COMMUNICATION
Functional Coupling of Phosphatidylinositol 4,5-Bisphosphate to Inositol 1,4,5-Trisphosphate Receptor*

Vitalie D. LupuDagger , Elena KaznacheyevaDagger §, U. Murali Krishna, J. Russell Falck, and Ilya BezprozvannyDagger parallel

From the Departments of Dagger  Physiology and  Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

The inositol 1,4,5-trisphosphate receptor (InsP3R) plays a key role in intracellular Ca2+ signaling. InsP3R is activated by InsP3 produced from phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C cleavage. Using planar lipid bilayer reconstitution technique, we demonstrate here that rat cerebellar InsP3R forms a stable inhibitory complex with endogenous PIP2. Disruption of InsP3R-PIP2 interaction by specific anti-PIP2 monoclonal antibody resulted in 3-4-fold increase in InsP3R activity and 10-fold shift in apparent affinity for InsP3. Exogenously added PIP2 blocks InsP3 binding to InsP3R and inhibits InsP3R activity. Similar results were obtained with a newly synthesized water soluble analog of PIP2, dioctanoyl-(4,5)PIP2, indicating that insertion of PIP2 into membrane is not required to exert its inhibitory effects on the InsP3R. We hypothesize that the functional link between InsP3R and PIP2 described in the present report provides a basis for a local, rapid, and efficient coupling between phospholipase C activation, PIP2 hydrolysis, and intracellular Ca2+ wave initiation in neuronal and non-neuronal cells.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

The inositol 1,4,5-trisphosphate receptor (InsP3R)1 plays a key role in intracellular Ca2+ signaling (1). InsP3R have been biochemically purified, cloned, and functional properties of these channels were extensively studied (1-4). Planar lipid bilayer reconstitution technique proved useful for studies of functional properties and modulation of InsP3R (4). Modulation of cerebellar InsP3R by cytosolic Ca2+ (5) and ATP (6) has been previously characterized by this method. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a major signaling role in cells (1). Hydrolysis of PIP2 by phospholipase C (PLC) leads to production of inositol 1,4,5-trisphosphate (InsP3) and Ca2+ release from intracellular stores via activation of InsP3R. PIP2 by itself can play a regulatory role in cells. Effects of PIP2 on structural organization of cellular cytoskeleton are well established (7). More recently direct functional interaction between skeletal muscle ryanodine receptor (8), Na+/Ca2+ exchanger (9), and inward rectifier K+ channels (10) has been demonstrated. The emerging paradigm is that PIP2 by itself may be a signaling messenger.

We set out to test the hypothesis that PIP2 may interact directly with the InsP3R. To investigate InsP3R-PIP2 interaction, we reconstituted rat cerebellar InsP3R into planar lipid bilayers and tested response of these channels to addition of specific monoclonal anti-PIP2 antibody (11), exogenous PIP2, and newly synthesized water-soluble PIP2 analog dioctanoyl-(4,5)PIP2. Based on our data, we concluded that cerebellar InsP3R forms a stable inhibitory complex with PIP2 that can be disrupted by anti-PIP2 antibody. Membrane insertion of PIP2 is not required for interaction with the InsP3R. We concluded that PIP2 most likely interacts with InsP3 binding site of the InsP3R. A novel model of compartmentalized Ca2+ signaling is proposed based on results obtained in the study.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Planar Lipid Bilayer Experiments-- Rat (Sprague-Dawley; 4-5 weeks old) cerebellar microsomes were isolated essentially as described previously for canine preparation (5, 6, 12) and stored frozen at -70 °C. Planar lipid bilayers were formed from PE:PS (3:1) synthetic lipid mixture in decane on the small (100-200 µm in diameter) hole in Teflon film separating two chambers 3 ml each (cis and trans). Prior to formation of the bilayer the hole was prepainted with PC:PS mixture (3:1). Rat cerebellar InsP3R were incorporated into the bilayer by microsomal vesicle fusion as described previously for canine preparation (5, 6, 12). Single channel currents were recorded at 0 mV transmembrane potential using 50 mM Ba2+ dissolved in HEPES (pH 7.35) in the trans (intraluminal) side as a charge carrier (12). In most of the experiments (standard recording conditions of InsP3R activity), the cis (cytosolic) chamber contained 110 mM Tris dissolved in HEPES (pH 7.35), 0.2 µM free Ca2+ (5) buffered with 1 mM EGTA and 0.7 mM CaCl2, and 1 mM Na2ATP (6). Unless indicated otherwise InsP3R were activated by addition of 2 µM InsP3 to the cis chamber. In some experiments 2 µM of ruthenium red was added to the cis chamber to block cerebellar RyanR present in the bilayer (5) and facilitate InsP3R activity (Po = 5.8 ± 0.7% (n = 4)). All PIP2 blocking and most of PIP2Ab activating experiments were performed in the presence of 0.1 mM NaF in the cis chamber to chelate contaminating traces of heavy metal ions. Addition of NaF had no effect on InsP3R activity (n = 20). All additions were to the cis chamber from the concentrated stocks with at least 30 s stirring of solutions in both chambers.

