Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130.

Abstract

The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal alpha-helix and the short loop between the beta6-beta7 sheets of the PLCdelta(1)PH domain, in addition to its InsP(3)-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCdelta(1)PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

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Inositol Lipid Binding and Membrane Localization of Isolated
Pleckstrin Homology (PH) Domains
STUDIES ON THE PH DOMAINS OF PHOSPHOLIPASE C ?1AND p130*
Received for publication, October 5, 2001, and in revised form, May 17, 2002
Published, JBC Papers in Press, May 17, 2002, DOI 10.1074/jbc.M109672200
Pe ´ter Va ´rnai‡, Xuena Lin§¶, Sang Bong Lee?, Galina Tuymetova‡, Tzvetanka Bondeva‡,
Andras Spa ¨t**‡‡, Sue Goo Rhee?, Gyo ¨rgy Hajno ´czky§¶, and Tamas Balla‡§§
From the ‡Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health,
Bethesda, Maryland 20892, the §Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107, ?Laboratory of Biochemistry, NHLBI, National Institutes of Health,
Bethesda, Maryland 20892, and **Department of Physiology and Laboratory of Cellular and Molecular Physiology,
Semmelweis University Medical School, H-1444 Budapest, Hungary
The relationship between the ability of isolated pleck-
strin homology (PH) domains to bind inositol lipids or
soluble inositol phosphates in vitro and to localize to
cellular membranes in live cells was examined by com-
paring the PH domains of phospholipase C?1(PLC?1)
and the recently cloned PLC-like protein p130 fused to
the green fluorescent protein (GFP). The prominent
membrane localization of PLC?1PH-GFP was paralleled
with high affinity binding to inositol 1,4,5-trisphosphate
(InsP3) as well as to phosphatidylinositol 4,5-bisphos-
phate-containing lipid vesicles or nitrocellulose mem-
brane strips. In contrast, no membrane localization was
observed with p130PH-GFP despite its InsP3and phos-
phatidylinositol4,5-bisphosphate-binding
being comparable with those of PLC?1PH-GFP. The N-
terminal ligand binding domain of the type I InsP3re-
ceptor also failed to localize to the plasma membrane
despite its 5-fold higher affinity to InsP3than the PH
domains. By using a chimeric approach and cassette
mutagenesis, the C-terminal ?-helix and the short loop
between the ?6–?7 sheets of the PLC?1PH domain, in
addition to its InsP3-binding region, were identified as
critical components for membrane localization in intact
cells. These data indicate that binding to the inositol
phosphate head group is necessary but may not be suf-
ficient for membrane localization of the PLC?1PH-GFP
fusion protein, and motifs located within the C-terminal
half of the PH domain provide auxiliary contacts with
additional membrane components.
properties
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2),1a minor
phospholipid component of the plasma membrane, is a key
regulator of several cellular processes. PI(4,5)P2is 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).
PI(4,5)P2is also phosphorylated by class I PI 3-kinases to form
PI(3,4,5)P3, which controls membrane recruitment and the
functions of several important signaling proteins (3). PI(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 (see Ref. 6), provid-
ing a link between the plasma membrane and the cortical
cytoskeleton (7). PI(4,5)P2has been implicated in several forms
of membrane remodeling events, including the fusion of secre-
tory vesicles with the plasma membrane (8), clathrin-mediated
endocytosis (9–11), and membrane recovery by endocytosis
during neurotransmitter release (12) (also see Ref. 13 for a
review). Such diverse functions must rely upon interaction of
the lipid with a large number of regulatory molecules. Most
proteins that bind PI(4,5)P2contain a sequence composed of
basic residues that provide electrostatic interaction with the
phosphate groups of the inositol ring (6). Recent advances
revealing the three-dimensional structures of several protein
motifs that bind phosphoinositides offer a deeper insight into
their molecular recognition (14). One of the first protein mod-
ules capable of binding membrane PI(4,5)P2was identified in
pleckstrin, the major protein kinase C substrate in platelets
(15). Pleckstrin homology (PH) domains have since been de-
scribed in a large number of signaling proteins, and they show
remarkable specificity in recognizing various forms of inosi-
tides. It is generally believed that at least one of the functions
of PH domains is to provide proper localization of proteins via
interactions with inositol phospholipids. However, some PH
domains have also been shown to bind proteins (16–18), and
their protein binding together with lipid binding may act in
concert to regulate the localization and/or the function of those
proteins. In addition to PH domains, additional motifs, such as
FYVE domains (19, 20) and the PX domains (21, 22), have been
shown to bind phosphoinositides, namely the lipid product of
the class III PI 3-kinases.
