Structural and Functional Analysis of the Putative Inositol 1,3,4,5-Tetrakisphosphate Receptors GAP1IP4BPand GAP1m

Bottomley, Joanna; Reynolds, Jon S.; Lockyer, Peter J.; Cullen, Peter J.

doi:10.1006/bbrc.1998.9179

Cited by 28 publications

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“…IP4BP resulted in a mutant protein that, although GAP1 IP4BP and the Regulation of Ca 2ϩ Signaling 48780 displaying a greatly reduced IP 4 binding activity, ϳ10% of that seen with wild-type protein (33) (Fig. 1A) nevertheless retained full Ras GAP activity when assayed in vitro (Fig.…”

Section: Generation Of a Constitutively Active And A Potential Dominamentioning

confidence: 99%

“…As described under "Experimental Procedures," a mouse monoclonal antibody (GP-3) was raised against a GST fusion of a GAP1 IP4BP mutant lacking the N-terminal tandem C 2 domains (33).…”

Section: Isolation Of a Monoclonal Antibody Capable Of Detecting Endomentioning

confidence: 99%

“…GAP1 IP4BP along with the related proteins GAP1 m , RASAL, and CAPRI (23-30) is composed of tandem N-terminal C 2 domains, a C-terminal pleckstrin homology (PH) domain adjacent to a Bruton's tyrosine kinase (Btk) motif, and a central catalytic Ras GAP-related domain. Associated with the plasma membrane through a complex interaction between its PH/Btk domain and the inner plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) (31, 32), GAP1 IP4BP functions as a dual Ras and Rap1 GAP (19,33). Importantly, at least in vitro, the Ras GAP activity of GAP1 IP4BP is regulated by IP 4 (19).…”

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confidence: 99%

“…IP4BP were transformed individually into the E. coli strain BL21(DE3) to express and purify as GST fusion proteins using the procedure described previously (33). A single colony of the transformed strain was inoculated into 5 ml of LB containing ampicillin (100 g/ml) and grown overnight at 37°C with shaking at 250 rpm.…”

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confidence: 99%

“…In Vitro Ras GAP Assays-These were performed under first order kinetics as described previously (33 Ras Pull-down Assays-GST-Ras-binding domain was purified as previously described previously (27,30). Dishes (100 mm) of cells (8 ϫ 10 5 cells) were transiently transfected by lipofection (FuGENE 6, Roche Molecular Biochemicals) with 2.5 g of H-Ras DNA.…”

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confidence: 99%

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Analyzing the Role of the Putative Inositol 1,3,4,5-Tetrakisphosphate Receptor GAP1IP4BP in Intracellular Ca2+ Homeostasis

Walker¹,

Kupzig²,

Lockyer³

et al. 2002

Journal of Biological Chemistry

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Despite many studies on the possible role of inositol 1,3,4,5-tetrakisphosphate (IP 4 ) 1 in cellular physiology, its function remains unclear and indeed there is still some debate over whether it has a function at all (1, 2). Formed by direct phosphorylation of inositol 1,4,5-trisphosphate (IP 3 ), a reaction catalyzed by a family of Ca 2ϩ -regulated IP 3 3-kinases (1, 3), IP 4 has been linked to a potential role in the regulation of intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) following cellular stimulation with agonists that activate phosphoinositide-specific phospholipase C (4 -7). Evidence for this finding has come from a number of sources. For example, in endothelial cells, there is direct evidence (8) and, in neurons, there is direct (9) and indirect evidence (10) that IP 4 can activate Ca 2ϩ influx channels in the plasma membrane. Furthermore, one of the first effects of IP 4 to be reported highlighted an ability of this compound to synergize with IP 3 to mobilize Ca 2ϩ and regulate subsequent store-operated Ca 2ϩ influx (11-13). However, the marked sensitivity of this particular system to experimental protocols (11, 14 -16) has also raised a significant degree of controversy over the role of IP 4 in intracellular Ca 2ϩ homeostasis. In recent years, we have taken the view that if IP 4 does indeed constitute a novel second messenger, one criterion that must be fulfilled is the presence within cells of protein(s) that specifically bind IP 4 , i.e. an IP 4 receptor. To this end, we have described the purification (17, 18) and cloning (19) of a highly specific IP 4 -binding protein termed GAP1IP4BP . This protein, which functions as a GTPase-activating protein for members of the Ras-like family of small GTPases, at present constitutes the most promising candidate IP 4 receptor.The Ras-like family includes H-Ras, N-Ras, and K-Ras4A and 4B, the R-Ras proteins, the Ral proteins, and the Rap proteins 1A, 1B, 2A, and 2B (20 -22). These are ubiquitously expressed, evolutionarily conserved proteins that couple extracellular signals to various cellular responses (20 -22). All of these proteins have the inherent ability to undergo conformational changes in response to the alternate binding of GDP and GTP. The GDP-bound "off" state and the GTP-bound "on" state recognize distinct effector proteins, thereby allowing these proteins to function as two-state molecular "switches." Importantly, cycling between the two forms does not occur spontaneously. Activation requires guanine nucleotide exchange factors to induce the dissociation of GDP to allow association of the more abundant GTP, and deactivation requires GTPase-activating proteins (GAPs) to bind to the GTP-bound form to enhance the rate of intrinsic GTPase activity (20 -22). GAP1 IP4BP along with the related proteins GAP1 m , RASAL, and CAPRI (23-30) is composed of tandem N-terminal C 2 domains, a C-terminal pleckstrin homology (PH) domain adjacent to a Bruton's tyrosine kinase (Btk) motif, and a central catalytic Ras GAP-related domain. Associated with th...