Monoclonal PIP2Ab (11) was reconstituted in phosphate-buffered saline (titer 1:1500) and added either to the cis chamber with stirring (30 µl) or directly to the bilayer without stirring (6 µl). In both cases similar effects on InsP3R activity were observed. Initial PIP2 stocks were made in chloroform or chloroform/methanol (19:1). An aliquot of the stock solution was transferred into a siliconized glass tube, and organic solvent was evaporated under a stream of argon. Nanopure water was added to the dried lipids, and PIP2 vesicles were prepared by vortexing and sonication. Only freshly made or 1-day-old vesicles were used for all experiments. A sample from each vesicle stock was taken to determine lipid concentration from the total phosphate content using spectrophotometric phosphorus assay. PIP2 stock concentrations (0.2-1 mM) determined by phosphorus assay were employed for data analysis. Hydrophilic ShPIP2 was solubilized in water to yield 0.2 mM stock as determined by the phosphorus assay.

Single Channel Data Analysis-- InsP3R single channel currents were amplified (Warner OC-725), filtered at 1 kHz by a low pass 8-pole Bessel filter, digitized at 5 kHz (Digidata 1200, Axon Instruments), and stored on a computer hard drive and recordable optical disks. For off-line computer analysis (pClamp 6.0.3, Axon Instruments) single channel data were filtered digitally at 500 Hz. Single channel open probability (Po) was determined by using half-threshold crossing criteria (t >=  2 ms) from records lasting at least 2 min. In most of the experiments (55 out of 70) multiple InsP3R were incorporated into the bilayer (probably due to InsP3R clustering). Po in multichannel experiments was calculated under the assumption that all active channels in the bilayer were identical and independent and using the binomial distribution: P1 = Np(1 - p)- 1, where P1 is the experimentally determined probability of the first open level, N is the number of channels in the bilayer, and p = Po. N in each experiment was estimated from the maximal number of simultaneously open channels.

[3H]InsP3 Binding Assay-- Specific [3H]InsP3 binding to rat cerebellar microsomes was measured with minor modifications of procedure described previously (13). Briefly, microsomes (5 µg of protein) were incubated on ice with 10 nM [3H]InsP3 in the binding buffer (50 mM Tris-HCl (pH 9.0), 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl) and precipitated with 12.5% polyethylene glycol and 1.2 mg/ml gamma -globulin at 14,000 × g. Precipitates were quickly washed with the binding buffer, dissolved in Soluene, and their [3H] content was determined by liquid scintillation counting. Nonspecific counts, determined in the presence of 25 µM nonlabeled InsP3, were subtracted from the total to yield specific binding. Various amounts of PIP2 vesicles, prepared as described above for bilayer experiments, were added to the binding buffer in [3H]InsP3 competition experiments.

