS2 domains in the C-terminal half of GBF1.
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gbf1 a cis golgi and vtcs localized arf gef is implicated in er to golgi protein traffic
Journal of Cell Science, 2006Co-Authors: Xinhua Zhao, Alejandro Claude, Justin Chun, David J Shields, John F Presley, Paul MelançonAbstract:The formation and maturation of membrane carriers that transport cargo from the ER to the Golgi complex involves the sequential action of the coat protein complexes COPII and COPI. Recruitment of COPI to nascent carriers requires activation of ADP-ribosylation factors by a BrefeldinA-sensitive guanine nucleotide exchange factor. Using new antisera and a GFP-tagged protein, we demonstrate that the exchange factor GBF1 localized to both Golgi membranes and peripheral puncta, near but separate from ER exit sites. Live cell imaging revealed that GFP-GBF1 associates dynamically with both membranes through rapid exchange with a large cytosolic pool. Treatment with BrefeldinA dramatically altered this rapid exchange, causing accumulation of GBF1 on both Golgi and peripheral puncta before eventual redistribution to the ER in a microtubule-dependent manner. Measurement of diffusion coefficients and subcellular fractionation confirmed this shift in GBF1 from cytosolic to membrane bound. BrefeldinA-induced accumulation of GBF1 coincided with loss of COPI from peripheral puncta. Furthermore, recruitment of GBF1 to cargo-containing peripheral puncta coincided with recruitment of COPI, but not COPII. Strikingly, microinjection of anti-GBF1 antibodies specifically caused dissociation of COPI from membranes. These observations strongly suggest that GBF1 regulates COPI membrane recruitment in the early secretory pathway.
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localization of large adp ribosylation factor guanine nucleotide exchange factors to different golgi compartments evidence for distinct functions in protein traffic
Molecular Biology of the Cell, 2002Co-Authors: Xinhua Zhao, Troy K R Lasell, Paul MelançonAbstract:Activation of several ADP-ribosylation factors (ARFs) by guanine nucleotide exchange factors (GEFs) regulates recruitment of coat proteins (COPs) on the Golgi complex and is generally assumed to be the target of brefeldin A (BFA). The large ARF-GEFs Golgi-specific BFA resistance factor 1 (GBF1) and BFA-inhibited GEFs (BIGs) localize to this organelle but catalyze exchange preferentially on class II and class I ARFs, respectively. We now demonstrate using quantitative confocal microscopy that these GEFs show a very limited overlap with each other (15 and 23%). In contrast, GBF1 colocalizes with the cis-marker p115 (86%), whereas BIGs overlap extensively with TGN38 (83%). Consistent with these distributions, GBF1, but not BIG1, partially relocalized to peripheral sites after incubation at 15°C. The new GBF1 structures represent peripheral vesicular tubular clusters (VTCs) because 88% of structures analyzed stained for both GBF1 and p115. Furthermore, as expected of VTCs, they rapidly reclustered to the Golgi complex in a microtubule-dependent manner upon warm-up. These observations suggest that GBF1 and BIGs activate distinct subclasses of ARFs in specific locations to regulate different types of reactions. In agreement with this possibility, COPI overlapped to a greater extent with GBF1 (64%) than BIG1 (31%), whereas clathrin showed limited overlap with BIG1, and virtually none with GBF1.
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gbf1 a novel golgi associated bfa resistant guanine nucleotide exchange factor that displays specificity for adp ribosylation factor 5
Journal of Cell Biology, 1999Co-Authors: Alejandro Claude, Bao Ping Zhao, Craig E Kuziemsky, Sophie Dahan, Scott J Berger, Jian Ping Yan, Adrian D Armold, Eric M Sullivan, Paul MelançonAbstract:Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged–GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the β-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.
Catherine L. Jackson - One of the best experts on this subject based on the ideXlab platform.
