From the Division of Cell Biology, The Netherlands
Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The
Netherlands and the ¶ Endocrinology and Reproduction Research
Branch, National Institutes of Health,
Bethesda, Maryland 20892-4510
Received for publication, August 8, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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Agonist-induced intracellular
Ca2+ signals following phospholipase C (PLC)
activation display a variety of patterns, including transient,
sustained, and oscillatory behavior. These Ca2+ changes
have been well characterized, but detailed kinetic analyses of PLC
activation in single living cells is lacking, due to the absence of
suitable indicators for use in vivo. Recently, green fluorescent protein-tagged pleckstrin homology domains have been employed to monitor PLC activation in single cells, based on (confocal) imaging of their fluorescence translocation from the membrane to the
cytosol that occurs upon hydrolysis of phosphatidylinositol bisphosphate. Here we describe fluorescence resonance energy transfer between pleckstrin homology domains of PLC One of the earliest effects of the addition of certain agonists to
quiescent cells is a rapid increase in cytosolic free Ca2+
concentration
([Ca2+]i).1
In most cases, these [Ca2+]i increases are due to
mobilization from internal Ca2+ stores, caused by
activation of PLC enzymes that cleave phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane to
generate the second messengers inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol. IP3-mediated release
of Ca2+ from internal endoplasmic reticulum stores
has been well characterized, since the availability of specific vital
dyes that allow monitoring of [Ca2+]i in single
living cells with high spatial and temporal resolution. These studies
have demonstrated a variety of different response kinetics, including
transient and sustained Ca2+ increases, oscillations and
Ca2+ waves (1). An important conclusion drawn from these
studies is that kinetic differences in [Ca2+]i
responses can have marked effects on downstream regulatory targets with
important physiological consequences (2).
Although Ca2+ signals are believed to reflect
IP3 increases and hence PLC activation, it was noted in
several studies that various agonists generate grossly different
amounts of inositol phosphates, only to achieve similar
[Ca2+]i responses (3). However, due to the lack
of suitable methods, activation and inactivation kinetics of PLC have
not been characterized in single cells. An exciting recent development is the utilization of GFP-tagged protein domains for the in
vivo visualization of inositide lipid dynamics. For example, the
pleckstrin homology domain of PLC While this approach has a number of obvious advantages, including
detection of changes in single living cells with time resolution in the
second range, it also has several limitations. First, it is hard to
obtain quantitative data from translocation studies using confocal
microscopy. Even minor focal drift and changes in cell morphology, that
often occur after stimulation, degrade signal-to-noise ratio, since
these will render it difficult to reliably assign measurement regions
(so-called regions of interest) that correspond to membrane and
cytosol. Second, it is difficult to see translocation in very flat
cells or in cellular subregions such as neurites and lamellipodia.
Third, at fast imaging rates, confocal imaging requires high excitation
intensities that can cause severe cell damage in a matter of minutes.
Therefore, there is a trade-off between sampling speed and duration of
the experiment. Moreover, single cell determinations are inherently
variable, and the imaging approach is not easily extended to cell populations.
To overcome these limitations, we have applied fluorescent resonance
energy transfer (FRET) as a way to improve the detection of PH domain
translocation. FRET, the radiationless transfer of energy from a
fluorescent donor to a suitable acceptor fluorophore, depends on
fluorophore spectral overlap and dipole alignment, and is a very steep
function of donor-acceptor distance (for review, see Refs. 8 and 9).
For the GFP mutants CFP and YFP, FRET can take place when the
fluorophores are within ~10 nm distance. Here we describe FRET
between CFP- and YFP-tagged PH domains of PLC Materials--
1-Oleoyl-LPA, histamine, bradykinin, phenylarsine
oxide, and quercetin were from Sigma; neurokinin A, caged
IP3 (catalog number 407135), and ionomycin were from
Calbiochem-Novabiochem Corp. (La Jolla, CA);
myo-[3H]inositol (60 Ci/mmol) was from
Amersham Pharmacia Biotech. Fura red (K salt) and bodipy-FL were from
Molecular Probes Inc. (Eugene, OR). All other chemicals were of
analytical grade.
DNA Constructs--
The pleckstrin homology domain was obtained
from the Superhiro PLC
YFP was obtained from yellow Cameleon 2.0 (a kind gift from A. Miyawaki
and R. Tsien) and subcloned into cloning vector PGEM3z (Promega), via
SacI and EcoRI, and subsequently into pcDNA3
(Invitrogen) via BamHI and EcoRI. Polymerase
chain reaction on YFP-pcDNA-3 with primers T7 (Promega) and GFP3;
5'-GGCTGAGACCCGGGAATTCGGCTTGTACAGCTCGTCCATG-3' was done to remove the
stop codon. The polymerase chain reaction product, taken between
primers PLC
For YFP-CAAX and
GFP-CAAX, the membrane
localization sequence of K-Ras (KMSKDGKKKKKKSKTKCVIM) was obtained by
polymerase chain reaction amplification from Bp180-CAAX
(GenBankTM accession numbers M54968 and M38506),
using primers CAAX3 5'-CCGAATTCCCGGGTCAAGATGAGCAAAGATGGTAAAAAG-3', containing an
EcoRI site, and CAAX2;
5'-CCTGCGGCCGCGGTACCGAGATCTTTACATAATTACACACTT-3', that contained
a NotI site behind the stop codon. The final constructs were
made by exchanging the PH domain from YFP-PH and GFP-PH for the
CAAX domain using EcoRI and NotI. All
clones were verified by sequence analysis. YFP-CAAX
contained a point mutation (Val instead of Gly in the
CAAX domain), but this did not influence its membrane
localization. The constitutively active mutants of G Cell Culture and Transfections--
N1E-115 neuroblastoma cells
were seeded in 6-well plates at ~25,000 cells per well on
25-mm glass coverslips, and cultured in 3 ml of Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum and
antibiotics. Unless otherwise indicated, constructs were transfected
for 6-12 h using calcium phosphate precipitate, at 0.8 µg of
DNA/well. Following transfection, cells were incubated in serum-free
Dulbecco's modified Eagle's medium for 12-48 h. For fluorescence
detections, coverslips with cells were transferred to a culture chamber
and mounted on an inverted microscope. All experiments were performed
in bicarbonate-buffered saline (containing, in mM, 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, with
10 mM HEPES added), pH 7.2, kept under 5% CO2, at 37 °C.
Inositol Phosphate Determinations--
Preparation, culture, and
labeling of bovine adrenal glomerulosa cells have been described
elsewhere (11). Cells labeled with
myo-[3H]inositol for 24-48 h were stimulated
by angiotensin II (30 nM) for the indicated times in a
medium containing either Sr2+ or Ca2+.