Because recognition of inositol lipids by PH domains is based
on the specific interaction of key surface residues with the
phosphate groups of the inositide head group (23), it is not
* This work was supported in part by Mobility Grant TeT 181/MO/01
from the United States-Hungarian Joint Fund (to P. V. and T. B.). The
costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “adver-
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
¶ Supported by National Institutes of Health Grants DK51526 and
GM59419 (to G. H.).
‡‡ Supported by the Hungarian National Science Foundation Grant
OTKA T-032270.
§§ To whom correspondence should be addressed: Rm. 6A35, Bldg. 49,
49 Convent Dr., National Institutes of Health, Bethesda, MD 20892-4510.
Tel.: 301-496-2136; Fax: 301-480-8010; E-mail: tambal@box-t.nih.gov.
1The abbreviations used are: PI(4,5)P2, phosphatidylinositol 4,5-
bisphosphate; Ang II, angiotensin II; AT1A, type IA angiotensin II
receptor; InsP3, inositol 1,4,5-trisphosphate; InsP4, inositol 1,3,4,5-tet-
rakisphosphate; InsP6, inositol hexakisphosphate, PLC?1, phospho-
lipase C ?1; PH domain, pleckstrin homology domain; GFP, green fluores-
cent protein; YFP, yellow fluorescent protein; PIP, phosphatidylinositol-
phosphate; NTA, nitrilotriacetic acid; FRET, fluorescence resonance energy
transfer.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 277, No. 30, Issue of July 26, pp. 27412–27422, 2002
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
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surprising that many (although not all) PH domains also bind
to the soluble inositol phosphate counterpart of their lipid
binding partner, a feature that is widely utilized to study their
binding properties (24). It is also known that hydrophobic in-
teractions also contribute to the membrane localization of PH
domains (25). In the case of several PH domains it is not quite
clear whether membrane localization via lipid binding or bind-
ing to soluble inositol phosphates is more important for their
regulation. For example, the membrane localization of PLC?1
via its PH domain and hence its activity is regulated by InsP3
levels (26). Rapid and large InsP3increases can translocate a
PLC?1PH-GFP fusion protein from the membrane to the cy-
tosol (27, 28). Also, the PH domain of the InsP4-binding protein,
GAP1IP4BP, is believed to be the target of InsP4(29), but it also
anchors the protein to the membrane via phosphoinositide
binding (30). These data prompted us to investigate the ques-
tion of whether binding to the inositol phosphate head group
(such as to InsP3) or to the lipid, PI(4,5)P2, is sufficient to
localize a PH domain to the cell membrane. While creating
protein domains with high InsP3binding affinity, we also ex-
plored the possibility whether such molecules could be used as
research tools to alter Ca2?signaling by sequestering InsP3.
Here we used the PH domains of PLC?1and the recently
cloned PLC-like protein, p130, as well as the InsP3binding
domain of the type I InsP3receptor to demonstrate that high
affinity binding to InsP3or even to PI(4,5)P2is not sufficient to
recruit these proteins to the plasma membrane. We also pro-
pose that regions other than those participating in recognition
of the inositide head group are important for the membrane
recruitment of the PLC?1PH domain and perhaps of other
similar protein modules.
EXPERIMENTAL PROCEDURES
Materials—Angiotensin II (human) was purchased from Peninsula
Laboratories. Ionomycin, 1,2-bis(2-aminophenoxy)ethane-N,N,N?,N?-
tetraacetic acid, InsP3, InsP4, and InsP6were obtained from Calbio-
chem. Phosphatidylinositol, phosphatidylserine, and phosphatidylcho-
line were all from Sigma. diC8-PI(4,5)P2and the PIP strips were
purchased from Echelon (Salt Lake City, UT). myo-[3H]Inositol (68
Ci/mmol) and [3H]InsP3(48 Ci/mmol) were from Amersham Bio-
sciences. All other chemicals were of high performance liquid chroma-
tography or analytical grade.