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identification of gbf1 as a cellular factor required for hepatitis e virus rna replication
Cellular Microbiology, 2018Co-Authors: Rayan Farhat, Catherine L. Jackson, Yves Rouillé, Jean Dubuisson, Nadjet Lebsir, Maliki Ankavay, Jerome Gouttenoire, C Wychowski, Darius Moradpour, Laurence CocquerelAbstract:The hepatitis E virus (HEV) genome is a single-stranded, positive-sense RNA that encodes three proteins including the ORF1 replicase. Mechanisms of HEV replication in host cells are unclear and only a few cellular factors involved in this step have been identified so far. Here, we used brefeldin A (BFA) that blocks the activity of the cellular Arf guanine nucleotide exchange factors GBF1, BIG1 and BIG2, which play a major role in reshuffling of cellular membranes. We showed that BFA inhibits HEV replication in a dose-dependent manner. The use of siRNA and Golgicide A identified GBF1 as a host factor critically involved in HEV replication. Experiments using cells expressing a mutation in the catalytic domain of GBF1 and overexpression of wildtype GBF1 or a BFA-resistant GBF1 mutant rescuing HEV replication in BFA-treated cells, confirmed that GBF1 is the only BFA-sensitive factor required for HEV replication. We demonstrated that GBF1 is likely required for the activity of HEV replication complexes. However, GBF1 does not colocalize with the ORF1 protein and its subcellular distribution is unmodified upon infection or overexpression of viral proteins, indicating that GBF1 is likely not recruited to replication sites. Together, our results suggest that HEV replication involves GBF1-regulated mechanisms.
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interaction between the triglyceride lipase atgl and the arf1 activator gbf1
PLOS ONE, 2011Co-Authors: Emy Njoh Ellong, Krishnakant G Soni, Mariepierre Golinellicohen, Rachid Sougrat, Catherine L. JacksonAbstract:The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets.
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poliovirus replication requires the n terminus but not the catalytic sec7 domain of arfgef gbf1
Cellular Microbiology, 2010Co-Authors: George A Belov, Catherine L. Jackson, Gennadiy Kovtunovych, Ellie EhrenfeldAbstract:Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP-ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism.
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gbf1 a guanine nucleotide exchange factor for arf is crucial for coxsackievirus b3 rna replication
Journal of Virology, 2009Co-Authors: Kjerstin Lanke, Ellie Ehrenfeld, Catherine L. Jackson, Hilde M Van Der Schaar, George A Belov, Qian Feng, Daniel Duijsings, Frank J M Van KuppeveldAbstract:The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.
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molecular determinants of the interaction between coxsackievirus protein 3a and guanine nucleotide exchange factor gbf1
Journal of Virology, 2007Co-Authors: Els Wessels, Catherine L. Jackson, Kjerstin Lanke, Daniel Duijsings, Willem J G Melchers, Frank J M Van KuppeveldAbstract:The 3A protein of coxsackievirus B3 (CVB3), a small membrane protein that forms homodimers, inhibits endoplasmic reticulum-to-Golgi complex transport. Recently, we described the underlying mechanism by showing that the CVB3 3A protein binds to and inhibits the function of GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factor 1 (Arf1), thereby interfering with Arf1-mediated COP-I recruitment. This study was undertaken to gain more insight into the molecular determinants underlying the interaction between 3A and GBF1. Here we show that 3A mutants that have lost the ability to dimerize are no longer able to bind to GBF1 and trap it on membranes. Moreover, we identify a conserved region in the N terminus of 3A that is crucial for GBF1 binding but not for 3A dimerization. Analysis of the binding domain in GBF1 showed that the extreme N terminus, the dimerization/cyclophilin binding domain, and the homology upstream of Sec7 domain are required for the interaction with 3A. In contrast to that of full-length GBF1, overexpression of a GBF1 mutant lacking its extreme N terminus failed to rescue the effects of 3A. Together, these data provide insight into the molecular requirements of the interaction between 3A and GBF1.
Peter J Lockyer - One of the best experts on this subject based on the ideXlab platform.
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tissue specific expression and endogenous subcellular distribution of the inositol 1 3 4 5 tetrakisphosphate binding proteins gap1 ip4bp and gap1 m
Biochemical and Biophysical Research Communications, 1999Co-Authors: Peter J Lockyer, Jon S Reynolds, Sara Vanlingen, Tracy J Mcnulty, Robin F Irvine, Jan B Parys, Peter J CullenAbstract:Abstract GAP1 IP4BP and GAP1 m belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP 4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1 IP4BP is primarily localised to the plasma membrane, whereas GAP1 m appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1 m and several Ca 2+ store markers, consistent with the lack of co-localisation between GAP1 m and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1 m does not reside at a site where it could regulate the ability of InsP 4 to release intracellular Ca 2+ . As GAP1 m is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane.