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Section: Generation Of a Constitutively Active And A Potential Dominamentioning

confidence: 99%

“…As described under "Experimental Procedures," a mouse monoclonal antibody (GP-3) was raised against a GST fusion of a GAP1 IP4BP mutant lacking the N-terminal tandem C 2 domains (33).…”

Section: Isolation Of a Monoclonal Antibody Capable Of Detecting Endomentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Analyzing the Role of the Putative Inositol 1,3,4,5-Tetrakisphosphate Receptor GAP1IP4BP in Intracellular Ca2+ Homeostasis

Walker¹,

Kupzig²,

Lockyer³

et al. 2002

Journal of Biological Chemistry

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Synthesis of the Enantiomers of 6-Deoxy-myo-Inositol 1,3,4,5-Tetrakisphosphate, Structural Analogues ofmyo-Inositol 1,3,4,5-Tetrakisphosphate

Horne

Potter

2001

Chem. Eur. J.

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D-myo-Inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] is produced rapidly from the established second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P4] in stimulated cells. Despite extensive investigations, in particular concerning its potential role in mediating cellular Ca2+ influx, no exact cellular function has been described for this inositol phosphate; however, binding sites have been identified in a number of tissues and it has been shown to act synergistically with Ins(1,4,5)P3. To assist in the elucidation of the mechanism of action and structural requirements within the Ins(1,3,4,5)P4 moiety that are necessary for recognition and activation of the receptor, structural analogues of this tetrakisphosphate are required. Routes for the synthesis of racemic 6-deoxy-myo-inositol 1,3,4,5-tetrakisphosphate [6-deoxy-DL-Ins(1,3,4,5)P4] and the chiral antipodes D- and L-6-deoxy-myo-inositol 1,3,4,5-tetrakisphosphate are described here. The racemic tetrakisphosphate was synthesised from DL-1,2-O-isopropylidene-myo-inositol in eight steps. Deoxygenation at C-6 was achieved following the Barton-McCombie procedure. Both chiral tetrakisphosphates were synthesised through resolution of racemic cis-diol 6-deoxy-1,4,5-tri-O-p-methoxybenzyl-myo-inositol with the chiral auxiliary (S)-(+)-O-acetylmandelic acid. Absolute configuration was confirmed by synthesis of the known D-6-deoxy-myo-inositol. Both D-6-deoxy-Ins(1,3,4,5)P4 and its enantiomer will be useful tools to unravel the enigmatic role of Ins(1,3,4,5)P4 in the polyphosphoinositide pathway of signal transduction.

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Dual‐Specificity R as/ R ap GTPase Activating Proteins

Sot

2018

Encyclopedia of Life Sciences

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The small GTP‐binding proteins Ras and Rap regulate important cellular events switching between an activated GTP‐bound form and an inactivated GDP‐bound state. Guanine exchange factors (GEFs) control the GDP to GTP‐bound cycle, while the conversion, through hydrolysis, of GTP to GDP is catalysed by GTPase‐activating proteins (GAPs). The GAPs complete Ras and Rap catalytic site for efficient GTP hydrolysis. Although Ras and Rap are highly homologous, they possess different residues in the catalytic site. In consequence, Ras and Rap GTPase‐activating proteins (RasGAPs and RapGAPs) are structurally unrelated and use different mechanisms. Surprisingly, there are several RasGAPs with dual Ras/Rap specificity: SynGAP; GAP1 family members GAP1 IP4BP , Rasal and Capri; and Plexins. This characteristic was an intriguing issue for years, but today structural and biochemical studies have enlightened the overall general mechanism of these dual GAPs. Key Concepts Small GTP‐binding proteins Rap and Ras control different cellular signalling switching between inactive GDP and active GTP‐bound states. Ras and Rap GTPase‐activating proteins (RasGAPs and RapGAPs) inactivate their downstream signalling catalysing the GTP hydrolysis, using RasGAP and RapGAP domains. RasGAPs–Ras‐GTP interaction positions Ras Gln61, situated in switch II loop, for nucleophilic attack, while the GAP contributes an Arg (arginine finger) to neutralise unfavourable negative charges of the reaction intermediate. Rap has a Thr in position 61, and RapGAPs catalyse GTP hydrolysis, contributing an Asn (Asn thumb) for nucleophilic attack. There are five RasGAPs with dual Rap/Ras specificity: SynGAP, Rasal, GAP1 IP4BP , Capri and Plexin. Dual GAPs need extra‐GAP domains to act as RapGAPs. Dual GAPs promote a specific orientation of Rap switch II, locating Gln63 as the catalytic residue. Plexin‐Rap X‐ray structure showed that residues of GAP domain and an extra‐GAP motif (juxtamembrane segment) are responsible for Gln63 correct orientation. SynGAP, Rasal, GAP1 IP4BP , Plexin and Capri do not share the same residues, and thus they still need to be found.

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Structural and Functional Analysis of the Putative Inositol 1,3,4,5-Tetrakisphosphate Receptors GAP1IP4BPand GAP1m

Cited by 28 publications

References 0 publications

Analyzing the Role of the Putative Inositol 1,3,4,5-Tetrakisphosphate Receptor GAP1IP4BP in Intracellular Ca2+ Homeostasis

Analyzing the Role of the Putative Inositol 1,3,4,5-Tetrakisphosphate Receptor GAP1IP4BP in Intracellular Ca2+ Homeostasis

Synthesis of the Enantiomers of 6-Deoxy-myo-Inositol 1,3,4,5-Tetrakisphosphate, Structural Analogues ofmyo-Inositol 1,3,4,5-Tetrakisphosphate

Dual‐Specificity R as/ R ap GTPase Activating Proteins

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