Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-4510
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Abstract |
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Phosphatidylinositol 4,5-bisphosphate
(PtdIns[4,5]P2) pools that bind pleckstrin homology
(PH) domains were visualized by cellular expression of
a phospholipase C (PLC) PH domain-green fluorescent protein fusion construct and analysis of confocal images in living cells. Plasma membrane localization of
the fluorescent probe required the presence of three
basic residues within the PLC
PH domain known to
form critical contacts with PtdIns(4,5)P2. Activation of
endogenous PLCs by ionophores or by receptor stimulation produced rapid redistribution of the fluorescent
signal from the membrane to cytosol, which was reversed after Ca2+ chelation. In both ionomycin- and agonist-stimulated cells, fluorescent probe distribution
closely correlated with changes in absolute mass of
PtdIns(4,5)P2. Inhibition of PtdIns(4,5)P2 synthesis by quercetin or phenylarsine oxide prevented the relocalization of the fluorescent probe to the membranes after
Ca2+ chelation in ionomycin-treated cells or during
agonist stimulation. In contrast, the synthesis of the
PtdIns(4,5)P2 imaged by the PH domain was not sensitive to concentrations of wortmannin that had been
found inhibitory of the synthesis of myo-[3H]inositol-
labeled PtdIns(4,5)P2. Identification and dynamic imaging of phosphoinositides that interact with PH domains
will further our understanding of the regulation of such
proteins by inositol phospholipids.
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Introduction |
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PLECKSTRIN homology (PH)1 domains are ~120 amino
acid-long protein modules that were first described
in pleckstrin, the major protein kinase C substrate
in platelets (16). PH domains have since been identified in
several key regulatory proteins with characteristic structural features that include two orthogonal sheets that
form a sandwich with an
helix at the COOH terminus, and variable loops that create a highly charged surface (11, 16). It has been generally accepted that PH domains provide a structural basis for the interaction of certain regulatory proteins with membranes (23). The search for proteins
that would bind and regulate proteins via PH domains has
generally been unsuccessful, although in some cases, such
as the
-adrenergic receptor kinase, the region that confers regulation by G protein
subunits overlaps with the
PH domain (28). On the other hand, evidence is accumulating to suggest that PH domains of several proteins interact with membrane phosphoinositides, and that this interaction is critical to the regulation of those proteins (11,
16, 23).
The PH domains of several proteins have been shown to
bind phosphoinositides in a specific manner with micromolar affinities in vitro (29, 31). For example, the PH domain of phospholipase C (PLC)1 binds inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) and associates with lipid vesicles
containing phosphatidylinositol 4,5-bisphosphate (PtdIns-
[4,5]P2), but only weakly binds other inositol phosphates
and PtdIns(4)P (24). Similarly, the PH domain of the Akt
protein kinase appears to bind PtdIns(3,4)P2 but not PtdIns(4,5)P2 (13). However, some PH domains (such as that
of dynamin) show much weaker and less specific interactions with inositol phospholipids (31), raising the question
of whether these binding forces alone are sufficient to anchor those proteins to membranes. Whereas in vitro binding studies have been valuable in determining the ability
of PH domains to associate with inositol phospholipids,
the dependency of the specificity of their binding on lipid
micelle composition and detergent environment (21) emphasizes the need to assess these interactions in intact
cells. Another question that is raised by studies on PH domain-phosphoinositide interactions is how the binding of
PH domains to the inositide headgroup affects the primary
signaling functions of PtdIns(4,5)P2, particularly its availability to PLC enzymes or to PtdIns 3 kinases. It is known
that other proteins that bind PtdIns(4,5)P2, such as profilin, greatly impair the ability of PLC enzymes to hydrolyze this lipid (14).
The present studies were designed to examine whether
specific association of the PLC PH domain (PHPLC
)with
PtdIns(4,5)P2 can be exploited to visualize the cellular distribution of phosphoinositides, that are capable of binding
PH domains in intact cells, and to follow their changes in
real time after stimulation. Our results, obtained using a
fusion protein of the PHPLC
coupled to the green fluorescent protein (GFP), provide imaging of the dynamics of
membrane PtdIns(4,5)P2 pools that interact with PH domains. They also reveal a prominent difference between
the regulation of these pools compared with those labeled
with myo-[3H]inositol.