Reactions were terminated with perchloric acid and inositol phosphates
were separated by high performance liquid chromatography essentially as
described previously (11).
Confocal Microscopy and Image Analysis--
For confocal
imaging, a Leica DM-IRBE inverted microscope fitted with a TCS-SP
scanhead was used. Excitation of enhanced GFP was with the
488-nm argon ion laserline, and emission was collected at 500-565 nm.
For translocation studies, series of confocal images were taken at
2-10-s intervals and stored on disc. Determination of the ratio of
membrane to cytosolic fluorescence by directly assigning regions of
interest for membrane and cytosol was hampered by the shape changes of
cells during experiments (see text). Using Qwin software (Leica) this
ratio was therefore calculated by post-acquisition automated regions of
interest assignment and analysis. In brief, a binary mask of the
transfected cell was lined out using a thresholding step on a smoothed
image. From this mask, the area corresponding to the membrane was
eroded by a user-selectable amount to delineate the membrane. Further
erosion was then applied to reliably separate membrane from cytosol
area, and the remaining area was taken to represent cytosol. This mask
was updated for each image in a series, and translocation was expressed
as ratio of the fluorescence values for membrane and cytosol area, to
correct for bleaching. This approach corrects fully for cell movements
and shape changes, and was able to reliably detect very minor
translocations (using e.g. diluted agonists) that went
unnoticed for the eye.
Fluorescence Determinations--
For FRET experiments, cells on
coverslips were placed on an inverted Zeiss Axiovert 135 microscope
equipped with a dry Achroplan ×63 (NA 0.75) objective. Excitation of
CFP was at 425 ± 5 nm, and emission was collected with a 460-nm
dichroic mirror. Emission of CFP and YFP was split using an additional
505-nm dichroic mirror and filtered with 475DF30 and 540DF40 band pass
filters, respectively. Detection was with PTI model 612 analog
photomultipliers, and for data acquisition, the FELIX software (PTI
Inc.) was used. FRET was expressed as ratio of CFP to YFP signals, the
value of which was set as 1.0 at the onset of the experiment. Changes
are expressed as percent deviation from this initial value of 1.0. For
detection of intracellular Ca2+, Yellow Cameleon 2.1 was
used (12) at the same wavelengths.
For sustained stimulation, agonists and inhibitors were added to the
medium from concentrated stocks. Stimulation with short pulses of NKA
was performed by placing a glass micropipette (tip Ø ~2 µm) at
about 25 µm from the cell using an Eppendorf microinjection system
and applying pulses of pressure for 10 s. It was verified using
Lucifer Yellow in the pipette that following termination of the
pressure pulse the concentration at the cell rapidly dropped toward zero.
Loading and Flash Photolysis of Caged
IP3--
Before electroporation, adherent cells grown on
coverslips were washed twice in intracellular buffer (containing in
mM: 70 KCl, 70 K glutamate, 2 MgCl2, 0 CaCl2, 5 phosphate buffer, pH 7.1) and then 70 µl of this
intracellular buffer was added to the cells with 20 µM
Fura red tetrapotassium salt and 1, 10, or 100 µM caged
IP3. Electroporation was achieved by a series of 15 high
frequency square wave pulses, (1-s spaced, amplitude 150 V, frequency
80 kHz, lasting 0.5 ms each) using 2 platina electrodes of 8 × 3 mm with 2.5-mm spacing. The efficiency of this method was assessed by
control permeabilizations that were performed on the stage of a
confocal microscope. This protocol caused complete permeabilization
(based on equilibration of intracellular calcein concentrations with
the extracellular buffer) of the cells in the area between the electrodes.
For photorelease of caged IP3, a single cell was
illuminated with a short pulse of UV light (340-410 nm) from a 100 W
HBO lamp using a shutter. The shutter open time was adjusted to give full release of caged IP3, that is no response being
observed with a subsequent illumination. For partial photolysis, the
flash intensity was adjusted by neutral density filters placed in the illumination pathway.
Quantitation of Expression Levels--
For quantitation of
expression levels of CFP-PH and YFP-PH, cellular fluorescence was
compared with the fluorescence of a solution of known concentration of
purified, bacterially expressed CFP-PH or YFP-PH, following the method
of Miyawaki et al. (37). In short, CFP-PH and YFP-PH
were expressed as glutathione S-transferase fusion proteins,
and purified on glutathione-Sepharose beads. Protein concentration (5.4 µM) was measured by the BCA Protein Assay (Pierce).
Calibration of the fluorescent brightness of this protein preparation,
using values for molar absorption and quantum yield of 36,500 and 0.63, respectively (13), against commercial standards indicated that
essentially 100% of proteins were fluorescent. The solution was then
introduced in a linear wedge-shaped chamber (0-170 µm thickness)
that was placed on the microscope (using NA 0.7 objective), and the
position of the chamber was adjusted to give a fluorescence readout
that matched that of a single, CFP or YFP expressing cell. The estimate
of the fluorescent protein concentration in the cell was obtained by
comparing the local thickness of the wedge to that of an average cell
(17 µm). Relative amounts of CFP-PH and YFP-PH expression in cells
were always determined under conditions of full cytosolic localization
of the constructs. Comparison of voxel intensities within cells and the
protein solution using a confocal microscope gave very similar results.
At the onset of each experiment, photomultiplier gains (high voltage)
were adjusted to give a standard 6 V output for the resting cell.
Noting that over an extended range of light input, every 2-fold change
in intensity corresponds to a 35 V change in cathode voltage, cell
intensities were measured. By comparing these intensities to the values
obtained with the GFP wedge, estimates of expression levels were obtained.
Fluorescence Recovery after Photobleaching (FRAP)--
For FRAP
experiments, cells were imaged using a Leica TCS-SP confocal microscope
equipped with ×63 (NA 1.3) oil immersion objective. The beam from a
external ArKr laser (25 mW) was coupled into the backfocal plane of the
objective via the epifluorescence excitation port, using a 30/70
beamsplitter, thus allowing simultaneous imaging and spot bleaching.
Spots of ~1.3 µm (full width half-maximum) were bleached (>95%)
in the basal membrane using a single 30-ms pulse from the ArKr laser
during data collection in linescan mode at 1000, 500, or 125 Hz. Data
were corrected for slight (<7%) background bleaching and fitted with
single exponents using Clampfit software (Axon Instruments).