Materials-- Monoclonal anti-PIP2 Antibody (PIP2Ab) (11) was from PerSeptive Biosystems. ShPIP2 was synthesized by following procedure similar to that previously described for (3,4)PIP2 water-soluble PIP2 analog (14). ShPIP2 was >95% pure based on NMR and TLC analysis. (1,4,5)InsP3 was from LC Laboratories. [3H]InsP3 was from Amersham Pharmacia Biotech. To form planar lipid bilayers dioleoyl-PS, dioleoyl-PE, and diphytanoyl-PC from Avanti Polar Lipids were used. Stearoyl-arachidonyl-(4,5)PIP2 (bovine brain) was from Boehringer Mannenheim. Dipalmitoyl-(4,5)PIP2 and dipalmitoyl-(3,4)PIP2 were from Matreya Inc. Stearoyl-arachidonyl-PC was from Avanti Polar Lipids. Ruthenium red and Na2ATP were from Calbiochem. Other chemicals were from Sigma or Intermountain Scientific Corp.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Anti-PIP2 Antibody Activates Cerebellar InsP3R-- Single-channel open probability (Po) of rat cerebellar InsP3R reconstituted in planar lipid bilayer is typically low (<5%), and channel openings are infrequent (Fig. 1A). We found that InsP3R activity was increased 3-4-fold by addition of monoclonal anti-PIP2 antibody (PIP2Ab) (11) to the cis (cytosolic) side of the membrane. On average, PIP2Ab increased Po of InsP3R from 3.5 ± 0.5% (n = 18) to 13.6 ± 1.7% (n = 14) (Fig. 1). In some experiments, PIP2Ab induced periods of InsP3R activity with Po of 30-40% Po. Addition of boiled PIP2Ab or irrelevant mouse IgG fraction had no effect on InsP3R in control experiments. InsP3R activated by PIP2Ab could be inhibited by addition of exogenous PIP2 vesicles (n = 3). We concluded that cerebellar InsP3R may form a stable inhibitory complex with PIP2 in vivo. During microsomal extraction and bilayer reconstitution procedure, preexisting InsP3R-PIP2 complex remains intact until it is disrupted by addition of PIP2Ab to the bilayer.


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Fig. 1.   PIP2Ab activate InsP3R. A, addition of 2 µM InsP3 induces openings of a single cerebellar InsP3R incorporated into planar lipid bilayer in standard recording conditions (0.2 µM Ca2+, 1 mM ATP). Addition of PIP2Ab into cis chamber facilitated InsP3R channel activity. Current records before and after addition of PIP2Ab are shown in compressed (50 s/trace) and expanded (2 s/trace) time scales. Demonstrated result is representative of at least 30 experiments with five different microsomal preparations. B, corresponding single channel open probability (Po) plot. Po was averaged over 5-s bin intervals and plotted against time in the experiment. Presence of InsP3 and PIP2Ab in the cytosolic chamber is indicated by bar diagrams.

Anti-PIP2 Antibody Increase an Apparent Affinity of InsP3R for InsP3-- To get insight into the mechanism of PIP2Ab action, we characterized the effect of PIP2Ab on InsP3R sensitivity to activation by InsP3. Fit of cerebellar InsP3R dose dependence (Fig. 2A, filled circles, n = 5) yielded apparent affinity (Kapp) of 1.5 µM InsP3, maximal Po (Pmax) of 6.6% and Hill coefficient nH of 1.4. In contrast, fit of the data obtained in the presence of PIP2Ab (Fig. 2B, open circles, n = 3) resulted in Kapp of 0.13 µM, Pmax of 13.4%, and nH of 2.0. Thus, on average PIP2Ab induced over 10-fold change in InsP3R apparent affinity and 2-fold change in Pmax.