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Materials and Methods |
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Materials
Angiotensin II (Ang II; human) was obtained from Pheninsula Laboratories, Inc. (San Carlos, CA) and PDGF (recombinant, AB) from Life Technologies, Inc. (Gaithersburg, MD). Thapsigargin, ionomycin, and 1,2-bis(2-aminophenoxy)ethane N,N,N',N-tetraacetic acid (BAPTA) were purchased from Calbiochem-Novabiochem, Corp. (La Jolla, CA), and wortmannin was a gift from Kyowa Hakko Laboratories (Tokyo, Japan). 2,3-Dimercaptopropanol (BAL), phenylarsine oxide, and quercetin were obtained from Sigma Chemical Co. (St. Louis, MO). Myo-[3H]inositol (68 Ci/mmol) and [3H]inositol-1,4,5-trisphosphate (48 Ci/mmol) was from Amersham Corp. (Arlington Heights, IL). All other chemicals were of HPLC or analytical grade.
Plasmid Constructs
The PH domains of PLC1 (1-170), Bruton's tyrosine kinase (1-177), Akt
protein kinase (1-167), and dynamin (508-652) were amplified with the
Advantage Klentaq polymerase mix (CLONTECH Labs, Inc., Palo Alto,
CA) from human cDNAs (marathon cDNA from brain and K562 leukemia cells; CLONTECH Labs, Inc.) with the following primer pairs:
PLC: 5'-GGCATGGACTCGGGCCGGGACTTCCTG-3',
5'-AAGATCTTCCGGGCATAGCTGTCG-3';
Btk: 5'-CCAAGTCCTGGCATCTCAATGCATCTG-3',
5'-TGGAGACTGGTGCTGCTGCTGGCTC-3';
Akt: 5'-GTCAGCTGGTGCATCAGAGGCTGTG-3',
5'-CACCAGGATCACCTTGCCGAAAGTGCC-3';
Dyn: 5'-ATGCTCAGCAGAGGAGCAACCAGATG-3',
5'-GAGTCCACAAGATTCCGGATGGTCTC-3'.
The amplified products were subcloned into the PGEM-Easy T/A cloning
vector (Promega Corp., Madison, WI) and sequenced with dideoxy sequencing (thermosequenase; Amersham Corp.). A second amplification
reaction was performed from these plasmids with nested primers that contained restriction sites for appropriate cloning into the pEGFP-N1 (PLC,
Btk, and Akt) or pEGFP-C1 (dynamin) plasmids (CLONTECH Labs,
Inc.) to preserve the reading frame. Plasmids were transfected into COS-7
cells or NIH-3T3 cells and cell lysates were resolved by SDS-PAGE followed by Western blot analysis for the presence of the GFP fusion proteins using a polyclonal antibody against GFP (CLONTECH Labs, Inc.).
Mutations were created in the PHPLC-GFP fusion plasmid by the
QuickChangeTM mutagenesis kit (Stratagene, La Jolla, CA). For practical
purposes, a SalI site was introduced into the PH domain sequence which
changed S34 to a T but this substitution did not change any characteristic
compared with the wild-type protein. All mutations were confirmed by
dideoxy sequencing and the expression of the fusion protein by Western
blot analysis.