Fluorescence Resonance Energy Transfer between Plasma
Membrane-localized PLC
The kinetics of BK-induced PLC activation in N1E-115 cells as detected
by FRET is characterized by a rapid onset, with translocation peaking
at 20-30 s after addition of the agonist. The decaying phase is
somewhat slower, usually returning to baseline within 1 to 4 min. This
time course is very similar to that deduced from confocal detection of
PLC
The above described kinetics with a fast and rather complete
translocation induced by BK, suggest that PI(4,5)P2
depletion after stimulation is quite extensive. While most reports of
agonist-induced PI(4,5)P2 hydrolysis, as detected
biochemically from [3H]inositol-labeled cells, show
slower and less pronounced decreases in phosphoinositide levels,
considerable agonist- and cell type-dependent variations
exist, e.g. Refs. 14-16. Where early time points were also
studied, rapid decreases in PI(4,5)P2 levels have been
detected (17-19). For example, significant bradykinin-induced
PI(4,5)P2 decreases were reported to occur within 10 s
in bovine aortic endothelial cells (20), and at 1 min in
bombesin-stimulated 3T3 cells (18). Rapid recovery toward basal levels
has also been found. Wijelath et al. (17) reported as much
as 85% hydrolysis of PI(4,5)P2 at 5 s after
stimulation of macrophages with interleukin, while
PI(4,5)P2 levels recovered to 50% at 60 s. Similar
fast recovery was also seen in other cell types (18, 19). Since biochemical analyses have to rely upon measurements on cell
populations, where not all cells give synchronized and identical
responses (and many cells may not respond at all), it is not surprising to find differences between the results of measurements with these two
alternative approaches.
Characterization of Fluorescence Signals--
During
agonist-induced translocation, several factors may affect the
fluorescent properties of these PH domain chimeras as well as the
transfer of fluorescent energy between them (21). For example, the move
away from a compartment adjacent to the lipophilic membrane could alter
fluorescent characteristics, and is also likely to alter FRET by
increasing the degree of rotational freedom. While the relative
influence of increased rotational freedom on the translocation-induced
decrease in FRET is difficult to assess in this model sytem, we
analyzed fluorescence changes in some further detail. Cells were
transfected with only one of the PLC
To assess the effects of construct concentrations on FRET, we compared
cells expressing various levels of the chimeric proteins. Intracellular
fluorescent protein concentrations were estimated by comparing the
emission intensities of individual cells to those of a solution of
bacterially expressed, purified protein of known concentration (37)
(see "Experimental Procedures"). Based on these estimates,
resonance could be observed in cells with expression levels between
about 0.8 and 80 µM, over a 100-fold concentration range.
However, FRET was not observed in cells expressing less than ~400
nM of each of the constructs. High expression levels, on
the other hand, appeared to be detrimental to the cells (as judged from
the appearance of membrane blebs, rounding and detachment of cells 2-3
days after transfection). For our analysis, we selected cells with
estimated expression levels between 0.8 and 12 µM, which corresponds to the range that is
used for confocal detection of translocation, the lower value being
just 2 to 3 times the autofluorescence of the cells (13). These data
also revealed that PLC
Can such estimates of CFP and YFP concentrations be used to calculate
lipid concentrations and molecular proximity in the cells studied? If
we model a typical attached N1E-115 cell with a pyramid of 20 × 20-µm base and 10-µm height (having 1.3 pl volume and 1100 µm2 surface), and assume that (i) the concentration of
both chimera is 10 µM; (ii) 50% of fluorophores are
located at the membrane (complete translocation roughly doubles the
fluorescence in the cytosol); (iii) the distribution of fluorophores is
homogenous along the membrane; and (iv) fluorophores are insensitive to
the local environment, then the calculated mean distance between
fluorophores is about 10 nm, which is close to the reported Forster
radius (50 Å) for FRET between this pair of fluorophores (21).
However, it should be emphasized that these assumptions are valid only as first approximations. For example, we and others (22) have noted
that GFP-PH is not homogeneously localized along the plasma membrane.
Also, as discussed above, the spectral properties of the fluorescent
proteins are sensitive to the microenvironment. Nevertheless, these
data set a lower limit for the density of PI(4,5)P2
molecules available for PH binding at the inner surface of the plasma membrane.
GFP-PH Rapidly Shuttles between Membrane and Cytosol--
Another
important characteristic we wanted to address was membrane association
and dissociation rates of the PH chimera. These rates directly
influence reliability of FRET in reporting rapid changes in PLC
activity, and are also relevant to the ability of PLC to hydrolyze
PI(4,5)P2 in cells that express high levels of the
PLC Widefield FRET Detection Allows Prolonged Monitoring Independent of
Cell Shape Changes--
Rapid confocal scanning of cells transfected
with PLC
In N1E-115 and other cells, addition of certain agonists causes rapid
and significant shape changes. For instance, LPA causes neurites to
retract and the cell soma to round up within 60 s (23). In
contrast, addition of BK has opposite effects, promoting a
differentiated phenotype (24). During confocal imaging, such shape
changes (as well as the slight drift in focal plane that inevitably
occurs over prolonged times) seriously complicate the quantification of
GFP-PH translocation. Since our FRET analysis uses the total integrated
emission from a cell, shape changes and focal drift do not present any problems.
In very flat and small cell structures such as neurites and
lamellipodia (below ~2 µm in thickness), confocal imaging cannot detect translocation due to its inherent limit in z axis
resolution. However, in such cases changes in FRET can still be
reliably detected as shown by the agonist-induced PLC activation
recorded over a single neurite (Fig. 4B). FRET can also be
recorded from cell populations (Fig. 4C) providing with an
average response that would need analysis of hundreds of single cell
recordings. Thus, detecting resonance between fluorescent
protein-labeled PH domains has several advantages to report on the
distribution of PH domains without the need for confocal detection.
Does FRET Report Changes in Membrane PI(4,5)P2 or
Increases in Cytosolic IP3?--
While PLC
While FRET analysis effectively monitors the result of PLC activation
regardless of whether it is the lipid decrease or the IP3
increase that is more important for the translocation response, we felt
that this question deserves a more detailed analysis. First we wanted
to determine whether intracellular applications of IP3 that
generate a Ca2+ signal comparable to that evoked by an
agonist would cause translocation of the PLC
Next, the effects of interfering with PI(4,5)P2 resynthesis
on the kinetics of translocation in N1E-115 cells was studied. PI(4,5)P2 resynthesis was inhibited by low concentrations
(5 µM) of phenylarsine oxide (Fig. 5B) or
quercetin (26), or by depletion of free inositol using prolonged
incubation in inositol-free medium (not shown). In phenylarsine
oxide-treated cells, BK induced a sustained translocation of
PLC
Moreover, when adrenal glomerulosa cells were stimulated with
angiotensin II in the presence of Sr2+, a condition under
which IP3 metabolism via Ins(1,3,4,5)P4 is greatly reduced (11), hence yielding significantly higher
Ins(1,4,5)P3- and diminished Ins(1,3,4)P3
increases (Fig. 5, C and D), the translocation of
PLC
Taking all these data together, we conclude that, at least for the
cells and agonists tested in the present study, PLC FRET Reveals Response Heterogeneity to Different GPCR Agonists That
Is Not Reflected in Ca2+ Mobilization--
Having
characterized the use of FRET between CFP- and YFP-tagged PH domains of
PLC
These agonists evoke very similar Ca2+ mobilizations in
N1E-115 cells, characterized by a fast onset and rapid termination well within 2 min (Fig. 6). Estimated peak
Ca2+ levels ranged from 0.6 to 2 µM, and,
again, showed no consistent differences between agonists. When the
effects of PLC activation were recorded by FRET analysis, using the
same agonists under identical conditions, quite unexpectedly several
distinct kinetic profiles were obtained (Fig. 6). First, both NKA and
BK caused fast and near complete translocation of the probe. This
response was transient, returning to baseline within 2-5 min.
