Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Phosphatidylinositol 4,5-bisphosphate
[PtdIns(4,5)P2], a minor phospholipid component of the plasma membrane, is
a key regulator of several cellular processes, and has
become the focus of research on intracellular signal
trans-duction. PtdIns(4,5)P2 is a precursor of important second
messengers, such as the diffusible
InsP3, which regulates Ca2+ release from intracellular
Ca2+ stores, and the protein kinase C activator,
diacylglycerol[1,2].
PtdIns(4,5)P2 is also phosphorylated by class I PtdIns 3-kinases to form
PtdIns(3,4,5)P3, which controls membrane recruitment and
the functions of several important signaling
proteins[3]. PtdIns(4,5)P2 itself is a regulator of a great variety of target
molecules, including ion
channels[4,5] and several proteins that regulate actin polymerization and the
cytoskeleton[6], providing a link between the plasma membrane and the cortical
cytoskeleton[7].
PtdIns(4,5)P2 has also been implicated in
several forms of membrane remodeling events, including the
fusion of secretory vesicles with the plasma
membrane[8], clathrin-mediated
endocytosis[9], and membrane recovery by
endocytosis during neurotransmitter
release[10]. Such diverse functions rely upon interaction of the lipid with a large
number of regulator molecules.
Pleckstrin homology (PH) domains have been described
in a large number of signaling proteins, and they show
remarkable specificity in recognizing various forms of
inositides[11]. The PH domain of phospholipase
Cd1 (PLCd1PH) binds with high affinity and selectivity to
PtdIns(4,5)P2[12]. Recently, a fusion construct of
PLCd1PH with enhanced green fluorescent protein (GFP)
(PLCd1PH-GFP) was developed as a probe to visualize
PtdIns(4,5)P2 in single cells because it
binds to PtdIns(4,5)P2 within the plasma and translocates to
the cytoplasm after receptor stimulation. Subsequently,
when PtdIns(4,5)P2 is resynthesized, fluorescence returns to
the membrane[13].
It has been demonstrated that neomycin, an
aminogly-coside antibiotic with a large positive charge (about +4.5),
binds with high affinity to
PtdIns(4,5)P2[14]. Later studies
also showed that neomycin bound to and neutralized the
negative charge of
PtdIns(4,5)P2[15].
Phospholipase C (PLC)-induced PtdIns(4,5)P2
hydrolysis is an important cell signaling mechanism. Many
membrane receptors couple to PLC, and thus regulate
PtdIns(4,5)P2 turnover and subsequent downstream cell
signaling[16]. A few PLC modulators have been developed and they play an
important role in understanding the cell signaling process
involving PLC and PtdIns(4,5)P2. Neomycin has long been
used as a blocker of PLC, although it actually binds to
PtdIns(4,5)P2 and presumably prevents
PtdIns(4,5)P2 from hydrolysis by
PLC[17]. Previous studies have demonstrated
that both PLCd1PH and neomycin bind
PtdIns(4,5)P2 through an electrostatic
interaction[12,14]. This similar nature of
interaction would indicate a similar consequence for
PtdIns(4,5)P2 hydrolysis by PLC. However, in the present study, we
demonstrate that although both PLCd1PH and neomycin bind
to PtdIns(4,5)P2, only neomycin blocks
PtdIns(4,5)P2 hydrolysis by PLC activation.
Materials and methods
Reagents and plasmids Acetylcholine (ACh),
bradykinin (BK), wortmannin, neomycin and Fluo 3-AM, the
calcium indicators, were purchased from Sigma-Aldrich (St
Louis, MO, USA). ACh, BK and neomycin were dissolved in
distilled water. U73122 was purchased from Calbiochem (San
Diego, CA, USA). U73122 and wortmannin were prepared as
stock solutions in dimethylsulfoxide
(Me2SO), with a final concentration of
Me2SO of 0.1%. Fetal bovine serum (FBS)
and Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) were
products of Hyclone (Logan, UT, USA). COS-7 was obtained
from the Institute of Biochemistry and Cell Biology, Chinese
Academy of Sciences. cDNA from the type 1 muscarinic
(M1) receptor (M1R) and bradykinin 2 receptor
(BK2R), pEGFP-N1(GFP) and the
PLCd1PH construct with GFP
(PLCd1PH-GFP) were gifts from Prof DE LOGOTHETIS (Mount Sinai Medical
School, NY, USA). Red fluorescent protein
(pDsRed-Express-C1, pDsRed) was purchased from Clontech (Mountain View,
CA, USA). All other chemicals were of high performance
liquid chromatography or analytical grade.