Transfection of Cells for Confocal Microscopy
Cells were plated onto poly-L-lysine-coated 30-mm-diam circular cover slips at a density of 5 × 104 cells/dish and cultured for 3 d before transfection with plasmid DNAs (1 µg/ml) using the Lipofectamine reagent (10 µg/ml; Life Technologies, Inc.) and OPTI-MEM (Life Technologies, Inc.). 48 h after transfection cells were washed twice with a modified Krebs-Ringer solution, containing (mM): NaCl 120, KCl 4.7, CaCl2 1.2, MgSO4 0.7, glucose 10, Na-Hepes 10, pH 7.4, and the coverslip was placed into a chamber that was mounted on a heated stage with the medium temperature kept at 33°C. Cells were incubated in 1 ml of the Krebs-Ringer buffer and the stimuli were added in 0.5 ml prewarmed buffer after removing 0.5 ml medium from the cells. Cells were examined in an inverted microscope under a 40× oil-immersion objective (Nikon, Inc., Melville, NY) and a BioRad laser confocal microscope system (MRC-1024) with the Lasersharp acquisition software (Bio-Rad Laboratories, Hercules, CA). All pictures presented are original recordings without postacquisition enhancing.
Analysis of Inositol Lipids
Inositol phospholipids were analyzed from either COS-7 cells transfected with the AT1a Ang II receptor together with selected GFP-PH domain fusion constructs, or from untransfected NIH-3T3 cells after labeling with myo-[3H]inositol for 24 h (COS-7 cells) or 48 h (NIH-3T3 cells) as previously described (4, 17). [3H]Inositol phosphates were analyzed by HPLC (17) or, in the COS cell experiments, by Dowex minicolumns. PtdIns(4,5)P2 mass was determined from NIH-3T3 cells and bovine adrenal glomerulosa (BAG) cells that were cultured on 12-well plates. After stimulation at 33°C, reactions were terminated with 400 µl ice-cold perchloric acid (5% final) followed by an acidic lipid extraction (3). Samples were taken to dryness with N2 stream and were subjected to alkaline hydrolysis to liberate Ins(1,4,5)P3 from PtdIns(4,5)P2 for quantitation in a radioreceptor assay, essentially as described in Challis (see reference 6) using bovine adrenocortical membranes (15) and [3H]Ins(1,4,5)P3 (Amersham Corp.).
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Results |
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Plasma Membrane Localization of the PLC
PH Domain Requires Interaction with PtdIns(4,5)P2
Among the known PH domains reported to interact with
PtdIns(4,5)P2, the PH domain of PLC1 has the highest affinity (12). Therefore, we chose this PH domain to create a
fluorescent probe for imaging purposes and fused it to the
NH2 terminus of GFP. Expression of GFP alone showed
no specific cellular localization and was present in the cytosol, as expected from a protein of its size (27 kD) lacking
a localization signal. Addition of the PH domain of PLC
1
(1-170) to the GFP (PHPLC
-GFP) was sufficient to localize the construct to the plasma membrane when transiently expressed in various cell types (Fig. 1). A small
amount of fluorescence was always present in the cytosol
and the nucleus, but very little in internal membranes
apart from some vesicular structures that could be membrane invaginations. Although this result was consistent with binding of the construct to membrane PtdIns(4,5)P2,
it did not exclude the possibility of other interaction(s) between the PH domain and some other membrane component(s). Therefore, we mutated each of three critical basic
residues, K30, K32, and R40, within the PH domain that
have been previously found, based on the crystal structure
of the molecule, to contribute to the high affinity binding
of PLC
to PtdIns(4,5)P2 (12). Each of the three mutations
(K30L, K32L, and R40L) prevented the plasma membrane localization of the construct, whereas mutation of a
non-charged residue in the same region (S34T) had no effect (Fig. 1). These results indicate that the plasma membrane localization of this fluorescent probe is based on its
interaction with PtdIns(4,5)P2 through the PLC
PH domain, and that the lipid is present predominantly in the
plasma membrane.
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PLC Activation by Ca2+ Releases the Fluorescent Probe from the Plasma Membrane into the Cytosol
If the distribution of PHPLC-GFP truly maps the cellular
PtdIns(4,5)P2 pools, then hydrolysis of these phospholipids by endogenous PLCs should change the distribution of
the fusion protein to report those phosphoinositide changes.