Stimulation with thrombin or LPA evoked a different type of response:
these translocations had slower onset and smaller amplitude, averaging 25% of BK response control values (n = 22). They also
returned to baseline at a slower rate. The response to histamine was
much slower and of small amplitude (40% of BK-induced peak values, n = 15), but it was long-lasting (at least for 15 min,
but often much longer).
Differences in degree of PI(4,5)P2 hydrolysis induced by
activation of different Gq-coupled receptors have also been
reported (15, 32) in biochemical studies. However, so far only
cytosolic Ca2+ responses could be used to analyze receptor
activation patterns at the single cell level. On the other hand, the
shape of the Ca2+ response is determined by several other
factors: it can be triggered at relatively low levels of
IP3 and its shape is also determined by the
Ca2+-induced Ca2+ release and inactivation
properties of the IP3-receptor channels, as well as by the
activities of the various Ca2+ sequestration mechanisms.
The present approach provides an opportunity to study a more upstream
receptor-mediated event, namely PLC activation, and its regulation in
detail at the single cell level.
PH Domain Translocation Kinetics Mirror Receptor
Activation--
These results thus suggest that PLC activation as
assessed by FRET is a more faithful index of receptor activity than the more distal Ca2+ transients. However, inactivation could
occur at various steps in the signal cascade, including at the levels
of receptor, G protein, and PLC and, conceivably, also by modulation
(up-regulation) of PI(4,5)P2 resynthesis. To test whether
there is desensitization at the level of PLC, G proteins were directly
activated using AlF4
To further determine whether the membrane association of PLC
In summary, in the present study, we describe a fluorescence
resonance-based detection scheme to follow the membrane localization of
tagged PLC
Our analysis of the translocation responses suggests that localization
of PLC1 tagged with cyan and
yellow fluorescent proteins as a sensitive readout of
phosphatidylinositol bisphosphate metabolism for use both in cell
populations and in single cells. Fluorescence resonance energy transfer
requires significantly less excitation intensity, enabling prolonged
and fast data acquisition without the cell damage that limits confocal experiments. It also allows experiments on motile or extremely flat
cells, and can be scaled to record from cell populations as well as
single neurites. Characterization of responses to various agonists by
this method reveals that stimuli that elicit very similar
Ca2+ mobilization responses can exhibit widely different
kinetics of PLC activation, and that the latter appears to follow
receptor activation more faithfully than the cytosolic Ca2+ transient.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 that binds to
PI(4,5)P2 has been used to monitor changes in the cellular
levels of this lipid (4, 5) and the PH domains of Btk or PKB were
employed to follow changes in 3-phosphorylated polyphosphoinositides
(6, 7). In these studies detection is based on imaging of the change in localization of GFP-PH by fluorescence microscopy. PLC
1PH-GFP, for
instance, is located at the membrane in resting cells, where it is
bound to PI(4,5)P2 (5), and upon agonist-induced
PI(4,5)P2 hydrolysis it translocates to the cytosol.
Subsequently, when PI(4,5)P2 is resynthesized, fluorescence
returns to the membrane. The transient translocation is usually
detected in single cells by confocal imaging and quantified by
post-acquisition image analysis (4, 6).
1 as a sensitive
readout for membrane localization that offers a number of the
advantages over confocal imaging. FRET detects agonist-induced
translocation of the fluorescent proteins from the membrane to the
cytosol essentially identical to what was reported in GFP-PH imaging
studies, without the need for optical sectioning, providing with a more
reliable quantitative measurement of membrane localization. In
addition, this method is applicable for cell populations and single
neurites, and allows analysis for extended periods of time with minimal
cell damage. Comparison of FRET responses in several cell types with
various agonists reveals significant differences between kinetics of
PLC activation triggered via various GPCRs, despite similar
[Ca2+]i responses. It is also shown that receptor
activity and desensitization, rather than post-G protein mechanism(s)
is a major determinant of the PLC activation pattern.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1PH construct (amino acids 1-174; a kind gift
from Dr. Tobias Meyer) and cloned into the eukaryotic expression
vector pECFP-C1 (CLONTECH, CA). Two primers
(PLC
PH1; 5'-CCTGCGGCCGCGGTACCGATATCAGATGTTGAGCTCCTTCAG-3' and
PLC
PH2; 5'-CCGAATTCCCGGGTCTCAGCCATGGACTCGGGCCGGGACTTC-3') were
designed to generate the PH domain in-frame behind the CFP followed by
a stop codon. The polymerase chain reaction product was cloned into the
pECFP plasmid with the restriction sites EcoRI and
EcoRV on EcoRI and SmaI, leading to
pECFP-PH.
PH1 and PLC
PH2, was cloned in-frame behind YFP with
EcoRI and NotI, leading to
pcDNA3YFPPH. To obtain
pcDNA3eGFPPH, YFP was swapped with enhanced
GFP, using primers T7 and GFP3 on pcDNA3eGFP and restriction
enzymes BamHI and EcoRI.
q and G
12 cloned into pcDNA3 vectors
were a kind gift from Dr. O. Kranenburg et al. (10).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1PH-CFP and PLC
1PH-YFP--
PH-CFP and
PH-YFP chimera were transiently transfected into N1E-115 mouse
neuroblastoma cells at a 1:1 molar ratio. After 1-2 days, cells were
transferred to an inverted epifluorescence microscope and assayed for
FRET by simultaneously monitoring the emission of CFP (475 ± 15 nm) and of YFP (530 ± 20 nm), while exciting CFP at 425 ± 5 nm. In resting cells, PH-CFP and PH-YFP reside at the plasma membrane
bound to PI(4,5)P2, and the two fluorophores remain within
resonance distance. Upon activation of PLC by the addition of BK,
PI(4,5)P2 is rapidly hydrolyzed and consequently PH domains
can no longer bind to the plasma membrane. The distance(s) between
fluorophores increase significantly, and therefore FRET no longer
occurs (Fig. 1). As a result, the donor (CFP) emission intensity increases, while the acceptor (YFP) emission decreases. By taking the ratio of CFP to YFP emission, the FRET signal
becomes essentially independent on excitation intensity fluctuations
and photobleaching.