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Fig. 2.   Apparent affinity of InsP3R is increased by PIP2Ab. A, plot of Po at different InsP3 concentrations in standard recording conditions (filled circles, n = 5; at 20 and 40 µM InsP3; n = 2) and in the presence of PIP2Ab (open circles, n = 4; at 50 nM, 20 µM, and 40 µM; n = 1). At each InsP3 concentration Po was calculated from current records at least 3 min long. Both sets of data were fit with the equation Po = Pmax [InsP3]nH/([InsP3]nH + KappnH). The values of parameters resulting in the best fit (smooth curves) are in the text. More thorough analysis of InsP3R dose response in the presence of PIP2Ab was prevented by significant destabilization of the bilayer by addition of antibody in most of the experiments. B, PIP2Ab-induced shift of InsP3R sensitivity to InsP3 observed in the same experiment. Planar lipid bilayer in this experiment was unusually resistant to destabilizing influence of PIP2Ab. Po is averaged over 5-s bin intervals and plotted versus time in the experiment. Presence of InsP3 (in concentrations as indicated) and PIP2Ab in the cis chamber is shown by bar diagrams. Following initial additions of 2 µM InsP3 and PIP2Ab, the cis chamber was perfused by the fresh recording buffer containing 0.2 µM Ca2+ and 1 mM ATP (standard recording conditions), and cis InsP3 concentration was raised from 100 nM to 2 µM in increment steps. Second addition of PIP2Ab was performed at the end of the experiment.

A similar shift in sensitivity to InsP3 induced by PIP2Ab can be observed for the same InsP3R (Fig. 2B). In this experiment 2 µM InsP3 initially activated channels to 3-5% Po and then PIP2Ab further increased InsP3R activity to 15% Po level. Following perfusion of the cis chamber InsP3R activity subsided. Addition of 100 nM InsP3 to the cis chamber induced InsP3R activity at 15% Po that could not be further elevated by increase in InsP3 concentration or addition of PIP2Ab (Fig. 2B). The shift in InsP3 sensitivity of the InsP3R induced by PIP2Ab led us to hypothesize that endogenous PIP2 may interact directly with the InsP3 binding site of the InsP3R.

PIP2 Inhibits InsP3 Binding to InsP3R-- Interaction between PIP2 and the InsP3 binding site of InsP3R was tested in competitive [3H]InsP3 binding assay performed with cerebellar microsomes and exogenously added (4,5)PIP2 vesicles (Fig. 3A). We found that stearoyl-arachidonyl-PIP2 (SA-PIP2, IC50 of 3.2 µM, filled circles) and dipalmitoyl-PIP2 (DP-PIP2, IC50 of 1.3 µM, filled triangles) inhibited [3H]InsP3 binding with IC50 in micromolar range, whereas SA-PC did not have an effect in concentrations as high as 100 µM (open diamonds). In another control, addition of PS in concentrations up to 100 µM had only a minor effect on InsP3 binding (closed diamonds), whereas PIP2:PS mixed vesicles inhibited binding proportionately to molar PIP2 content (open triangles). Thus, the inhibitory effect on InsP3 binding was specific for PIP2. Does PIP2 have to be inserted into the membrane to inhibit InsP3 binding? To answer this question, we performed experiments with the novel water-soluble synthetic PIP2 analog ShPIP2. ShPIP2 inhibited [3H]InsP3 binding to cerebellar microsomes with IC50 of 1.9 µM (Fig. 3A, IC50 of 1.9 µM, open circles), similar to lipophilic SA-PIP2 and DP-PIP2. Therefore, insertion of PIP2 into the membrane is not required to block InsP3 binding.