PLC was activated in intact NIH-3T3 cells by treatment
with ionomycin (10 µM) in the presence of external Ca2+
(1.2 mM) to allow a large increase in cytosolic Ca2+ concentration ([Ca2+]i) with sequential activation of PLC
isozymes (30). As shown in Fig. 2, this manipulation, indeed, caused rapid disappearance of fluorescence from the
plasma membrane and its simultaneous appearance in the
cytosol, consistent with PLC-mediated hydrolysis of the membrane PtdIns(4,5)P2 at high [Ca2+]i. Once released into the
cytosol, this 40-kD fusion construct only slowly appeared
in the nucleus. Preincubation of the cells with neomycin
(10 mM) for 10 min to inhibit the hydrolysis of PtdIns(4,5)P2 (8), completely prevented the release of the fluorescent signal from the membranes in the majority
(>90%) of cells (not shown).
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Subsequent chelation of external Ca2+ by either BAPTA or EGTA (2 mM) caused the slow reappearance of the fluorescence at the plasma membrane and, most notably, in some intracellular membrane structures (Fig. 2 B). In many cells, the first signs of relocalization were the appearance of bright foci at perinuclear membranes and in other intracellular structures that eventually translocated to the plasma membrane (Fig. 2 B). These changes were considered to reflect the resynthesis of the phosphoinositide pools as PLC activity declined in concert with the falling [Ca2+]i (see below).
To analyze whether Ca2+ release from intracellular stores without Ca2+ influx was sufficient to cause the breakdown of membrane phosphoinositides that bind PH domains, we applied ionomycin to the transfected cells in Ca2+-free medium. Under these conditions ionomycin failed to evoke a lipid signal that could be detected by the redistribution of the fluorescent construct (not shown). To calculate the concentration of Ca2+ that was required to activate PtdIns(4,5)P2 breakdown in the presence of ionomycin, we titrated extracellular Ca2+ in the presence of 2 mM BAPTA and measured [Ca2+]i under identical conditions in Fura-2-loaded NIH-3T3 cells. Based on these measurements, the concentration of Ca2+ that was required to activate PtdIns(4,5)P2 hydrolysis (assessed by the release of fluorescence from the membrane into the cytosol) was around 10 µM. The need for relatively high Ca2+ to trigger this process was also indicated by the finding that lower concentrations of ionomycin (1 µM), that evoke rapid Ca2+ release with consequential activation of the capacitive Ca2+ entry pathway, did not induce translocation of the fluorescent construct (not shown). Similarly, thapsigargin (100 nM) or PDGF (25-100 ng/ml), both of which evoked moderate [Ca2+]i increases in NIH-3T3 cells, had no effect on the distribution of the fusion protein (not shown).
Ca2+-induced Changes in Cellular
PtdIns(4,5)P2 Mass Closely Correlate with Plasma
Membrane Localization of PLC PH Domain
To examine how the cellular PtdIns(4,5)P2 pool is affected by the same manipulations that caused the redistribution of the fusion construct, we analyzed the total mass of this phospholipid from NIH-3T3 cells after ionomycin treatment and subsequent Ca2+ chelation. The total mass of PtdIns(4,5)P2 in cultured cells was measured by lipid extraction followed by alkaline hydrolysis to liberate Ins(1,4,5)P3, which was then quantitated by a radioreceptor assay (6). As shown in Fig. 2 C, ionomycin (10 µM) caused rapid breakdown of PtdIns(4,5)P2, and upon Ca2+ chelation the level of this phospholipid rapidly returned nearly to its original value within 5 min.
To analyze the correlation between PtdIns(4,5)P2 changes and the redistribution of fluorescence, we needed to quantify membrane localization. Dividing the fluorescence of the membrane with that of the adjacent cytosol (values were taken from line intensity histograms on series of images) proved to be a useful index of membrane-association of the construct. As shown in Fig. 2 D, PtdIns(4,5)P2 changes and membrane localization of the fluorescent construct correlated remarkably closely during manipulation of intracellular Ca2+, except for a clear delay observed in the plasma membrane reappearance of the fluorescence. This, however, was preceded by localization of the construct at intracellular sites (Fig. 2 D), indicating that the earliest sites of PtdIns(4,5)P2 synthesis are at intracellular membrane compartments.