DNA Constructs—The PH domain of human PLC-?1(GenBank ac-
cession number U09117) (residues 1–170), its mutant R40L, as well as
its truncated version (residues 1–135) has been described previously
(31). The PH domain of the p130 protein (GenBank accession number
D45920) (residues 95–233) was amplified from rat brain cDNA (Quick-
clone, CLONTECH, Palo Alto, CA) using the primers 5?-ACAGA AT-
TCA CCATG GTGTC TTTCA GCAGC ATGCC ATC-3? and 5?-CAGTG
GATCC ATAAA GTCAA GTGGT TGCTT ACTGC GAG-3?. After diges-
tion with EcoRI and BamHI the product was ligated into the pEGFP-N1
plasmid (CLONTECH) digested with the same enzymes. p130PH was
also cloned into the pEGFP-C1 plasmid (CLONTECH) after amplifica-
tion with the following primer pairs: 5?-CTTCCTCGAGT GTCTT
TCAGC AGCAT GCCA-3? and 5?-TAAGA ATTCA CATAA AGTCA
AGTGG TTGCT-3? and digestion with XhoI and EcoRI. Chimeric PH
domains were created by introducing an EcoRV site to PLC?1PH-GFP
at residues 71–72 (silent) or at 111–112 (L110I) and to p130PH-GFP at
residues 163–164 (A163D) or 204–205 (L204I) for swapping the C-
terminal sequences (starting from either the ?5 or the ?7 strands,
respectively) between the two molecules. These conservative mutations
alone did not alter the cellular localization of the proteins. The short
8-amino acid loop located between the ?6 and ?7 strands of PLC?1PH-
GFP was substituted for the same segment in p130PH-GFP with the
following primers: 5?-ctc ttc aag gac caa cgc aac act ctg gac cta gttgc-3?
and 5?-gtc cag agt gtt gcg ttg gtc ctt gaa gag tat gga ga-3?. All mutations
were verified with dideoxy sequencing.
The InsP3binding domain (residues 1–610) of the human type I
InsP3receptor (GenBank accession number D26070) was amplified
from human brain cDNA (Quickclone, CLONTECH) with the following
primer pairs: 5?-GCAAC AGAGT GCCTG ACCCA GGTCA G-3? and
5?-CTTTC GCACC AGGCT GACAA ATGTG TC-3?. The PCR product
was subcloned into the pGEM-Easy T/A cloning plasmid (Promega,
Madison, WI). After sequencing, two clones were identified, one con-
taining (SII?) and the other lacking (SII?), the linker region located
between the two domains involved in InsP3binding (32). By using the
SII? splice variant as template, the InsP3binding domain (residues
224–605) was amplified with nested primers containing XhoI and
EcoRI restriction sites, and the PCR product was subcloned into the
pEGFP-C1 vector. All constructs were sequenced with dideoxy sequenc-
ing. The integrity and expression levels of the fusion proteins were
assessed by Western blot analysis from cells lysates prepared from
COS-7 or NIH 3T3 cells transfected with the constructs, using a poly-
clonal antibody against GFP (CLONTECH).
Transfection of Cells for Confocal Microscopy—Cells were plated onto
polylysine-coated 30-mm diameter circular glass coverslips at a density
of 5 ? 104cells/35-mm dish and cultured for 3 days before transfection
with plasmid DNAs (1 ?g/ml) using the LipofectAMINE reagent (10
?g/ml, Invitrogen) and Opti-MEM (Invitrogen). One day after transfec-
tion, cells were washed twice with a modified Krebs-Ringer buffer (120
mM NaCl, 4.7 mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose,
Na-Hepes 10 mM, 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
stimuli were added in 0.5 ml of prewarmed buffer after removing 0.5 ml
of medium from the cells. Cells were examined in an inverted micro-
scope under a 40? oil immersion objective (Nikon) and a Bio-Rad laser
confocal microscope system (MRC-1024) with the Lasersharp acquisi-
tion software (Bio-Rad).
