Determination of Specificity of a High-Affinity Inositol 1,3,4,5-Tetrakisphosphate Binding Site at a 42 kDa Receptor Protein, p42IP4: Comparison of Affinities of All Inositoltris-, -Tetrakis-, and -Pentakisphosphate Regioisomers

Stricker, Rolf; Chang, Young Tae; Chung, SK; Reiser, Georg

doi:10.1006/bbrc.1996.1703

Cited by 20 publications

(15 citation statements)

References 31 publications

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“…1A Table 1). The inhibitory constant (K i ) for d/l-Ins(3,4,5,6)P 4 was virtually identical to the value found with the protein purified from pig cerebellum [27] whereas the affinity obtained with Ins(1,3,5,6)P 4 was approximately five times lower.…”

Section: Q Febs 1999supporting

confidence: 68%

“…Binding of Ins(1,3,4,5)P 4 to the recombinant protein was determined as described previously for the purified receptor protein from pig cerebellum [27]. In brief, purified recombinant protein (70±500 ng) was incubated for 20 min on ice in a total volume of 280 mL of 25 For the analysis of the binding data, K d values and the number of binding sites were estimated by using the radlig dataanalysis computer program (Biosoft) as described previously [28].…”

Section: Binding Assaysmentioning

confidence: 99%

“…Properties of the recombinant protein were compared to the data obtained in earlier studies in which protein purified from pig cerebellar membranes [27] or from rat brain was used [18]. The displacement of [ 3 H]Ins(1,3,4,5)P 4 by different inositoltrisphosphate, tetrakisphosphate and pentakisphosphate regioisomers was measured.…”

Section: Q Febs 1999mentioning

confidence: 99%

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Recombinant p42^IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P₄ receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P₃

Hanck

Stricker

Krishna

et al. 1999

European Journal of Biochemistry

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We have recently cloned the cDNA of p42 IP4 , a membrane-associated and cytosolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hu Èlser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. & Reiser, G. (1997) FEBS Lett. 405, 229±236.] p42 IP4 is a protein of 374 amino acids with M r of 42 kDa. The p42 IP4 protein has a zinc finger motif at its N-terminus, followed by two pleckstrin homology domains. To characterize further the biochemical and functional properties of p42 IP4 , it was expressed as a glutathione-S-transferase fusion protein in Sf9 cells using a recombinant baculovirus vector. The protein was affinity adsorbed on glutathione beads, cleaved from glutathione-S-transferase with the protease factor-Xa and purified on heparin agarose. The recombinant purified protein is active because it shows binding affinities similar to those of the native p42 IP4 , purified from pig cerebellum or rat brain (K i for inositol(1,3,4,5)P 4 of 4.1 nm and 2.2 nm, respectively). Moreover the ligand specificity of the recombinant protein for various inositol polyphosphates is similar to that of the native protein purified from brain. Importantly, we show here that p42 IP4 binds phosphatidylinositol(3,4,5)P 3 specifically, as the recombinant protein can associate with lipid membranes (vesicles) containing phosphatidylinositol(3,4,5)P 3 ; this binding occurs in a concentration-dependent manner and is blocked by inositol(1,3,4,5)P 4 . This specific association and the possibility that endogenous p42 IP4 can be converted from a membrane-associated state to a soluble state support the hypothesis that p42 IP4 might be redistributed between cellular compartments upon hormonal stimulation.Keywords: baculovirus; inositolP 4 ; membrane interaction; pleckstrin homology domain; phosphatidylinositolP 3 ; receptor; vesicle association.Two second messengers, inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol, are produced from phosphatidylinositol 4,5-bisphosphate [Ptd-Ins(4,5)P 2 ]. The generation of Ptd-Ins(4,5)P 2 and its cleavage by phospholipase C (PLC) are tightly regulated signal transduction pathways [1]. Ins(1,4,5)P 3 mobilizes Ca 2+ from intracellular stores whereas diacylglycerol activates protein kinase C [2]. During the last few years, the putative second messengers Ins(1,3,4,5)P 4 and Ptd-Ins(3,4,5)P 3 , which are generated by phosphorylating the D3 position of the inositol ring, have attracted increasing interest. The functions of Ins(1,3,4,5)P 4 and Ptd-Ins(3,4,5)P 3 are much less clear than that of Ins(1,4,5)P 3 [2]. Ptd-Ins(3,4,5)P 3 is the product of class I phosphoinositide kinases. Phosphoinositide-3-kinase (PI3-kinase) a is regulated by membrane-bound receptor tyrosine kinases [3] whereas PI3-kinase g is activated by heterotrimeric G-proteins [4]. Ins(1,4,5)P 3 can be phosphorylated to Ins(1,3,4,5)P 4 by Ins(1,4,5)P 3 -3-kinase [5].The physiological role of Ins(1,3,4,5)P 4 is still widely disputed [6]. Ptd-Ins(3,4,5)P 3 has been demonstrated to be an important regulator of me...