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Fig. 3.   Exogenous PIP2 inhibits InsP3R. A, schematic drawing of chemical structures of stearoyl-arachidonyl-(4,5)PIP2 (SA-PIP2), dipalmitoyl-(4,5)PIP2 (DP-PIP2), and water-soluble D-myo-dioctanoyl-(4,5)PIP2 (ShPIP2). Added PIP2 blocks specific [3H]InsP3 binding to cerebellar microsomes. Specific [3H]InsP3 binding at each PIP2 concentration was normalized to binding obtained in the absence of PIP2 or at 0.01 µM PIP2 in the same series of experiments. Smooth curves were generated by fitting normalized data with the equation B = knH/([PIP2]nH + knH) that yielded IC50 (k) and Hill coefficient (nH) for each PIP2 analog tested: SA-PIP2 (n = 4, filled circles, IC50 = 3.2 µM, nH = 2.1); DP-PIP2 (n = 1, closed triangles, IC50 = 1.3 µM, nH = 1.9); ShPIP2 (n = 2, open circles, IC50 = 1.9 µM, nH = 1.3). PIP2 concentration in PIP2:PS mixture is used to plot the corresponding set of data (open triangles, n = 1). Control experiments were performed with 20 and 100 µM concentrations of stearoyl-arachidonyl-PC (open diamonds, n = 1) and dioleoyl-PS (closed diamonds, n = 2). B, PIP2 blocks InsP3R in planar lipid bilayer reconstitution experiments. InsP3R were initially activated by 2 µM InsP3 in standard recording conditions and then SA-PIP2 concentration in the cis chamber was elevated in increment steps until complete channel block. Po in the experiment is averaged over 5-s bin intervals and plotted versus time. Concentrations of InsP3 and PIP2 in the cis chamber are indicated by bar diagrams. InsP3 concentration was raised to 20 µM at the end of the experiment, which in come cases resulted in partial recovery of channel activity. C, block of InsP3R in planar lipid bilayer experiments by water-soluble ShPIP2. The experiment was performed and analyzed as described for SA-PIP2 (B).

PIP2 Inhibits InsP3R in Planar Lipid Bilayers-- Exogenous PIP2 inhibited InsP3R activated by 2 µM InsP3 in planar lipid bilayer reconstitution experiments (Fig. 3, B and C). SA-PIP2 and DP-PIP2 inhibited InsP3R in micromolar concentration range (Fig. 3B), whereas PC and PS vesicles did not block channels in concentrations as high as 100 µM. After complete channel block by PIP2, we increased InsP3 concentration to 20 µM, which resulted in partial recovery of InsP3R activity (Fig. 3B). Block of InsP3R by PIP2 also could be reversed by application of PIP2Ab (n = 4). ShPIP2 exerted a similar inhibitory effect on InsP3R (Fig. 3C), indicating that insertion of PIP2 into the membrane is not required to block InsP3R.

Topology of InsP3R-PIP2 Interaction-- Topology of InsP3R-PIP2 interaction appear to be unique when compared with all reported cases of functional effects of PIP2 on channels and transporters. Indeed, PIP2 and effector protein have to be located in the same membrane for effects on Na+/Ca2+ exchanger (9), inward rectifier K+ channels (10), and ryanodine receptor (data not shown). In contrast, our experiments with water-soluble PIP2 analog dioctanoyl-(4,5)PIP2 indicated that PIP2 insertion into the membrane is not required to block InsP3R. If PIP2 is not in the membrane, how can PIP2 interact with the InsP3R?

Several lines of evidence point to InsP3 binding site of the InsP3R as likely site of interaction with PIP2: 1) extraction of endogenous PIP2 from the complex with the InsP3R by anti-PIP2 antibody resulted in 10-fold increase in the apparent affinity of InsP3R for InsP3 (Fig. 2). 2) In micromolar concentration range exogenous PIP2 inhibits specific InsP3 binding to cerebellar microsomes and blocks InsP3R in planar lipid bilayers (Fig. 3, A and B). 3) Water-soluble dioctanoyl-(4,5)PIP2 analog exerts similar effects on InsP3 binding and InsP3R channel activity (Fig. 3, A and C).

Quantitatively similar competition between InsP3 and PIP2 for a binding site has been reported for the pleckstrin homology (PH) domain of PLCdelta 1 (15-17). Thus, by analogy with PLCdelta 1-PH domain and based on our data, we hypothesize that InsP3 binding domain of InsP3R is also responsible for PIP2 binding.