Recombinant Proteins and InsPx Binding Assays—For bacterial ex-
pression of the GFP-fused protein domains, the coding sequences were
amplified from the T/A cloning plasmids (see above) and were subcloned
into the pET-23b bacterial expression vector (Novagen) using the XhoI/
EcoRI (for p130PH-GFP and PLCd1PH-GFP) and the NcoI/EcoRI re-
striction sites (for the GFP-InsP3R-(224–605) construct). The resulting
plasmids were used to transform the BL-21-DE3 strain of Escherichia
coli (Novagen). Bacterial cells were grown to A6000.6 at 37 °C and
induced with 300 ?M isopropyl-1-thio-?-D-galactopyranoside at 18–
20 °C for 7 h. Bacterial lysates were prepared by sonication in lysis
buffer (20 mM NaCl and 20 mM Tris, pH 8.0) followed by centrifugation
at 10,000 ? g for 30 min at 4 °C. The supernatant was incubated with
Ni2?-NTA-agarose beads (Qiagen) in the presence of 5 mM imidazole for
1 h at 4 °C. The beads were washed three times with lysis buffer, and
the recombinant proteins were eluted with the same buffer containing
1 M imidazole. Protein samples were concentrated and stored in phos-
phate-buffered saline containing 5 mM dithiothreitol and 20% glycerol
at ?20 °C. The concentration of recombinant proteins was assessed by
quantifying the bands of Coomassie Blue-stained SDS gel containing
the recombinant proteins and bovine serum albumin as a standard.
The incubation buffer of the in vitro InsP3binding assay contained 50
mM Na-Hepes, pH 7.4, 50 mM KCl, 0.5 mM MgCl2, and 0.01 mM CaCl2.
About 0.2 ?g of soluble recombinant proteins was incubated in 50 ?l of
this buffer with 0.74 kBq (0.5 nM) [3H]Ins(1,4,5)P3and the various
unlabeled inositol phosphates or short side-chained inositol lipids for 10
min on ice. The binding reaction was terminated by adding 5 ?l of
human ?-globulin (10 mg/ml) and 50 ?l of polyethylene glycol 6000
(30%) (24). Tubes were left on ice for 5 min and were centrifuged at
10,000 ? g for 10 min. The precipitates were dissolved in 0.1 ml of 2%
SDS, and the radioactivity was counted in a liquid scintillation counter.
Binding assays were also performed on proteins still bound to Ni2?-
NTA beads, where separation of the protein-bound ligand (on the beads)
from unbound ligand was achieved by adding 100 ?l of a mixture of
bis(3,5,5-trimethylhexyl)-phthalate and dimethyl phthalate (density,
1.010 g/ml) and centrifugation at 15,000 ? g at 4 °C for 5 min.
For binding of recombinant proteins to lipids on PIP strip mem-
branes (33), 100 pmol of recombinant protein was incubated in 5 ml of
binding buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 3% bovine serum
albumin (lipid-free), 2 mM sodium pyrophosphate, and 0.1% Tween 20)
overnight at 4 °C, after blocking the strips with the same buffer for 90
min at room temperature. After washing, GFP was visualized by West-
ern blotting using the polyclonal anti-GFP antibody from CLONTECH
essentially as described previously (34).