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Fig. 1.
Fluorescence resonance detection of PH domain
translocation. A, schematic representation of
FRET occurring between CFP-PH and YFP-PH bound to the membrane. Upon
hydrolysis of PI(4,5)P2, PH domains translocate to the
cytosol and FRET ceases. B, emission signals of
CFP and YFP collected at 475 and 530 nm respectively, and their ratio
(530/475), recorded from a single N1E-115 cell stimulated with
bradykinin (BK, 1 µM). Signals were low-pass filtered at
2 Hz and sampled at 3 Hz. Scale bar for ratio signal shows
percent deviation from baseline. C, confocal
detection of GFP-PH translocation, depicted on the same scale. Images
were collected once per 10 s, and the ratio of fluorescence
intensities in membrane and cytosol (PM/Cyt) was
deduced for each image by post-acquisition automated image analysis
(see "Experimental Procedures" for details).
1PH-GFP translocation recorded under identical conditions (Fig.
1C). In this latter case, the data were extracted from a
time series using post-acquisition automated image analysis (see
"Experimental Procedures"). Similar translocation responses can be
obtained by FRET in other cell types, including A431 epidermoid
carcinoma cells, HEK293 embryonal kidney cells, and COS monkey kidney
cells stimulated with a variety of ligands to Gq-coupled receptors.
1PH-CFP or PLC
1PH-YFP
constructs. After stimulation, a small but consistent transient
fluorescence decrease was observed with either the CFP or the
YFP-tagged PH domains (Fig. 2). The original green construct (PLC
1PH-GFP) displayed similar behavior (not shown). This transient decrease is likely caused by fluorophore displacement from the membrane, since it is not observed in cells that
express a more stably membrane-anchored GFP-CAAX, nor is it
seen in cells that express a mutated PLC
1PH-GFP (R40L) (5) that
cannot bind PI(4,5)P2 and, therefore, is cytosolic
throughout the experiment. The precise mechanism that causes this
decrease of emission upon cytosolic translocation is unknown; however, influence of the local microenvironment (e.g.
hydrophobicity, charged groups, changing ion concentrations etc.) on
the spectral properties of GFP seems to be the likely reason (21). When
recording FRET, this downward "displacement" effect will be added
to the translocation-induced decrease of the YFP signal, while it will lessen the simultaneous CFP increase, likely explaining why the translocation-induced decrease in YFP signal is somewhat larger than
the increase in CFP fluorescence. However, expressing FRET as an
emission ratio largely eliminates this effect. As expected, FRET could
also be measured in cells that coexpress PLC
1PH-CFP with
YFP-CAAX (not shown); however, using this pair, ratioing does not cancel the above mentioned displacement effect.
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Fig. 2.
Characterization of fluorescence
emission. Cells expressing the constructs as indicated were
stimulated with 1 µM bradykinin and fluorescence emission
was detected at the indicated wavelength. See the text for further
details.
1PH-CFP expression levels (detected in fully
translocated cells) did not differ more than about 2-fold from those of
PLC
1PH-YFP in most cells. It is interesting to note that we could
not see signs of saturation of the membrane-binding sites for the
fluorescent PH domains, and could not establish a trend in which higher
PH domain expression would be associated with altered translocation kinetics. Whether cells respond to the expression of these probes with
compensatory increases in PI(4,5)P2 levels remains to be investigated. Nevertheless, possible interference of the expressed PH
domains with cellular signals including PLC activation per se, needs to be kept in mind when using such analyses.
1PH-GFP protein. We, therefore, performed FRAP experiments to estimate the binding and dissociation kinetics of PLC
1PH-GFP in
the membrane. Fig. 3 shows representative
results from such FRAP experiments in N1E-115 cells. In panels
A and B, the recovery rates are depicted for
GFP-CAAX and PLC
1PH(R40L)-GFP, constructs that are
delimited to the plasma membrane and the cytosol, respectively. The
former presents the extreme of slow, purely membrane-delimited diffusion (2.81 ± 0.31 s, n = 15), and the
latter of fast cytosolic diffusion (0.201 ± 0.022 s,
n = 15). Since FRAP of membrane-localized PLC
1PH-GFP
is significantly faster than that of the membrane-delimited GFP-CAAX (1.22 ± 0.23 s, n = 40;
p < 0.005; compare panels A and C), its recovery has to be partially through the cytoplasma.
Thus, PI(4,5)P2-PH binding is a dynamic process, with
on-off rates in the order of seconds. In support of this notion, FRAP
further decreased during agonist-induced partial translocation, when
association rates are increased due to the raised cytosolic GFP-PH
levels (panel D). The rapid shuttling between membrane and
cytosol of individual PLC
1PH-GFP molecules could explain why
PI(4,5)P2 is still available for PLC-mediated hydrolysis or
for binding of other proteins in cells expressing these chimeras.
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Fig. 3.
FRAP reveals dynamic movements of GFP-PH
between cytosol and membrane. Spots (~1.3 µm full-width half
maximum) were completely bleached in the basal membrane (or in the
cytosol for B) with a 30-ms pulse of 488 nm laser light, and
recovery was monitored in line scan mode in a confocal microscope. FRAP
of membrane-delimited YFP-CAAX (A); cytosolic
PLC 1PH(R40L)-GFP mutant that cannot bind PI(4,5)P2
(B); PLC
1PH-GFP in a resting cell (C); and
PLC
1PH-GFP in a cell that has agonist-induced partial translocation
of fluorescence (D). Insets show confocal images
for the distribution of these constructs, taken from representative
cells.
1PH-GFP leads to severe phototoxic damage (often within 100 frames), manifested as membrane blebbing and loss of membrane integrity within minutes. Using wide field optical detection and integrating emission from an entire cell (or even clusters of cells) allowed excitation intensity to be dimmed by as much as 100 to >1000-fold, while still retaining acceptable signal-to-noise ratio. Thus, FRET can
be followed in single cells for extended periods of time without
detectable cell damage. This permits recording of complex stimulation
protocols, as shown in Fig.
4A. Here, a trace obtained from a single N1E-115 cell that is repeatedly stimulated with short
pulses of neurokinin A (NKA) from a puffer pipette indicates repeated
PLC activation. The response to NKA displays incremental partial
homologous desensitization of PLC activation, while the response to
subsequently added BK is unaltered. Optimizing for low excitation
intensity, recordings of several hours can be obtained with sub-second
resolution.
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Fig. 4.