Ca2+-mobilizing Hormones Can Also Initiate
the Breakdown of the PtdIns(4,5)P2 Pool That Binds
PLC PH Domain
To investigate whether the PtdIns(4,5)P2 pools that are visualized by PHPLC-GFP show any change after agonist
stimulation, we examined the effect of the calcium mobilizing hormone, Ang II, on the redistribution of the fluorescent probe in various cells. The fluorescent construct
was transfected together with the AT1a Ang II receptor
into COS-7 or NIH-3T3 cells, or alone into primary cultures of adrenal glomerulosa cells that express endogenous AT1 receptors. Stimulation of the cells with Ang II caused a rapid release of the fluorescent signal from the
plasma membrane to the cytosol in each of the three cell
types (Figs. 3 and 4). This process was very rapid and, in
contrast to ionomycin stimulation, was only transient with
the construct partially relocalizing to the plasma membrane in spite of the sustained stimulation. Simultaneous
changes in PtdIns(4,5)P2 mass measured in BAG cells showed again a close correlation between changes in membrane-associated fluorescence and the absolute amounts
of this lipid (Fig. 4, B and C). In contrast to the effects of
ionomycin, the effect of Ang II on PHPLC
-GFP distribution was still observed in a medium nominally free of Ca2+,
although this effect was very short-lived, the fluorescence being relocalized to the membrane within 2 min (not
shown). These results indicate that stimulation of a G protein-coupled receptor can cause the breakdown of the PtdIns(4,5)P2 pool(s) that bind PH domains.
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Inhibition of the Resynthesis of PtdIns(4,5)P2
Prevents Relocalization of the PLC PH Domain to the
Plasma Membrane
To further examine the interaction between the PLC PH
domain and the plasma membrane phosphoinositides, we
used inhibitors that have been reported to inhibit the formation of PtdIns(4,5)P2 from PtdIns by inhibiting PtdIns 4 and PtdIns(4)P 5 kinases (34). These inhibitors were tested
for their ability to prevent the resynthesis of PtdIns(4,5)P2
after ionomycin treatment and subsequent Ca2+ chelation.
As shown in Fig. 5, both quercetin (100 µM) and phenylarsine oxide (PAO; 100 µM) were each able to prevent the
resynthesis of PtdIns(4,5)P2 as measured by the total mass
of this phospholipid. The inhibitory effect of PAO was
completely reversed by DTT, only partially by BAL and
not by
-mercaptoethanol (1 mM each). These same treatments equally affected the relocalization of the fluorescent probe to the plasma membrane upon Ca2+ chelation after
ionomycin treatment: both quercetin and PAO prevented the reappearance of the fluorescent signal in membranes,
and DTT but not
-mercaptoethanol, were each able to
antagonize the inhibitory effect of PAO (Fig. 5 B). Interestingly, although BAL only partially restored PAO-inhibited PtdIns(4,5)P2 resynthesis, it fully restored the translocation of PHPLC
-GFP. In bovine glomerulosa cells that
were pretreated with quercetin (or with PAO), Ang II induced a more permanent release of the fluorescent construct into the cytosol instead of the transient translocation observed in control cells, indicating that once the
resynthesis of the PtdIns(4,5)P2 is inhibited, the Ca2+-mobilizing agonist causes a more complete degradation of
membrane PtdIns(4,5)P2 (Fig. 4, B and C).
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The Resynthesis of PtdIns(4,5)P2 That Binds PH Domains Is Not Sensitive to WT Inhibition
Interestingly, high concentrations of wortmannin (WT; 10 µM) shown to inhibit type III PtdIns 4-kinases (27) did not affect relocalization of the fluorescent construct after Ca2+ chelation in ionomycin-treated cells, although it partially inhibited PtdIns(4,5)P2 resynthesis measured by the mass assay (Fig. 5). Similarly, 10 µM WT had no effect on the Ang II-induced transient translocation of the fluorescent signal (not shown). This result contrasted our previous finding that the metabolically labeled hormone-sensitive PtdIns(4,5)P2 pools are maintained by WT-sensitive PtdIns 4-kinase(s) in several agonist-stimulated cells and that stimulation of these cells in the presence of 10 µM WT leads to the depletion of these phosphoinositide pools in myo-[3H]inositol-labeled cells (27).