N-terminal InsP3 binding domain of InsP3R is on the opposite end of the molecule from the C-terminal transmembrane domain (18). Thus, we postulate that in vivo PIP2 interacts with the InsP3R in trans configuration, that is PIP2 and InsP3R are located in juxtaposed membrane leaflets. Immunolocalization data support the existence of trans InsP3R-PIP2 complexes in cells. InsP3R are highly concentrated in compact endoplasmic reticulum cisternal stacks and peripheral cisternal membrane structures in Purkinje cerebellum cells (19-21) and in vas deferens smooth muscle cells (22). These membrane structures are sufficiently compact to allow formation of the complex between InsP3R from one endoplasmic reticulum membrane leaflet and PIP2 in the juxtaposed membrane. InsP3R-PIP2 complex at the plasma membrane may form in caveolae (23), which are highly enriched in both PIP2 (24) and InsP3R (25, 26). This hypothesis agrees well with the emerging role of caveolae as a specialized messenger center of the cell (27, 28).

InsP3R-PIP2 Signal Transduction Model-- Based on our in vitro functional data and discussed above in vivo morphological findings, we would like to postulate a novel compartmentalized coupling model in which InsP3R is linked directly to PIP2 (Fig. 4). We propose that in unstimulated cells, some fraction of InsP3R is constitutively inhibited due to interaction with PIP2 in the juxtaposed membrane (resting state). We propose that these InsP3R are unable to open because of the spatial constraint imposed by the topology of InsP3R-PIP2 interaction. Agonist stimulation leads to activation of PLC, cleavage of InsP3R-tethered PIP2, and release of the spatial clamp on the InsP3R (signal transduction step). PLC simultaneously removes the inhibitor (PIP2) and generates the activator (InsP3) of the InsP3R, leading to Ca2+ wave initiation. We propose that this novel compartmentalized signaling mechanism is responsible for Ca2+ wave initiation in specialized trigger zones, whereas Ca2+ wave propagation through the cell is sustained by direct Ca2+ feedback on the InsP3R (5, 29, 30). Preferential coupling between PLC-linked hormonal receptors and InsP3R have been demonstrated previously in some intact cell preparations (31-33). It appears that integrity of this preferential coupling depends on correct spatial arrangement between intracellular Ca2+ release stores and PLC-linked receptors, which is maintained by intact actin cytoskeleton network (34). Our direct coupling model (Fig. 4) is in agreement with the phenomenon of compartmentalized Ca2+ signaling reported in these papers.


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Fig. 4.   Model of InsP3R-PIP2 functional coupling. In unstimulated cells InsP3R are constitutively inhibited due to interaction with PIP2 in the juxtaposed membrane (resting state). Hormonal stimulation leads to activation of PLC, which cleaves InsP3R-tethered PIP2, simultaneously removing inhibitor (PIP2) and generating activator (InsP3) of the InsP3R (signal transduction step). PIP2 cleavage and InsP3 liberation results in immediate InsP3R opening and Ca2+ release (Ca2+ wave initiation).

    ACKNOWLEDGEMENTS

We are grateful to D. W. Hilgemann for continuous encouragement to work on this project and freely sharing his expertise and reagents. We thank D. W. Hilgemann, C.-L. Huang, S. Muallem, E. Ross, J. Stull, T. C. Südhof, and H. Yin for insightful discussions, reagents, and comments on the manuscript. I. B. is thankful to S. Bezprozvannaya for tremendous support and encouragement of his work.

    FOOTNOTES

* This work was supported by the American Heart Association, the Robert A. Welch Foundation, and start-up funds from University of Texas Southwestern Medical Center (to I. B.) and National Institutes of Health (to J. R. F).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ On leave from the Institute of Cytology Russian Academy of Sciences.

parallel To whom correspondence should be addressed: Dept. of Physiology, K4.112, University of Texas Southwestern Medical Center, Dallas, TX 75235-9040. Tel.: 214-648-6737; Fax: 214-648-8685; E-mail: bezprozv{at}utsw.swmed.edu.

1 The abbreviations used are: InsP3R, inositol 1,4,5-trisphosphate receptor(s); PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; ShPIP2, D-myo-dioctanoyl-(4,5)PIP2; PIP2Ab, monoclonal anti-PIP2 antibody; SA-PIP2, stearoyl-arachidonyl-PIP2; DP-PIP2, dipalmitoyl-(4,5)PIP2; PH, pleckstrin homology.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

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