Using FRET to record PLC activation in single
cells, neurites, or in cell populations. A, a single
N1E-115 cell was stimulated repeatedly with NKA as indicated by the
lines (dashes, 10-s pulses of 100 µM NKA from a puffer pipette; solid line,
addition of 1 µM final concentration to the culture
dish). The response indicates repeated PLC activation, detected as
decreases in FRET, and exhibits partial desensitization. Translocation
induced by subsequently added BK (1 µM) was not
desensitized by NKA pretreatment. For calibration, maximal
translocation was induced by adding 5 µm ionomycin + 2 mM
extra Ca2+ (5). B, FRET recording from a single
neurite of a neuroblastoma cell, differentiated by culturing in
serum-free medium for 48 h. Area of measurement (3.5 × 9 µm) is indicated in the micrograph. For this experiment, excitation
bandwidth was increased to 20 nm. C, FRET recording from a
cluster of about 15 transfected cells demonstrates improved
signal-to-noise ratio and averaged kinetics (note the same scale for
B and C).
1PH-GFP has
been introduced as an indicator of membrane PI(4,5)P2 (4,
5), it also displays high affinity to IP3 (25), which may
significantly exceed its affinity to PI(4,5)P2, although it
is difficult to accurately measure the latter as it is displayed
in vivo. Based on such relative affinity estimates, Hirose
and co-workers (25) recently suggested that PLC
1PH-GFP actually
monitors IP3 increases rather than the changes in lipid levels in Madin-Darby canine kidney cells. They reported that microinjection of IP3 in Madin-Darby canine kidney cells
was sufficient to cause displacement of PLC
1PH-GFP from the membrane
to the cytosol through competition for binding of the fluorescent
construct to membrane PI(4,5)P2. They also showed that
expression of an IP3 5-phosphatase completely blocked the
agonist-induced translocation of the fluorescent protein and concluded
that PI(4,5)P2 changes do not make a significant
contribution to the translocation response during stimulation.
1PH that is similar to
what is caused by agonist stimulation. N1E-115 cells were loaded with
20 µM of the calcium indicator Fura red and 100 µM caged IP3 by in situ high
frequency electroporation. Unlike microinjection, this technique allows
setting of the final concentration of caged IP3 in the
cytosol with high precision (see "Experimental Procedures"), as
confirmed by the observation that upon electroporation, intracellular
and extracellular fluorescence levels were equal. As shown in Fig.
5A, UV flash photolysis of 1 µM caged IP3 rapidly mobilized
Ca2+ from internal stores, with no visible translocation of
PLC
1PH-GFP to the cytosol. Subsequent release of 10 µM
caged IP3 caused a higher Ca2+ response and a
small translocation. Only high IP3 concentrations that
evoked a large and prolonged Ca2+ increase were able to
displace PLC
1PH-GFP from the plasma membrane. In contrast, BK
stimulation caused a larger translocation response than the highest
amounts of IP3 with a Ca2+ signal that was
comparable to that induced by the smallest amount of IP3
(Fig. 5A). In cells electroporated with no caged
IP3 in the electroporation buffer, intense UV flashes did
not influence intracellular Ca2+ levels, membrane
localization of the chimera, or any of the BK-induced changes herein
(not shown).
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Fig. 5.
The PH domain of
PLC 1 reports changes in PI(4,5)P2
rather than in IP3. A, cells expressing GFP-PH
were loaded with both Fura red (20 µM) and caged
IP3 (100 µM) by in situ high
frequency electroporation. Shown is the response of a single cell,
assayed simultaneously for GFP translocation and Ca2+
mobilization induced by flash photolysis of caged IP3.
Arrows indicate photolysis of 1, 10, and 90 µM
as detailed under the "Results." For comparison, bradykinin (1 µM) was added afterward. Representative trace from 16 similar experiments. B, FRET response to bradykinin detected
in a single cell, pretreated with 5 µM phenylarsine oxide
for 10 min. C and D, time course of
Ins(1,4,5)P3 and Ins(1,3,4)P3 formation in
adrenal glomerulosa cells prelabeled with [3H]inositol,
after stimulation with angiotensin II (Ang, 1 µM) in the presence of 2 mM Sr2+
(red) or Ca2+ (black). E,
angiotensin II-induced translocation as quantitated by analysis of
serial confocal images of glomerulosa cells in the presence of
Sr2+ (red) or Ca2+
(black). Data points represent mean ± S.E.,
n = 5. F, bradykinin-induced translocation,
with and without Sr2+, as detected by FRET in N1E-115
cells.
1PH-GFP to the cytosol, while IP3 increases in such
cells are only transient (27). In control experiments, these
pretreatments did not influence signaling events such as BK-induced
Ca2+ signaling (peak Ca2+ values of 870 ± 130 nM in control cells, and 845 ± 114 nM, in phenylarsine oxide-pretreated cells,
n = 6, mean ± S.E.) or the thrombin- and
lysophosphatidate-induced actinomyosin contraction (23, 28). Similar
observations were made in HEK293 cells (not shown), suggesting that the
translocation of PH domains under these conditions reports the depleted
PI(4,5)P2 pool rather than the transient IP3 increase.
1PH-GFP was not significantly different (Fig. 5E) from that observed in the presence of Ca2+. Translocation
responses of N1E-115 cells in response to BK were also similar in the
presence of Ca2+ or Sr2+ (Fig.
5F).
1PH-GFP translocation primarily reports changes in membrane
PI(4,5)P2 content and not the IP3 increases.
The reason for the apparently stronger binding of PLC
1PH to
membranes observed in live cells compared with the reported low
in vitro affinity (25) to PI(4,5)P2 containing
lipid vesicles or BiaCore surface (29), is unclear at present, but may
indicate a more complex interaction of the PLC
1PH domain with the
native membranes that is not mimicked by the in vitro
experiments. However, we did confirm the finding (25) that high
IP3 levels can make significant contributions to the
translocation response. Whether such high levels or IP3 occur under the experimental conditions used with intact cells remains
to be elucidated. Nevertheless, possible interference from large
IP3 increases should be kept in mind during interpretations of the results of such translocation experiments.
1 to record the effects of PLC activation, we next set out to
compare the kinetics of responses to a set of calcium-mobilizing GPCR
agonists. Included in this panel were the peptide agonists BK and NKA,
as well as the bioactive lipid LPA, the protease thrombin, and the
bioactive amine, histamine. Thrombin and LPA, in addition to inducing
Ca2+ mobilizations from internal stores, are also strong
inducers of Rho-dependent remodeling of the actin
cytoskeleton in these cells (23, 28). Histamine, on the other hand,
does not induce Rho-dependent actin remodeling, but is
known to induce Ca2+ oscillations in several cell types,
e.g. Refs. 30 and 31.
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Fig. 6.