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Section: Q Febs 1999supporting

confidence: 68%

Section: Binding Assaysmentioning

confidence: 99%

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Recombinant p42^IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P₄ receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P₃

Hanck

Stricker

Krishna

et al. 1999

European Journal of Biochemistry

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“…The samples were incubated for 1 h on ice followed by centrifugation (50 000 g , 10 min, 4 °C, Beckmann Avanti 30 centrifuge) and the resulting supernatants were analyzed by SDS/PAGE (12.5% separation gel), transfer to poly(vinyl difluoride) membranes and Western blotting with an anti‐peptide antiserum directed against a 19 amino acid peptide comprising amino acids 353–371 of p42 IP4 [19]. SDS/PAGE, immunoblotting, other standard procedures and materials used were described previously [27–29]. For determining the time course of the release of p42 IP4 and the stability of Ins(1,3,4,5) P 4 in the presence of Mg 2+ , membranes were resuspended in buffer pH 7.5, as described above, with varying concentrations of Mg 2+ , and incubated either in the absence or presence of 10 µ m Ins(1,3,4,5) P 4 on ice.…”

Section: Methodsmentioning

confidence: 99%

Translocation between membranes and cytosol of p42^IP4, a specific inositol 1,3,4,5‐tetrakisphosphate/phosphatidylinositol 3,4,5‐trisphosphate‐receptor protein from brain, is induced by inositol 1,3,4,5‐tetrakisphosphate and regulated by a membrane‐associated 5‐phosphatase

Stricker

Adelt

Vogel

et al. 1999

European Journal of Biochemistry

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The highly conserved 42-kDa protein, p42IP4 was identified recently from porcine brain. It has also been identified similarly in bovine, rat and human brain as a protein with two pleckstrin homology domains that binds Ins(1,3,4,5)P 4 and PtdIns(3,4,5)P 3 with high affinity and selectivity. The brain-specific p42 IP4 occurs both as membrane-associated and cytosolic protein. Here, we investigate whether p42 IP4 can be translocated from membranes by ligand interaction. p42 IP4 is released from cerebellar membranes by incubation with Ins(1,3,4,5)P 4 . This dissociation is concentration-dependent (. 100 nm), occurs within a few minutes and and is ligand-specific. p42IP4 specifically associates with PtdIns(3,4,5)P 3 -containing lipid vesicles and can dissociate from these vesicles by addition of Ins(1,3,4,5)P 4 . p42 IP4 is only transiently translocated from the membranes as Ins(1,3,4,5)P 4 can be degraded by a membrane-associated 5-phosphatase to Ins(1,3,4)P 3 . Then, p42IP4 re-binds to the membranes from which it can be re-released by re-addition of Ins(1,3,4,5)P 4 . Thus, Ins(1,3,4,5)P 4 specifically induces the dissociation from membranes of a PtdIns(3,4,5)P 3 binding protein that can reversibly re-associate with the membranes. Quantitative analysis of the inositol phosphates in rat brain tissue revealed a concentration of Ins(1,3,4,5)P 4 comparable to that required for p42 IP4 translocation. Thus, in vivo p42 IP4 might interact with membranes in a ligand-controlled manner and be involved in physiological processes induced by the two second messengers Ins(1,3,4,5)P 4 and PtdIns(3,4,5)P 3 .Keywords: p42 IP4 ; pH domain; centaurin; inositol; 5-phosphatase.Phosphoinositides and inositol polyphosphates are central players in cellular development and intracellular signal transduction. A well characterized pathway in phosphoinositide signalling is the hydrolysis of PtdIns(4,5)P 2 by several distinct receptor-activated phospholipase C isoforms, yielding Ins(1,4,5)P 3 , which releases Ca 2+ from intracellular stores and diacylglycerol which activates protein kinase C [1]. The Ins(1,4,5)P 3 signal is terminated by specific enzymatic dephosphorylation of Ins(1,4,5)P 3 in position 5. Alternatively, Ins(1,4,5)P 3 is phosphorylated by a Ins(1,4,5)P 3 3-kinase to the putative second messenger Ins(1,3,4,5)P 4 . The function of Ins(1,3,4,5)P 4 (reviewed in [2]) is not yet clarified, but has been implicated in regulation of the intracellular Ca 2+ concentration ([3] and references therein). In endothelial cells, an Ins(1,3,4,5)P 4 -sensitive Ca 2+ channel has been demonstrated [4]. In hippocampal pyramidal neurons, Ins(1,3,4,5)P 4 was described to promote Ca 2+ accumulation after ischaemia which resulted in neuronal cell death [5]. We have recently demonstrated that Ins(1,3,4,5)P 4 injected into hippocampal CA1 neurons enhanced long-term potentiation by stimulating Ca 2+ entry either via activating N-type Ca 2+ channels or via enhancing Ins(1,4,5)P 3 -sensitive Ca 2+ release [6]. In order to clarify the physiological role of Ins(...