Cell culture and transfection COS-7 cells were seeded
in 24-well plates on 12-mm glass coverslips, and cultured in
0.3 mL of DMEM supplemented with 10%
(v/v) FBS, 100 μg/mL streptomycin, and 100 U/mL of penicillin at 5%
CO2 and 37 °C. When they were 60%-70% confluent, the cells
were transiently transfected with DNA constructs for 8 h
using calcium phosphate precipitate, with 1 μg of DNA and
equal proportions for each kind of plasmid per well.
Following transfection, cells were incubated in 10% FBS DMEM
for 12-48 h. For fluorescence detection, cells were washed
twice with a modified Krebs-Ringer buffer containing (in
mmol/L): 120 NaCl, 4.7 KCl, 0.7 MgSO4, 1.2
CaCl2, 10 glucose, with 10
N-2-hydroxyethylpiperazine-N¡¯-2-ethanesulfonic acid
(HEPES) added (pH 7.4). The coverslips were placed into a
flow-through chamber and mounted on an inverted microscope.
Confocal microscopy and image analysis For confocal
imaging, a Leica (Wetzlar, Germany) DM-IRBE inverted mi
croscope with a 20×objective (numerical aperture 0.7) and
fitted with a TCS-SP2 scanhead was used. Excitation of
PLCd1PH-GFP and Fluo 3-AM was achieved with a 488 nm
argon ion laserline, and emissions were collected at 500-565
nm. pDsRed fluorescence was visualized with excitation at
543 nm and a 570-600 nm emission filter. For translocation
studies, a series of confocal images were taken at 3-10 s
intervals and stored on disk. Determination of the ratio of
membrane to cytosolic fluorescence was carried out by
assigning regions of interest for membrane and cytosol.
TCS-SP2 confocal software (Leica) was used to analyze data off-line.
Measurement of intracellular Ca2+
([Ca2+]i) of single cells
Cells were seeded onto sterile 12-mm borosilicate coverslips
in 35-mm Petri dishes, and incubated with 5
mmol/L fluo 3-AM at 37 °C for 45 min. After loading, cells were washed
twice and maintained in modified Krebs-Ringer buffer until
assay. [Ca2+]i changes were represented by relative
fluorescence intensity calculated by using the equation
DF/F0, where DF and
F0 are the change in fluorescence intensity
before and after treatment, and the initial fluorescence
intensity, respectively[18,19].
Statistics Data were analyzed by using the Chi-square
test. P<0.05 was considered to be a statistically significant
difference. All data shown are the mean value of at least 5
experiments and are expressed as mean±SD.
Results
Activation of M1R and BK2R induced
PtdIns(4,5)P2 hydrolysis and a reversible translocation of
PLCd1PH-GFP Both M1R, and PLCd1PH-GFP or both
BK2 and PLCd1PH-GFP were expressed in COS-7 cells. To follow the localization of
PLCd1PH-GFP within intact cells, we used GFP as a control.
In unstimulated cells, expressed GFP was found to be
cytosolic and also present in the nucleus (data not shown).