These findings suggest that although the myo-[3H]inositol-labeled, agonist-sensitive PtdIns(4,5)P2 pools are formed by WT-sensitive PtdIns 4-kinase(s), additional PtdIns(4,5)P2 pools that bind PH domains are synthesized by WT-insensitive mechanisms that are only inhibited by less-specific PI kinase inhibitors, such as quercetin or PAO (34).
Relationship between Ca2+- and Agonist-responsive PtdIns(4,5)P2 Pools That Bind PH Domains
The transient nature of the release of PHPLC-GFP from
the plasma membrane after Ang II stimulation indicated the
resynthesis of the membrane PtdIns(4,5)P2 during the
action of the hormone. We examined whether such newly
synthesized PtdIns(4,5)P2 having reassociated with PHPLC
-
GFP is available for Ca2+-induced hydrolysis. As shown in
Fig. 6 A, ionomycin caused further and complete hydrolysis of PtdIns(4,5)P2 in Ang II-stimulated cells as assessed
by the release of PHPLC
-GFP from the membrane. Similar results were obtained when Ang II action was terminated by the AT1 receptor antagonist, losartan (10
5) before the addition of ionomycin (not shown). The reciprocal experiment was also performed; bovine glomerulosa cells
expressing PHPLC
-GFP were subjected to one round of
treatment with ionomycin (10 µM) and Ca2+ chelation (5 mM EGTA) to allow complete hydrolysis of PtdIns(4,5)P2 and to allow its resynthesis to take place in the presence of the fluorescent construct. After the removal of ionomycin,
(by several washes with a solution containing BSA), such
cells still showed an Ang II-stimulated redistribution of
fluorescence that did not appear to be significantly different from the changes observed in naive cells (Fig. 6 B).
These results indicated that the Ca2+- and agonist-responsive pools of PtdIns(4,5)P2 that are available for binding to
PH domains are largely overlapping and that the resynthesized PtdIns(4,5)P2 is still available for agonist-induced hydrolysis.
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PH Domains Can Inhibit the Agonist-induced Hydrolysis of Myo-[3H]inositol-labeled PtdIns(4,5)P2
Since PH domains bind to and therefore cover the inositol
phosphate headgroup of phosphoinositides, they can hinder its accessibility and hydrolysis by the agonist-regulated
PLC enzymes. Alternatively, PH domains may interfere
with the membrane localization of the PLC enzymes that
are activated by an agonist. Although all of the known
PLC isoenzymes contain a PH domain, its affinity and specificity toward inositides has not been analyzed in every case (10). To test the possibility that PH domains interfere with agonist-induced Ins(1,4,5)P3 formation from
[3H]inositol-labeled PtdIns(4,5)P2, we overexpressed various PH domain-GFP constructs in COS-7 cells together
with the AT1a Ang II receptor and measured Ang II-stimulated [3H]inositol phosphate formation in cells prelabeled by myo-[3H]inositol. Fig. 7 A shows that PLC PH
domain greatly inhibited Ang II-stimulated formation of
[3H]inositol phosphates and that other PH domains with
low affinity for PtdIns(4,5)P2, such as that of the Bruton's
tyrosine kinase or the Akt protein kinase, as well as that of
dynamin (13, 18, 31), showed no similar inhibitory effect.
Fluorescent constructs containing these PH domains did
not show the same membrane localization as those with
the PLC
PH domain (not shown). Also, mutations within
the PH domain of PLC
that prevented its interaction with PtdIns(4,5)P2, and hence its membrane localization, failed
to inhibit Ang II-induced inositol phosphate production
(Fig. 7 B). The corollary of this finding is that fluorescent
PH domain constructs with high enough affinity to "label"
PtdIns(4,5)P2 pools are most likely to also interfere with
the agonist-sensitive phosphoinositide pools, since their
binding to PtdIns(4,5)P2 impedes their access to the relevant PLC enzymes.