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structural and functional analysis of the putative inositol 1 3 4 5 tetrakisphosphate receptors gap1ip4bpand gap1m
Biochemical and Biophysical Research Communications, 1998Co-Authors: Joanna R Bottomley, Jon S Reynolds, Peter J Lockyer, Peter J CullenAbstract:Abstract Previously we have purified and cloned a high affinity isomerically specific inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 )-binding protein which, because it is clearly a member of the GAP1 family of Ras GTPase-activating proteins (GAP), we have termed GAP1 IP4BP . Here we show that expressed full-length GAP1 IP4BP binds Ins(1,3,4,5)P 4 with an affinity and specificity similar to that of the originally purified protein, a binding activity which is dependent on a functional PH/Btk domain. Furthermore, we highlight a fundamental distinction between GAP1 IP4BP and its homologue GAP1 m , namely that both proteins function as Ras GAPs but only GAP1 IP4BP displays Rap GAP activity.
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distinct subcellular localisations of the putative inositol 1 3 4 5 tetrakisphosphate receptors gap1ip4bp and gap1m result from the gap1ip4bp ph domain directing plasma membrane targeting
Current Biology, 1997Co-Authors: Peter J Lockyer, Joanna R Bottomley, Jon S Reynolds, Tracy J Mcnulty, K Venkateswarlu, Barry V L Potter, Christopher E Dempsey, Peter J CullenAbstract:Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.
Jon S Reynolds - One of the best experts on this subject based on the ideXlab platform.
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tissue specific expression and endogenous subcellular distribution of the inositol 1 3 4 5 tetrakisphosphate binding proteins gap1 ip4bp and gap1 m
Biochemical and Biophysical Research Communications, 1999Co-Authors: Peter J Lockyer, Jon S Reynolds, Sara Vanlingen, Tracy J Mcnulty, Robin F Irvine, Jan B Parys, Peter J CullenAbstract:Abstract GAP1 IP4BP and GAP1 m belong to the GAP1 family of Ras GTPase-activating proteins that are candidate InsP 4 receptors. Here we show they are ubiquitously expressed in human tissues and are likely to have tissue-specific splice variants. Analysis by subcellular fractionation of RBL-2H3 rat basophilic leukemia cells confirms that endogenous GAP1 IP4BP is primarily localised to the plasma membrane, whereas GAP1 m appears localised to the cytoplasm (cytosol and internal membranes) but not the plasma membrane. Subcellular fractionation did not indicate a specific co-localisation between membrane-bound GAP1 m and several Ca 2+ store markers, consistent with the lack of co-localisation between GAP1 m and SERCA1 upon co-expression in COS-7 cells. This difference suggests that GAP1 m does not reside at a site where it could regulate the ability of InsP 4 to release intracellular Ca 2+ . As GAP1 m is primarily localised to the cytosol of unstimulated cells it may be spatially regulated in order to interact with Ras at the plasma membrane.
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structural and functional analysis of the putative inositol 1 3 4 5 tetrakisphosphate receptors gap1ip4bpand gap1m
Biochemical and Biophysical Research Communications, 1998Co-Authors: Joanna R Bottomley, Jon S Reynolds, Peter J Lockyer, Peter J CullenAbstract:Abstract Previously we have purified and cloned a high affinity isomerically specific inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 )-binding protein which, because it is clearly a member of the GAP1 family of Ras GTPase-activating proteins (GAP), we have termed GAP1 IP4BP . Here we show that expressed full-length GAP1 IP4BP binds Ins(1,3,4,5)P 4 with an affinity and specificity similar to that of the originally purified protein, a binding activity which is dependent on a functional PH/Btk domain. Furthermore, we highlight a fundamental distinction between GAP1 IP4BP and its homologue GAP1 m , namely that both proteins function as Ras GAPs but only GAP1 IP4BP displays Rap GAP activity.
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distinct subcellular localisations of the putative inositol 1 3 4 5 tetrakisphosphate receptors gap1ip4bp and gap1m result from the gap1ip4bp ph domain directing plasma membrane targeting
Current Biology, 1997Co-Authors: Peter J Lockyer, Joanna R Bottomley, Jon S Reynolds, Tracy J Mcnulty, K Venkateswarlu, Barry V L Potter, Christopher E Dempsey, Peter J CullenAbstract:Inositol 1,3,4,5-tetrakisphosphate (IP4), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP4 receptor, termed GAP1(IP4BP)[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1(IP4BP), called GAP1(m), has been identified [2] and here we describe the cloning of a GAP1(m) cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1(m), in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP4 with a similar affinity and specificity to that of the corresponding GAP1(IP4BP) mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1(IP4BP) is located solely at the plasma membrane, GAP1(m) seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP4.