PLCd1PH-GFP, on the other hand, accumulated strongly at
the plasma membrane and had a low and homogenous
distribution in the cytosol (Figure 1A, 1B, left panel), consistent
with the hypothesis that the large pool of
PtdIns(4,5)P2 exists in the plasma
membrane[13]. Next we examined the effects of ACh and BK, acting through their respective G
protein-linked receptors, and subsequent activation of
phospholipase Cb and hydrolysis of
PtdIns(4,5)P2, on the fluorescence distribution of the GFP and
PLCd1PH-GFP. COS-7 cells were transfected with the GFP or
PLC1PH-GFP together with the cDNA encoding the
M1R or BK2R. After stimulation with
either ACh (5 μmol/L) or BK (0.1
μmol/L), there was a decrease of
PLCd1PH-GFP fluorescence in the plasma membrane
and a concomitant increase in cytosolic fluorescence (Figure 1A, 1B, Table 1). The kinetics of ACh- or BK-induced
PLCd1PH-GFP fluorescence translocation were characterized
by a rapid onset, with translocation peaking at approximately
30-60 s and returning to baseline approximately 5-8 min
after washout (Figure 1C). No significant change in
fluorescence were seen in cells transfected with GFP only (data not
shown). To exclude the effects of the laser, we used
modified Krebs-Ringer buffer solution as a solvent control of
ACh. As shown in Table 1, there was no change in the
relative fluorescence ratios
(Fm/Fc) in the solvent control
group during perfusion. To examine whether ACh- or
BK-induced translocation of PLCd1PH-GFP is due to the
hydrolysis of PtdIns(4,5)P2, we utilized 2 different
PtdIns(4,5)P2 resynthesis and hydrolysis blockers: wortmannin and
U73122. Wortmannin is known to be able to block the PtdIns
3-kinase at low concentrations and block the PtdIns 4-kinase,
so therefore block the formation of
PtdIns(4,5)P2 from phosphatidylinositol (PI), at high
concentrations[20]. As shown in Figure 2, in COS-7 cells expressing
PLCd1PH-GFP and the M1R, after the cell was pre-incubated with wortmannin
(at 10 μmol/L, a concentration known to block PtdIns 4-
kinase) for 20 min, ACh induced a similar translocation of
fluorescence from the plasma membrane to the cytosol, which
lasted for more than 10 min after washout of ACh (Figure
2A). When the cells were pre-incubated with U73122 (10
μmol/L), a relatively specific PLC inhibitor, for 5 min, ACh
failed to induce transient translocation of the fluorescence
signal (Figure 2B). These data strongly suggest that
PLCd1PH-GFP translocation induced by membrane receptor
activation is indeed due to
PtdIns(4,5)P2 hydrolysis.
Expression of PLCd1PH-GFP inhibited the effects of
neomycin on PtdIns(4,5)P2 hydrolysis
Neomycin binds PtdIns(4,5)P2 with high affinity and has often been used as
an inhibitor of PLC. The blocking effect of neomycin on PLC
is believed to be indirect, the result of neomycin binding to
PtdIns(4,5)P2, the substrate of
PLC[15]. As we shown earlier,
PLCd1PH bound to
PtdIns(4,5)P2 but did not block
receptor-mediated PLC activation, or
PtdIns(4,5)P2 hydrolysis. Because both
PLCd1PH and neomycin bind
PtdIns(4,5)P2 in a similar way (electrostatic interaction, see Introduction), we
thought this difference between PLCd1PH and neomycin was
interesting, and worthy of further investigation. To
determine whether the binding of PLCd1PH-GFP to
PtdIns(4,5)P2 can disrupt the effects of neomycin on
PtdIns(4,5)P2 hydrolysis, COS-7 cells expressing
PLCd1PH-GFP and M1R were stimulated with ACh in the absence or presence of
neomycin. Preincubation of the cells with neomycin (5
mmol/L) for 40 min failed to prevent the release of the fluorescence
signal from the membrane to the cytosol upon the applica
tion of ACh (Figure 3). Thus in the presence of
PLCd1PH, neomycin could not block hydrolysis of
PtdIns(4,5)P2 induced by PLC.