Analysis of Cytosolic Ca2?Responses in Individual COS-7 Cells—
COS-7 cells were plated onto polylysine-coated coverslips and cultured
for 3 days prior to experiments. Cells were transfected with plasmid
DNAs (see above) and were loaded with the fluorescent Ca2?indicator,
fura-2, 24–48 h after transfection. Loading was achieved by incubating
cells with 5 ?M fura-2/AM for 25–30 min at room temperature in a
medium containing 121 mM NaCl, 5 mM NaHCO3, 4.7 mM KCl, 1.2 mM
KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose, 10 mM Na-Hepes,
pH 7.4, and 2% bovine serum albumin supplemented with 0.003%
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pluronic acid and sulfinpyrazone (200 ?M). After loading, cells were
washed with the same medium containing 0.25% bovine serum albumin
supplemented with sulfinpyrazone but without fura-2/AM. Coverslips
were then mounted on the thermostated stage (35 °C) of an Olympus
IX70 inverted microscope coupled to a high quantum efficiency cooled
CCD camera driven by a customized computer program that also con-
trolled a scanning monochromator (DeltaRAM, PTI) to select multiple
excitation wavelengths (35). Fura-2 fluorescence was measured (340
FIG. 1. Cellular localization of protein modules that bind InsP3(A) and their effect on angiotensin II-stimulated InsP production
(B). The PH domains of PLC?1or of the PLC-related protein, p130, were fused to the N terminus of enhanced GFP, whereas the InsP3binding
domain of the type I InsP3receptor was fused to the C terminus of enhanced GFP. The resulting constructs were transfected into NIH 3T3 cells,
and the cells were examined by confocal microscopy. The same constructs, tagged with a His6tag at their C terminus, were also created for
expression in E. coli, and the expressed proteins were purified on Ni2?-NTA columns (see “Experimental Procedures” for details). The recombinant
fluorescent proteins retained their fluorescence after SDS-PAGE (without boiling) and could be analyzed in a PhosphorImager (lanes 1–4 represent
p130PH-GFP, PLC?1PH-GFP, R40LPLC?1PH-GFP, and GFP-IP3R-(224–605), respectively). B, inositol phosphate production was assessed in
[3H]inositol-labeled COS-7 cells transiently expressing the AT1Aangiotensin receptor and the indicated GFP proteins. Labeled cells were
stimulated with angiotensin II for 30 min after a 30-min preincubation with 10 mM LiCl (37). InsP3and InsP2fractions obtained from Dowex AG
1-X8 columns were combined. Values are expressed as percent of the control response (2–3-fold), and the results are the means ? S.E. of 4–5
experiments performed in duplicate.
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and 380 nm excitations) simultaneously with enhanced GFP fluores-
cence (490 nm excitation) using a 510-nm long pass dichroic mirror and
a 520-nm long pass emission filter. Cells were stimulated with 50 ?M
ATP, which evokes a Ca2?signal via the endogenous P2yreceptor of
COS-7 cells. Fluorescence signals were calculated for the total area of
individual cells, and the background fluorescence obtained over cell-free
regions of each image was subtracted prior to calculation of the fluo-
rescence ratios. Recordings obtained from all transfected cells on the
field were averaged for comparison in each experiment. Experiments
were performed on 3–4 separate cell transfections.
Analysis of Inositol Phosphates—Inositol phosphates were analyzed
from COS-7 cells transfected with cDNA encoding the rat AT1Aangio-
tensin receptor, together with selected GFP-PH domain fusion con-
structs as described previously (36). One day after transfection, cells
were labeled with myo-[3H]inositol for 24 h. After washing with inositol-
free M199 and 30 min of preincubation in the presence of 10 mM LiCl,
cells were stimulated with Ang II (1 ?M) for 30 min. Reactions were then
terminated, and
separated by Dowex minicolumns as described previously (37).
Measurements of Binding to PI(4,5)P2Containing Lipid Vesicles—
Phospholipid binding was performed with mixed lipid vesicles. 110 ?g
of PI(4,5)P2(Roche Molecular Biochemicals) and 1.4 mg of phosphati-
dylethanolamine (bovine liver; Avanti) were mixed and dried under a
nitrogen stream followed by high vacuum, and the dried mixtures were
suspended to a final total lipid concentration of 2 mM in 20 mM Hepes,
pH 7.2, 100 mM NaCl, 2 mM EGTA, 0.1 ?g/ml bovine serum albumin by
bath sonication. 5 ?l of the purified GFP fusion protein (1 ?g) and 5 ?l
of inositol 1,4,5-trisphosphate stock solution were added to 90 ?l of
phospholipid vesicles. As a precaution, proteins were subjected to ul-
tracentrifugation (85,000 ? g for 20 min at 4 °C) prior to the assays to
remove possible protein aggregates, although protein preparations
were used freshly when they had no significant aggregation. The reac-
3H-labeled inositol phosphates were extracted and
tion mixture was incubated at 30 °C for 10 min followed by ultracen-
trifugation at 85,000 rpm for 20 min at 4 °C. The 100-?l supernatant
was mixed with 30 ?l of 5? Laemmli buffer, and the pellet was resus-
pended in 100 ?l of incubation buffer followed by the addition of 30 ?l
of 5? Laemmli buffer. After vortexing, 40 ?l of each fraction was loaded
onto a 12% Tris glycine gel without boiling and separated by SDS-PAGE
at 4 °C. After electrophoresis, gels were analyzed in a Storm 860 (Am-
ersham Biosciences) PhosphorImager using blue fluorescence screening
for quantitation of the GFP fusion protein band on the gel. Western blot
analysis was also performed on parallel samples using the purified
polyclonal antibody against GFP (CLONTECH).