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“…Moreover, we recently found that the purified native receptor protein from porcine cerebellum recognizes not only InsP4 (G 2.2 nM) but also the water soluble analogue of phosphatidylinositol-(3,4,5)-trisphosphate (PtdInsP3) glycero-phosphatidylinositol-( 3,4,5)-trisphosphate with the same high affinity (Stricker et al, 1996). All other inositol polyphosphate regioisomers were significantly less potent.…”

Section: Introductionmentioning

confidence: 99%

Expression and Subcellular Localization of p42^IP4/ Centaurin‐α, a Brain‐specific, High‐affinity Receptor for lnositol 1,3,4,5‐Tetrakisphosphate and Phosphatidylinositol 3,4,5‐Trisphosphate in Rat Brain

Kreutz¹,

Böckers

Sabel³

et al. 1997

Eur J of Neuroscience

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Recently emerging evidence suggests important roles for inositol polyphosphates and inositol phospholipids in neuronal Ca2+ signalling, membrane vesicle trafficking and cytoskeletal rearrangement. A prerequisite for a detailed physiological characterization of the signalling of both potential second messengers inositol-(1,3,4,5)-tetrakisphosphate (InsP4) and phosphatidylinositol-3,4,5-trisphosphate (PtdInsP3) in the nervous system is the precise cellular localization of their receptors. Based on the cDNA sequence of a recently cloned brain-specific receptor with high affinity for both InsP4 and PtdInsP3 (InsP4-PtdInsP3R), p42IP4/centaurin-alpha, we localized the mRNA and the protein in rat brain. In situ hybridization revealed a widespread expression of the InsP4-PtdInsP3R with prominent labelling in cerebellum, hippocampus, cortex and thalamus, which moreover is developmentally regulated. Using peptide-specific antibodies, the immunoreactivity was localized in the adult brain in the vast majority of neuronal cell types and probably also in some glial cells. Prominent immunoreactivity was found in axonal processes and in cell types characterized by extensive neurites. In the hypothalamus a subpopulation of parvocellular neurons in the peri- and paraventricular nuclei was most heavily labelled. This was confined by strong immunoreactivity in the lamina externa of the median eminence in close proximity to portal plexus blood vessels. Electron microscopy revealed that the InsP4-PtdInsP3R was frequently associated with presynaptic vesicular structures. Further studies should identify the role of the InsP4-PtdInsP3R in cellular neural processes.

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Determination of Specificity of a High-Affinity Inositol 1,3,4,5-Tetrakisphosphate Binding Site at a 42 kDa Receptor Protein, p42IP4: Comparison of Affinities of All Inositoltris-, -Tetrakis-, and -Pentakisphosphate Regioisomers

Cited by 20 publications

References 31 publications

Recombinant p42^IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P₄ receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P₃

Recombinant p42^IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P₄ receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P₃

Translocation between membranes and cytosol of p42^IP4, a specific inositol 1,3,4,5‐tetrakisphosphate/phosphatidylinositol 3,4,5‐trisphosphate‐receptor protein from brain, is induced by inositol 1,3,4,5‐tetrakisphosphate and regulated by a membrane‐associated 5‐phosphatase

Expression and Subcellular Localization of p42^IP4/ Centaurin‐α, a Brain‐specific, High‐affinity Receptor for lnositol 1,3,4,5‐Tetrakisphosphate and Phosphatidylinositol 3,4,5‐Trisphosphate in Rat Brain

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Determination of Specificity of a High-Affinity Inositol 1,3,4,5-Tetrakisphosphate Binding Site at a 42 kDa Receptor Protein, p42IP4: Comparison of Affinities of All Inositoltris-, -Tetrakis-, and -Pentakisphosphate Regioisomers

Cited by 20 publications

References 31 publications

Recombinant p42IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P3

Recombinant p42IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P3

Translocation between membranes and cytosol of p42IP4, a specific inositol 1,3,4,5‐tetrakisphosphate/phosphatidylinositol 3,4,5‐trisphosphate‐receptor protein from brain, is induced by inositol 1,3,4,5‐tetrakisphosphate and regulated by a membrane‐associated 5‐phosphatase

Expression and Subcellular Localization of p42IP4/ Centaurin‐α, a Brain‐specific, High‐affinity Receptor for lnositol 1,3,4,5‐Tetrakisphosphate and Phosphatidylinositol 3,4,5‐Trisphosphate in Rat Brain

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Recombinant p42^IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P₄ receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P₃

Recombinant p42^IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P₄ receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P₃

Translocation between membranes and cytosol of p42^IP4, a specific inositol 1,3,4,5‐tetrakisphosphate/phosphatidylinositol 3,4,5‐trisphosphate‐receptor protein from brain, is induced by inositol 1,3,4,5‐tetrakisphosphate and regulated by a membrane‐associated 5‐phosphatase

Expression and Subcellular Localization of p42^IP4/ Centaurin‐α, a Brain‐specific, High‐affinity Receptor for lnositol 1,3,4,5‐Tetrakisphosphate and Phosphatidylinositol 3,4,5‐Trisphosphate in Rat Brain