Heterogeneity of translocation responses to
different GPCR agonists. Single N1E-115 cells expressing CFP-PH
and YFP-PH were stimulated with 1 µM BK, 1 µM NKA, 50 µM thrombin-receptor activating
peptide (TRP), 1 µM LPA, or 10 µM histamine (HIS). In black, PLC
activation as assayed by FRET. In red, intracellular
Ca2+ recordings for these agonists detected ratiometrically
using Yellow Cameleon 2.1 in separate experiments. Changes in
fluorescence ratio are expressed as percentage of resting values. Shown
are representative examples of experiments performed at least 10 times.
(Fig.
7A). While the onset of
ALF4
-induced translocation was slow, no desensitization
was observed in any of these experiments. Similarly, when viewed using
confocal microscopy, cells expressing a constitutively active
G
q mutant showed mostly cytosolic localization of
PLC
1PH-GFP domains for at least 2 days (Fig. 7B). Cells
transfected with activated G
12, which does not activate
PLC, showed normal membrane localization of PLC
1PH-GFP and agonists
could still induce translocation of the fluorescence to the cytosol. At
lower expression levels, the activating mutant G
q
induced sustained partial translocation that also persisted for several
days. These experiments suggested that no significant desensitization
occurs downstream of Gq and PLC. In line with this notion,
we did not observe significant heterologous desensitization between
sequentially added agonists (compare e.g. Fig. 4 and 6,
last panel), whereas prolonged exposure of cells to each
individual agonist induced complete (homologous) desensitization.
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Fig. 7.
FRET inactivation kinetics mirror receptor
inactivation. A, FRET recording from a single N1E-115
cell stimulated with 1 µM NKA or 1 mM
aluminum fluoride (AlF4 ). B,
confocal micrographs of cells taken 56 h after transfection with
PLC
1PH-GFP (5 µg of DNA/well) together with different amounts of
constitutively active G
q subunit (Gq*, 0.8 µg/well, and Gq* 1:10, 0.08 µg/well) or with
constitutively active G
12 at 0.8 µg/well
(G12*). C, PLC activation, as detected
by FRET, in a single neuroblastoma cells (left panel) that
expresses wild-type NKA receptors, stimulated with 10-s pulse from a
puffer pipette with 100 µM NKA; and cells stimulated by
prolonged addition of NKA (1 µM) to the medium,
expressing either wild-type receptors (middle panel) or a
mutant, truncated at its C terminus (33) (right panel).
Recordings are all to the same scale. D, kinetics
of FRET after NKA addition in a N1E-115 cell transfected with the
C-terminal truncated NK2 receptors on an extended time scale.
1PH as
assessed by FRET truly reports on receptor activity (i.e. the coupling and uncoupling between receptors and G proteins), we
compared the responses of N1E-115 cells expressing either the wild-type
NK2 receptors or a C-terminal truncated form, which is greatly impaired
in its ability to desensitize (33). As shown earlier, after stimulation
of the wild-type human NK2 receptors the translocation response decays
toward baseline within minutes (average 50% recovery time 83 ± 38 s, n = 25; compare Fig. 6 and 7C).
Application of short pulses of the agonist using a puffer pipette
resulted in an incomplete desensitization of the translocation response
as shown by the small decrease of the peak amplitudes. These individual
responses decayed significantly faster (45 ± 7 s,
n = 60, Fig. 7C) between applications of
stimuli than the response to a sustained stimulation, reflecting the
rapid dissociation of the ligand from the receptor (34). In contrast,
stimulation of a C-terminal truncated mutant human NK2 receptor, that
was reported to be transforming in Rat-1 fibroblasts, and which has been found to display prolonged coupling to PLC (33, 35,
36), induced a prolonged cytosolic translocation as assessed
by our FRET analysis (Fig. 7C). However, remarkably, in the
majority of cells, the FRET signal eventually slowly returned to
baseline (Fig. 7D; note the different time scale), with an
average 50% recovery time of 1365 ± 599 s
(n = 19) in the truncated receptor. This finding
indicates the existence of an alternative and much slower
desensitization mechanism that functions even in NK2 receptors that
lack the C terminus. The kinetics of this slow desensitization closely
paralleled those of receptor internalization (not shown), suggesting
that one of the main determinants for termination of NKA-induced PLC
signaling could be receptor internalization. Analysis of receptor
activity by monitoring PLC activity by FRET will greatly aid further
studies addressing these questions in more detail.
1 PH domains for analysis of activation-inactivation kinetics of PLC in single cells with high temporal resolution. This
method can be used at comparable expression levels of the PH domains
that are used for confocal detection, but has a number of significant
advantages over the latter. These include: (i) a significant decrease
in excitation intensity allowing prolonged experiments or very fast
sampling with little phototoxicity and photobleaching; (ii) suitability
for very flat cells such as fibroblasts and motile cells; (iii)
extendibility to record from cell populations as well as from small
subregions such as neurites; and (iv) a simpler detection hardware. The
FRET assay produces a fairly robust response that can be routinely
obtained in a variety of cell types.
1PH-GFP mainly reports on PI(4,5)P2 dynamics, although at high concentrations IP3 can also contribute to
translocation of the PH domains to the cytosol. Comparison of the
Ca2+ and FRET-recorded responses of several agonists of
GPCRs suggests that detecting PLC
1PH translocation by FRET provides
a more faithful reflection of receptor activity than the
Ca2+ signal and that little if any "desensitization" or
"uncoupling" occurs beyond the level of G proteins. FRET analysis
should be a very useful tool not only to explore the activation
patterns of PLC in individual cells but also for the dissection of
various mechanisms of receptor desensitization.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. W. Moolenaar, N. Divecha, J. Hallstead (Department of Biochemistry), and members of the Department of Cell Biology for discussions and comments on the manuscript, and Dr. F. Postma for comments and help with the figures. We also thank Drs. T. Meyer, J. Alblas, O. Kranenburg, A. Miyawaki, and R. Y. Tsien for plasmids.
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FOOTNOTES |
---|
* 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.
To whom correspondence should be addressed. Tel.:
31-20-512-1933; Fax: 31-20-512-1944; E-mail: kees@nki.nl.
§ Current address: Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan 320, 1098SM Amsterdam.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M007194200
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ABBREVIATIONS |
---|
The abbreviations used are:
[Ca2+]i, intracellular Ca2+;
PI(4, 5)P2, phosphatidylinositol 4,5-bisphosphate;
IP3, inositol 1,4,5-trisphosphate;
FRET, fluorescence
resonance energry transfer;
BK, bradykinin;
CFP, cyan fluorescent
protein;
CFP-PH, PLC1PH-CFP;
FRAP, fluorescence recovery after
photobleaching;
GFP, green fluorescent protein;
GFP-PH, PLC
1PH-GFP;
GPCR, G protein-coupled receptor;
LPA, lysophosphatidic acid;
NKA, neurokinin A;
PLC, phospholipase C;
PH, pleckstrin homology;
YFP, yellow fluorescent protein;
YFP-PH, PLC
1PH-YFP.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Thomas, A. P.,
Bird, G. S.,
Hajnoczky, G.,
Robb-Gaspers, L. D.,
and Putney, J. W., Jr.