FRET Measurements—To have a quantitative measure of membrane
localization of the various constructs, the CFP and YFP variants of all
fusion proteins have been created. These were co-transfected into
COS-7 cells that were cultured in 10-cm culture dishes. One day after
transfection, cells were removed from the dishes by mild trypsinization,
washed, and centrifuged. Cells (about 3–5 million) were then resus-
pended in 2 ml of the Krebs-Ringer solution described above and placed
in the thermostated cuvette holder of a fluorescence spectrophotometer
used for ratiometric Ca2?measurements. Recordings were made using
an excitation of 425 nm and calculating a ratio from the emissions
detected at 525 and 475 nm (20 nm bandwidth each). Ionomycin (10 ?M)
was used to activate endogenous phospholipase C to hydrolyze the
phospholipids, and the decrease in the 525:475 ratio was taken as an
index of translocation of the domains from the membrane to the cytosol
(see Refs. 28 and 31 for details concerning the FRET approach and
ionomycin manipulation, respectively).
RESULTS
Cellular Distribution of Protein Domains Capable of Binding
Ins(1,4,5)P3—The PH domain of PLC?1binds PI(4,5)P2of the
plasma membrane and has been used as a tool to visualize
changes in the level of this lipid in living cells in the form of a
GFP fusion protein (31, 38). However, it has also been noted
that PLC?1PH-GFP does not bind to other intracellular mem-
branes despite extensive biochemical and immunocytochemical
(using anti-PI(4,5)P2antibodies) evidence that PI(4,5)P2is also
present in other cellular membranes such as the Golgi and the
nucleus (39–41). These observations prompted us to investi-
gate whether additional PH domains found in related proteins
could recognize PI(4,5)P2in other cellular compartments. Dur-
ing these efforts we isolated the PH domain (residues 95–233)
of the recently described PLC-like protein, p130, which was
initially described as an InsP3-binding protein with binding
features very similar to those of PLC?1(42). We also isolated
the InsP3-binding region of the type I InsP3receptor (residues
224–605) to examine whether its high affinity binding to InsP3
also allows it to bind to membrane PI(4,5)P2.
As shown in Fig. 1A, neither p130PH-GFP nor GFP-IP3R-
(224–605) showed membrane localization when expressed in
NIH 3T3 cells. This was in contrast to the well documented
membrane localization of PLC?1PH-GFP. Expression of the
constructs in a variety of mammalian cells (COS-7, Madin-
Darby canine kidney, and HEK-293) yielded no membrane
localization of either of the two proteins (not shown). The lack
of binding of p130PH-GFP to membrane PI(4,5)P2in COS-7
cells was also confirmed by analysis of inositol phosphate pro-
duction in response to Ang II stimulation. As documented pre-
viously, expression of PLC?1PH-GFP interferes with PLC acti-
vation, a property that closely correlates with the ability of the
protein to bind PI(4,5)P2of the plasma membrane (31). As
shown in Fig. 1B, expression of p130PH-GFP had no inhibitory
effect on the Ang II-stimulated InsP response, and if anything,
it slightly enhanced the response.
InsP3and PI(4,5)P2Binding to Recombinant Protein Do-
mains—To investigate the inositol lipid and inositol phosphate-
binding properties of these proteins, we created the same con-
structs for bacterial expression. Proteins were purified on Ni2?
columns, and in vitro binding assays were performed using
[3H]InsP3as the tracer. As shown in Fig. 2 and Table I,
p130PH-GFP was able to bind InsP3with an affinity that was
FIG. 2. Inositol phosphate binding characteristics of recombi-
nant InsP3binding domains fused to GFP. Proteins were expressed
in E. coli and were purified by Ni2?-NTA chromatography (see Fig. 1,
panel d). Binding assays were performed using [3H]InsP3in the pres-
ence of the indicated concentrations of the respective unlabeled ligand
(InsP3, filled circles), (InsP4, filled triangles), and (InsP6, inverted filled
triangles) as detailed under “Experimental Procedures.” The specific
binding (100%) was between 3000 and 6000 cpm in these experiments.