Effects of neomycin on PLC activation in the absence of
PLCd1PH-GFP To further confirm that binding of
PLCd1PH-GFP to PtdIns(4,5)P2
excludes the binding of neomycin to
PtdIns(4,5)P2, thus blocking neomycin¡¯s inhibitory effects
on PLC, we used [Ca2+]i as an indicator to reveal the effects
of neomycin on PLC in the absence of
PLCd1PH-GFP. One of the downstream products of
PtdIns(4,5)P2 hydrolyzed by PLC is
IP3, which acts to release intracellular
Ca2+[1,2]. Thus [Ca2+]i would serve as a good indicator of PLC activation
upon membrane receptor (M1R) stimulation. ACh induced a
significant increase in [Ca2+]i in COS-7 cells expressing the
M1R alone and pretreated with modified Krebs-Ringer buffer
solution for 40 min (Figure 4A, 4B); pDsRed was
co-transfected with M1R as a transfection tag (Figure 4A). However,
when cells were pretreated with 5 mmol/L neomycin (40 min),
no change was seen upon application of ACh (Figure 4A,
4B). Similar results were seen with BK as activator of PLC in
cells expressing B2R (data not shown). Thus, in the absence
of PLCd1PH, neomycin was able to block activation of PLC.
To further confirm these findings, we next examined
whether neomycin could also exert its inhibitory effects on
[Ca2+]i in the presence of
PLCd1PH-GFP. In this section of the study, we imaged the whole-cell fluorescence intensity
changes. GFP and Fluo 3-AM were excited and imaged at
the same wavelength. However, as shown in Figure 1C, the
total GFP signal from one cell did not change during
translocation, thus we were able to see an additional
fluorescence signal from Fluo 3-AM
(Ca2+) when Ca2+ was released
from the store by IP3. Figure 5 shows COS-7 cells
transfected with PLCd1PH-GFP and the
M1R. Three types of cells, presumably representing different transfection results, can
be identified. Cells designated a and b (Figure 5A) represent
those cells that had been transfected with both
PLCd1PH-GFP and the M1R, giving a clear and dominant localization of
the GFP signal on the cell membrane (Figure 5A), which
translocated into the cytosol upon application of ACh (Figure
5A); cell c represents cells that had only been transfected
with M1R, with no visible localization of the
PLCd1PH-GFP signal on the cell membrane, and a clear rising in
[Ca2+]i signal seen upon application of ACh; cell d represents cells that
had been transfected with PLCd1PH-GFP but not the
M1R, so that the clear PLCd1PH-GFP signal was not released from the
membrane, and neither could an increase in
[Ca2+]i signal be seen upon application of ACh (Figure 5A). When these
cells were pretreated with neomycin, only the response of
cell c to ACh was blocked, whereas the responses of cells a
and b were unaffected. These results strongly suggest that
neomycin blocks PLC activation only in the absence of
PLCd1PH-GFP.
Discussion
The main finding of the present study was that in the
cells expressing PLCd1PH-GFP, neomycin could not exhibit
its inhibitory effects on PtdIns(4,5)P2 hydrolysis by PLC.
There is increasing interest in understanding the actions of
inositol phospholipids, especially
PtdIns(4,5)P2, in living
cells[21]. PtdIns(4,5)P2 participates in many cellular functions,
including exocytosis, cytoskeletal function and membrane
transporter and ion channel
functions[4]. Many molecules have been found to be able to bind to phospholipids, and
more specifically to PtdIns(4,5)P2, which forms the basis of
modulation by this lipid[22]. The PH domain of
PLCd1 is one of these molecules that are believed to selectively bind to
PtdIns(4,5)P2[12]. Recently, a fusion construct of
PLCd1PH with enhanced green fluorescent protein
(PLCd1PH-GFP) was developed as a probe to visualize
PtdIns(4,5)P2 in single cells. This novel methodology allowed imaging and analysis of
spatiotemporal changes in PtdIns(4,5)P2 in single living cells,
and has been used increasingly in efforts to understand the
role PtdIns(4,5)P2 plays in cell signaling, and protein
function regulation[13]. The central idea behind this
methodology is that the GFP signal that has been linked to
PLCd1PH will faithfully follow the dynamic changes of
PtdIns(4,5)P2 during its metabolism, including during hydrolysis by PLC.