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Discussion |
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The present experiments demonstrate that PH domains
which bind PtdIns(4,5)P2 with high affinity and specificity,
such as that of PLC1, can be used to visualize certain PtdIns(4,5)P2 pools in single living cells. Decreasing the affinity of the PH domain by mutation of any one of three basic
residues known to form contacts with the inositol phosphate headgroup (12, 35) was sufficient to eliminate membrane localization of the construct. Also, PH domains with
lower affinity for PtdIns(4,5)P2 such as that of the GTP-binding protein, dynamin, or with different specificity such
as those of the Bruton's tyrosine kinase and the protein kinase, Akt, did not show the clear plasma membrane localization that was observed with the PLC
PH domain. The
high concentration of the fluorescent probe at the plasma
membrane was consistent with previously established
views that PtdIns(4,5)P2 is most abundant in the plasma
membrane (26). The lack of localization to intracellular membranes, including the nuclear membrane where a separate inositide system has been described (7), suggests that
in quiescent cells these membranes either contain only
small amounts of this lipid, or that such pools are not accessible to the expressed PH domain. Since the interaction
of the PH domain is formed with the inositol phosphate
headgroup of PtdIns(4,5)P2, hydrolysis of the lipid by PLC
is reflected in the release of the fluorescent probe from the
plasma membranes to the cytosol. Such change in localization was dramatically demonstrated when endogenous
PLC was activated by Ca2+ influx via Ca2+ ionophores.
After Ca2+ chelation, when PLC activity subsided and PtdIns(4,5)P2 resynthesis began to take place, the appearance
of bright spots at perinuclear structures preceded the localization of the fluorescence to the plasma membrane, indicating that some PtdIns(4,5)P2 synthesis also occurs in
intracellular membranes.
This novel methodology, which allowed analysis of the
regulation of cellular PtdIns(4,5)P2 pools from the viewpoint of PH domains showed that stimulation of a G protein-coupled Ca2+-mobilizing receptor, the AT1a Ang II
receptor, was also able to activate the hydrolysis of PH domain-tagged PtdIns(4,5)P2. In a recent report, Stauffer et
al. (see reference 32) reported that stimulation of PAF receptors (another G protein-coupled receptor) in a basophilic leukaemia cell line also caused a transient translocation of PLC PH domain using a similar methodology.
Interestingly, activation of PLC
by PDGF in NIH-3T3
cells (in this study), or by FC
RI stimulation in RBL cells
(33) did not cause any visible change in the distribution of
a PH domain-GFP construct (the PH domain of pleckstrin
was used in reference 33). Whether this reflects the inability of PLC
to hydrolyze PtdIns(4,5)P2 when it is covered
by a PH domain remains to be determined.
Overexpression of the PLC PH domain was also found
to inhibit agonist-induced formation of myo-[3H]inositol-
labeled inositol phosphates in transfected COS-7 cells, and
this effect was closely correlated with the ability of the construct to localize to the membrane. Similar finding was
presented in a recent report, in which pleckstrin, via its PH
domain, was shown to inhibit [3H]inositol phosphate formation in transfected COS-1 or HEK 293 cells regardless
of the type of PLC that was activated (1). These results
suggest that expressed PH domains also can interfere with
the agonist-regulated PLC activation mechanism, either
by masking the PtdIns(4,5)P2 headgroup from the enzyme's catalytic site or by competing with the PH domains
of the PLC enzymes to inhibit their localization to the
membranes. These results also emphasize that the PLC activity detected in cells is greatly influenced by membrane
components that interact with phosphoinositides, and that
the use of cell-free systems and artificial substrates cannot reveal this aspect of PLC regulation.