(1996)
FASEB J.
10,
1505-1517 |
2. | Dupont, G., and Goldbeter, A. (1998) Bioessays 20, 607-610[CrossRef][Medline] [Order article via Infotrieve] |
3. | Chau, L. Y., Lin, T. A., Chang, W. T., Chen, C. H., Shue, M. J., Hsu, Y. S., Hu, C. Y., Tsai, W. H., and Sun, G. Y. (1993) J. Neurochem. 60, 454-460[Medline] [Order article via Infotrieve] |
4. | Stauffer, T. P., Ahn, S., and Meyer, T. (1998) Curr. Biol. 8, 343-346[Medline] [Order article via Infotrieve] |
5. |
Varnai, P.,
and Balla, T.
(1998)
J. Cell Biol.
143,
501-510 |
6. |
Varnai, P.,
Rother, K. I.,
and Balla, T.
(1999)
J. Biol. Chem.
274,
10983-10989 |
7. | Gray, A., Van Der, K. J., and Downes, C. P. (1999) Biochem. J. 344, 929-936[CrossRef][Medline] [Order article via Infotrieve] |
8. | Lankiewicz, L., Malicka, J., and Wiczk, W. (1997) Acta Biochim. Pol. 44, 477-489[Medline] [Order article via Infotrieve] |
9. | Pollok, B. A., and Heim, R. (1999) Trends Cell Biol. 9, 57-60[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Kranenburg, O.,
Poland, M.,
van Horck, F. P.,
Drechsel, D.,
Hall, A.,
and Moolenaar, W. H.
(1999)
Mol. Biol. Cell
10,
1851-1857 |
11. |
Balla, T.,
Nakanishi, S.,
and Catt, K. J.
(1994)
J. Biol. Chem.
269,
16101-16107 |
12. | Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Nature 388, 882-887[CrossRef][Medline] [Order article via Infotrieve] |
13. | Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R., and Piston, D. W. (1997) Biophys. J. 73, 2782-2790[Abstract] |
14. | Tilly, B. C., van Paridon, P. A., Verlaan, I., de Laat, S. W., and Moolenaar, W. H. (1988) Biochem. J. 252, 857-863[Medline] [Order article via Infotrieve] |
15. | van der Bend, R. L., de Widt, J., van Corven, E. J., Moolenaar, W. H., and van Blitterswijk, W. J. (1992) Biochem. J. 285, 235-240[Medline] [Order article via Infotrieve] |
16. | Zhang, C., Schmidt, M., Eichei-Streiber, C., and Jakobs, K. H. (1996) Mol. Pharmacol. 50, 864-869[Abstract] |
17. | Wijelath, E. S., Kardasz, A. M., Drummond, R., and Watson, J. (1988) Biochem. Biophys. Res. Commun. 152, 392-397[Medline] [Order article via Infotrieve] |
18. | Divecha, N., Banfic, H., and Irvine, R. F. (1991) EMBO J. 10, 3207-3214[Abstract] |
19. | Stephens, L., Jackson, T. R., and Hawkins, P. T. (1993) Biochem. J. 296, 481-488[Medline] [Order article via Infotrieve] |
20. | Myers, D. E., and Larkins, R. G. (1989) Cell Signal. 1, 335-343[Medline] [Order article via Infotrieve] |
21. | Tsien, R. Y. (1998) Annu. Rev. Biochem. 67, 509-544[CrossRef][Medline] [Order article via Infotrieve] |
22. | Tall, E. G., Spector, I., Pentyala, S. N., Bitter, I., and Rebecchi, M. J. (2000) Curr. Biol. 10, 743-746[CrossRef][Medline] [Order article via Infotrieve] |
23. | Jalink, K., Eichholtz, T., Postma, F. R., van Corven, E. J., and Moolenaar, W. H. (1993) Cell Growth & Differ. 4, 247-255[Abstract] |
24. | van Leeuwen, F. N., van Delft, S., Kain, H. E., van der Kammen, R. A., and Collard, J. G. (1999) Nat. Cell Biol. 1, 242-248[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Hirose, K.,
Kadowaki, S.,
Tanabe, M.,
Takeshima, H.,
and Iino, M.
(1999)
Science
284,
1527-1530 |
26. | Wiedemann, C., Schafer, T., and Burger, M. M. (1996) EMBO J. 15, 2094-2101[Abstract] |
27. |
Hunyady, L.,
Merelli, F.,
Baukal, A. J.,
Balla, T.,
and Catt, K. J.
(1991)
J. Biol. Chem.
266,
2783-2788 |
28. | Jalink, K., and Moolenaar, W. H. (1992) J. Cell Biol. 118, 411-419[Abstract] |
29. | Lemmon, M. A., Ferguson, K. M., O'Brien, R., Sigler, P. B., and Schlessinger, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10472-10476[Abstract] |
30. |
Paltauf-Doburzynska, J.,
Frieden, M.,
Spitaler, M.,
and Graier, W. F.
(2000)
J. Physiol.
524,
701-713 |
31. |
Zhu, D. M.,
Tekle, E.,
Huang, C. Y.,
and Chock, P. B.
(2000)
J. Biol. Chem.
275,
6063-6066 |
32. | Tilly, B. C., Tertoolen, L. G., Lambrechts, A. C., Remorie, R., de Laat, S. W., and Moolenaar, W. H. (1990) Biochem. J. 266, 235-243[Medline] [Order article via Infotrieve] |
33. |
Alblas, J.,
van Etten, I.,
Khanum, A.,
and Moolenaar, W. H.
(1995)
J. Biol. Chem.
270,
8944-8951 |
34. |
Vollmer, J. Y.,
Alix, P.,
Chollet, A.,
Takeda, K.,
and Galzi, J. L.
(1999)
J. Biol. Chem.
274,
37915-37922 |
35. | Alblas, J., van Etten, I., and Moolenaar, W. H. (1996) EMBO J. 15, 3351-3360[Abstract] |
36. |
Alblas, J.,
van Corven, E. J.,
Hordijk, P. L.,
Milligan, G.,
and Moolenaar, W. H.
(1993)
J. Biol. Chem.
268,
22235-22238 |
37. | Miyawaki, A., Llopis, J., Mizuno, H., Jalink, K., and Tsien, R. (2000) Calcium Signaling: A Practical Approach , pp. 3-16, Oxford University Press, Oxford, in press |