However, because PLCd1PH also binds to
IP3, a downstream product of
PtdIns(4,5)P2 hydrolysis, with higher affinity, some
have proposed that rather than being a faithful
PtdIns(4,5)P2 follower,
PLCd1PH-GFP molecules during
PtdIns(4,5)P2 hydrolysis are more likely to bind to newly produced
IP3[18]. But van der Wal
et al showed that physiological increases in
IP3 (10-100 μmol/L) on activation of PLC could not be solely
responsible for the translocation of
PLCd1PH-GFP[23]. Our data presented in Figure 2 are in agreement with the results
of van der Wal et al. In the cells expressing
PLCd1PH-GFP as well as the
BK2 or M1 receptors, BK or ACh induced the
reversible translocation of PLCd1PH-GFP from the plasma
membrane to the cytosol. Thus, although it bound to
PtdIns(4,5)P2, PLCd1PH-GFP did not interfere with cleavage
of PtdIns(4,5)P2 by PLC. On the other hand, neomycin, a
commonly used PLC blocker, is believed to block PLC
cleavage of PtdIns(4,5)P2 by preventing
PtdIns(4,5)P2 from
accessing PLC[17]. It is interesting to note that whereas a
smaller molecule such as neomycin would mask
PtdIns(4,5)P2 from PLC cleavage, a much bigger molecule such as
PLCd1PH-GFP would allow the cleavage to happen. It is also
interesting to consider that the expression of
PLCd1PH-GFP blocked the action of neomycin (Figure 3), suggesting that
PLCd1PH-GFP and neomycin bind to the same sites on
PtdIns(4,5)P2. Previous studies have demonstrated that both
PLCd1PH-GFP and neomycin interact with
PtdIns(4,5)P2 in an electrostatic
way[12,14]. Thus the charged inositide head group of
PtdIns(4,5)P2 is the binding site for both
PLCd1PH-GFP and neomycin[24], yet binding of
PLCd1PH-GFP or neomycin to
PtdIns(4,5)P2 has very different consequences for PLC
hydrolysis of PtdIns(4,5)P2. Although it is less likely, it needs
to be noted that the GFP, rather than
PLCd1PH, may block the binding of neomycin to
PtdIns(4,5)P2 through a spatial blocking effect. For many cellular proteins that have been known
to interact with, and whose functions are regulated by,
PtdIns(4,5)P2, the molecular basis for the interaction remains
to be elucidated. Less clear is the mechanism for
PtdIns(4,5)P2 hydrolysis by PLC. The present study provides interesting
and stimulating information for further understanding
protein-PtdIns(4,5)P2 interactions and
PtdIns(4,5)P2 hydrolysis by PLC. We are currently investigating the mechanism
underlying the different consequences of
PtdIns(4,5)P2 binding to
PLCd1PH-GFP or neomycin with respect to its
hydrolysis by PLC.
Acknowledgements
We thank Diomedes E LOGOTHETIS (Mount Sinai School
of Medicine, New York University, NY, USA), for providing
us with plasmids of the M1R,
BK2R and PLCd1PH-GFP, and members of the Logothetis laboratory for helpful
discussions on this work.
References
1 Takano M, Kuratomi S. Regulation of cardiac inwardly
rectifying potassium channels by membrane lipid metabolism. Prog
Biophys Mol Biol 2003; 81: 67-9.
2 Nishizuka Y. The molecular heterogeneity of protein kinase C
and its implications for cellular regulation. Nature 1988; 34:
661-5.
3 Cantley LC. The phosphoinositide 3-kinase pathway. Science
2002; 296: 1655-7.
4 Zhang H, He C, Yan X, Mirshaki T, Logothetis DE. Activation
of inwardly rectifying K+ channels by distinct
PtdIns(4,5)P2 interactions. Nat Cell Biol 1999; 1: 183-8.
5 Hilgemann DW, Feng S, Nasuhoglu C. The complex and
intriguing lives of PIP2 with ion channels and transporters. Sci STKE
2001; 111: RE 19.
6 Berridge PA, Xian W, Flanagan LA. Controlling cytoskeleton
structure by phosphoinositide-protein interactions:
phosphoino-sitide binding protein domains and effects of lipid packing. Chem
Phys Lipids 1999; 101: 93-7.