Means ? S.E. of 4–5 experiments performed in duplicate are shown.
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indistinguishable from that of PLC?1PH-GFP. Mutant forms of
either proteins (R40L-PLC?1PH-GFP and R134L-p130PH-
GFP) showed no [3H]InsP3binding, indicating that InsP3bind-
ing was specific to the recombinant wild-type proteins and not
to any impurities present in the preparations (not shown). The
GFP-IP3R-(224–605) protein showed higher affinity to InsP3
than either of the two PH domains, and its affinity was com-
parable with that of the intact InsP3receptor or with a similar
binding domain produced in E. coli as a GST fusion protein
(43). All three proteins discriminated between InsP3and InsP4,
but the receptor was significantly more selective in this re-
spect. InsP6was almost as effective as InsP4in displacing
[3H]InsP3from both p130PH-GFP and PLC?1PH-GFP, indicat-
ing that the inositol phosphate binding characteristics of the
two PH domains are very similar. These findings suggested
that the inability of p130PH-GFP or GFP-IP3R-(224–605) to
localize to cellular membranes in intact cells is not due to their
inability to recognize the inositol phosphate head group.
To investigate the relative affinities of these proteins to
inositol lipids rather than inositol phosphates, we used three
different approaches. First, a water soluble, short side-chain
analogue of PI(4,5)P2(diC8-PI(4,5)P2) was used as a competitor
in the [3H]InsP3binding assays. No difference was found be-
tween the affinities of PLC?1PH-GFP and p130PH-GFP to
diC8-PI(4,5)P2in these experiments (Fig. 3A and Table I). Both
proteins showed significantly lower affinities to the soluble
lipid derivatives than to InsP3. Second, the binding of
PLC?1PH-GFP and p130PH-GFP to lipid vesicles containing
PI(4,5)P2was compared. Both proteins were able to bind to
such vesicles in this assay; however, a larger fraction of the
PLC?1PH-GFP was found vesicle-bound, and higher concentra-
tions of InsP3were needed to displace it from the vesicles than
in the case of the p130PH-GFP (Fig. 3B). The R40L mutant of
PLC?1PH-GFP showed no specific localization to lipid vesicles
that would be displaced by InsP3(data not shown). A third
approach to compare the lipid binding of these proteins was to
study the binding of the recombinant proteins to lipids spotted
on nitrocellulose membranes as described previously (33, 34).
As shown in Fig. 4, PLC?1PH-GFP and p130PH-GFP showed
very similar inositol-lipid binding specificity and p130PH-GFP
only a slightly lower affinity to PI(4,5)P2based on these assays.
The R40L mutant PLC?1PH-GFP showed no binding under the
same conditions (Fig. 4).
Protein Domains That Bind Ins(1,4,5)P3Can Interfere with
Agonist-induced Ca2?Signaling—Whereas the in vitro binding
data clearly showed that the proteins made in bacteria are able
to bind InsP3and PI(4,5)P2in vitro, we also wanted to examine
whether their InsP3binding is manifested in intact cells. For
this, we used COS-7 cells in which the various GFP fusion
proteins were expressed. Expression of a protein that binds
InsP3with high enough affinity is expected to buffer InsP3
increases with consequences on Ca2?signaling when cells are
stimulated by an agonist that stimulates phospholipase C. To
test this, we used ATP to stimulate the endogenous P2yrecep-
tors of COS-7 cells expressing the various constructs. As shown
in Fig. 5, expression of both p130PH-GFP and GFP-IP3R-(224–
605), but not GFP alone, had a marked effect on the ATP-
evoked Ca2?signal in the COS-7 cells. The most prominent
FIG. 3. Inositol lipid binding of recombinant InsP3binding
domains fused to GFP. Proteins were expressed in E. coli and puri-
fied by Ni2?-NTA chromatography. A, [3H]InsP3binding was examined
in the presence of increasing concentrations of water-soluble forms of
PI(4,5)P2(diC8-PI(4,5)P2). B, binding of recombinant proteins to lipid
vesicles containing PI(4,5)P2in the presence of increasing concentra-
tions of InsP3. S and P represent the soluble (unbound) and pellet-
associated (bound) GFP fusion protein, respectively, analyzed by a
PhosphorImager after SDS-PAGE. Lower panel shows the summary of
six similar experiments (mean ? S.E.).