An important finding of the present study was the inability of WT (at µM concentrations that inhibit type III
PtdIns 4 kinases) to prevent the resynthesis of the PtdIns(4,5)P2 pools available for binding of the PLC PH domain in both ionomycin- and agonist-stimulated cells. The
same treatment completely prevented the synthesis of myo-[3H]inositol-labeled PtdIns(4,5)P2 in Ang II-stimulated adrenal glomerulosa cells (27), and also blocks the
resynthesis of [3H]PtdIns(4,5)P2 after chelation of Ca2+ in
ionomycin-treated NIH-3T3 cells (Varnai, P., and T. Balla, unpublished observations). This finding raised the possibility that the agonist-sensitive myo-[3H]inositol-labeled
PtdIns(4,5)P2 pools are not completely identical to those
that are imaged by the fluorescent PH domain of PLC
, although both respond to agonist stimulation. Heterogeneity of phosphoinositide pools have been described earlier
in a few reports. These studies indicated that some of the
metabolically labeled PtdIns and PtdIns(4,5)P2 pools are
not sensitive to agonist stimulation (22) and conversely,
some PtdIns(4,5)P2 that is not labeled metabolically is still
subject to PLC-mediated hydrolysis (20). The existence
of a metabolically hyperactive hormone-sensitive PtdIns(4,5)P2 pool, however, could not be substantiated (25). Our results indicate that, unlike the [3H]inositol-labeled
PtdIns(4,5)P2 pools, the PtdIns(4,5)P2 pool(s) that bind
PH domains are not synthesized by WT-sensitive type III
PtdIns 4 kinases. The highly abundant, tightly membrane-bound type II PtdIns 4 kinase (5), which is not sensitive to
even high concentrations of WT (9) is a good candidate for
synthesizing the inositides that bind PH domains. Clearly,
more experiments will be required to find the explanation
for the apparent discrepancy between the regulation of
myo-[3H]inositol-labeled and PH domain-imaged PtdIns(4,5)P2.
An additional possibility to be considered in understanding the current results is that the Ins(1,4,5)P3 that is
formed after agonist stimulation competes for the PH domain of PLC (and of other proteins), and contributes to
the release of these proteins from the plasma membrane,
especially when PtdIns(4,5)P2 levels are decreasing. Such
competition by Ins(1,4,5)P3 with PtdIns(4,5)P2 for the PH
domain of PLC
has been demonstrated and also found to
inhibit the catalytic efficiency of the enzyme (2, 19). Since
the only known major source of Ins(1,4,5)P3 is PtdIns(4,5)P2, this competing effect of Ins(1,4,5)P3 would distort the imaging results if there were substantial amounts
of Ins(1,4,5)P3 formed from PtdIns(4,5)P2 pools that did
not bind PH domains in their initial steady-state before
stimulation. Nevertheless, this is an open possibility and a
better assessment of such a displacing effect of Ins(1,4,5)P3
requires additional studies with GFP-fused PH domains.
In summary, the present studies demonstrate that isolated PH domains fused to the GFP are capable of recognizing phosphoinositides in living cells with remarkable specificity. These interactions allow visualization of the spatiotemporal changes in phosphoinositides at the single cell level. Analysis of cellular PtdIns(4,5)P2 with this method reveals that the pools that bind PH domains my not be identical to those that can be metabolically labeled with myo-[3H]inositol. Studies on the receptor-mediated control of the PtdIns(4,5)P2 pools that bind PH domains will help to understand the manner in which phosphoinositides regulate various signaling processes via protein PH domains.
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Footnotes |
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Received for publication 4 May 1998 and in revised form 6 August 1998.
The skillful technical work of Ms. Y. Zhang is greatly appreciated.
Address all correspondence to T. Balla, National Institutes of Health,
Bldg. 49, Rm. 6A35, 49 Convent Drive, Bethesda, MD 20892-4510. Tel.:
(301) 496-2136. Fax: (301) 480-8010. E-mail: tambal{at}box-t.nih.gov
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Abbreviations used in this paper |
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Ang II, angiotensin II; BAG, bovine adrenal glomerulosa; BAL, 2,3-dimercaptopropanol; BAPTA, 1,2-bis(2-aminophenoxy)ethane N,N,N',N-tetraacetic acid; GFP, green fluorescent protein; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; PAO, phenylarsine oxide; PH, pleckstrin homology; PLC, phospholipase C; PtdIns, phosphatidylinositol; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; WT, wortmannin.
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