7 Raucher D, Stauffer T, Chen W, Shen K, Guo S, York JD,
et al. Phosphatidylinositol 4,5-bisphosphate functions as a second
messenger that regulates cytoskeleton-plasma membrane
adhesion. Cell 2000; 100: 221-8.
8 Hay JC, Fisette PL, Jenkins GH, Fukami K, Takenawa T,
Anderson RA, et al. ATP-dependent inositide phosphorylation
required for Ca2+-activated secretion. Nature 1995; 374: 173-7.
9 Jost M, Simpson F, Kavran JM, Lemmon MA, Schmid SL.
Phosphatidylinositol-4,5-bisphosphate is required for endocytic
coated reside formation. Curr Biol 1998; 8: 1399-402.
10 Martin TF. PtdIns(4,5)P2 regulation of surface membrane traffic.
Curr Opin Cell Biol 2001; 13: 493-9.
11 Harlan JE, Hajduk PJ, Yoon HS, Fesik SW. Pleckstrin homology
domains bind to phosphatidylinositol-4,5-bisphosphate. Nature
1994; 371: 168-70.
12 Cullen PJ, Cozier GE, Banting G, Mellor H. Modular
phosphoino-sitide-binding domains: their role in signaling and membrane
trafficking. Curr Biol 2001; 11: R882-93.
13 Varnai P, Rother KI, Balla T. Phosphatidylinositol
3-kinase-dependent membrane association of the Bruton's tyrosine
kinase pleckstrin homology domain visualized in single living cells.
J Biol Chem 1999; 274: 10983-9.
14 Schacht J. Inhibition by neomycin of polyphosphoinositide
turnover in subcellular fractions of guinea-pig cerebral cortex
in vitro. J Neurochem 1976; 27: 1119-24.
15 Gabev E, Kasianowicz J, Abbott T, McLaughlin S. Binding of
neomycin to phosphatidylinositol 4,5-bisphosphate
(PIP2). Biochim Biophys Acta 1989; 979: 105-2.
16 Lei Q, Jones MB, Talley EM, Garrison JC, Bayliss DA.
Molecular mechanisms mediating inhibition of G protein-coupled
inwardly-rectifying K+ channels. Mol Cell 2003; 15: 1-9.
17 Cockcroft S, Howell TW, Gomperts B. Two G-proteins act in
series to control stimulus-secretion coupling in mast cells: use of
neomycin to distinguish between G-proteins controlling
polypho-sphoinositide phosphodiesterase and exocytosis. J Cell Biol 1987;
105: 2745-50.
18 Hirose K, Kadowaki S, Tanabe M, Takeshima H, Lino M.
Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that
underlies complex Ca2+ mobilization patterns. Science 1999; 284:
1527-30.
19 Halet G, Tunwell R, Balla T, Swann K, Carroll J. The dynamics
of plasma membrane PtdIns(4,5)P2 at fertilization of mouse eggs.
Cell Sci 2002; 115: 2139-49.
20 Nakanishi S, Catt kJ, Balla T. A wortmannin-sensitive
phosphati-dylinositol 4-kinase that regulates hormone-sensitive pools of
inositolphospholipids. Proc Natl Acad Sci USA 1995; 92: 5317-21.
21 Martin TF. PI (4,5)P (2) regulation of surface membrane traffic.
Curr Opin Cell Biol 2001; 13: 493-9.
22 Hurley JH, Meyer T. Subcellular targeting by membrane lipids.
Curr Opin Cell Biol 2001; 13: 146-52.
23 van der Wal J, Habets R, Varnai P, Balla T, Jalink K. Monitoring
agonist-induced phospholipase C activation in live cells by
fluorescence resonance energy transfer. J Biol Chem 2001; 276:
15337-44.
24 Ferguson KM, Lemmon MA, Schlessinger J, Sigler PB. Crystal
structure at 2.2Å resolution of the pleckstrin homology domain
from human dynamin. Cell 1994; 79: 199-209.
|