TABLE I
IC50values (in nM) for the displacement of [3H]Ins(1,4,5)P3from the various GFP-tagged Ins(1,4,5)P3binding domains and for the ability of
Ins(1,4,5)P3to inhibit their association with PI(4,5)P2-containing lipid vesicles
Means ? S.E. are shown with the number of observations in parentheses. ND, not determined.
PLC?1PH-GFP
p130PH-GFPGFP-IP3R (224–605)
[3H]Ins(1,4,5)P3binding displaced by
Ins(1,4,5)P3
Ins(1,3,4,5)P4
InsP6
Di(C8)PI(4,5)P2
17 ? 0.4 (8)
219 ? 60 (4)
528 ? 276 (4)
91 ? 24 (4)
22 ? 8 (9)
308 ? 88 (3)
764 ? 422 (3)
91 ? 17 (4)
4 ? 2 (6)
868 ? 496 (2)
ND
ND
Binding to PI(4,5)P2displaced by
Ins(1,4,5)P3
1000 ? ?50 (6) 300 ? ?15 (6) ND
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effect was an increase in the lag time (the time that is required
to reach half-maximal Ca2?response) from the time of ATP
addition. Because expression of p130PH-GFP did not inhibit
agonist-induced PLC activation (and presumably InsP3produc-
tion, see above), the increased lag time was consistent with the
buffering of InsP3by the expressed domains. Under these con-
ditions, after stimulation of the P2yreceptors by ATP, it takes
a longer time to reach the level of InsP3where the coordinated
action of InsP3and Ca2?triggers Ca2?release (44). This assay
was sensitive enough to detect the InsP3affinity difference
between the constructs, because GFP-IP3R-(224–605) had a
significantly bigger effect on the lag time than p130PH-GFP
(Fig. 5, lower panel).
Analysis of Cellular Distribution of Chimeric Proteins Con-
structed from the PH Domains of PLC?1and p130—Together
these data indicated that although p130PH is capable of bind-
ing InsP3as well as PI(4,5)P2, this binding is not sufficient to
recruit the protein to the plasma membrane. Given the level of
similarity between the two amino acid sequences (Fig. 6), we
decided to create chimeras from the two proteins to investigate
which part of the PLC?1PH accounts for its ability to localize to
the plasma membrane. Because most of the contacts with InsP3
in PLC?1PH are found within the N-terminal part of the mol-
ecule (containing the ?1–?4 sheets) (45) (see also Fig. 6), first
we designed a chimera in which the N-terminal halves of the
PH domains were exchanged between the two proteins. These
FIG. 4.
PLC?1PH-GFP and p130PH-GFP to
various phosphoinositides.
were expressed in E. coli and purified by
Ni2?-NTA chromatography. PIP strips
were incubated with 100 pmol of the re-
combinant proteins overnight at 4 °C as
detailed under “Experimental Procedures.”
After washing, GFP was visualized by
Western blotting using the polyclonal anti-
GFP antibody from CLONTECH essen-
tially as described previously (34). A shows
binding of the recombinant proteins to the
various lipids (300 pmol/spot) and the spec-
ificity of both PH domains to PI(4,5)P2. B
shows binding of the recombinant proteins
to decreasing amounts (in pmol/spot) of the
lipids, revealing very similar affinities of
PLC?1PH-GFP and
PI(4,5)P2.
Bindingof recombinant
Proteins
p130PH-GFP to
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chimeras were then expressed in either NIH 3T3 or COS-7
cells, and their cellular distribution was observed. No plasma
membrane localization was observed with the chimera contain-
ing the N-terminal InsP3-binding half of PLC?1PH fused to the
C terminus of the p130PH-GFP (PLC?1-(1–71)/p130-(166–
233)-GFP) (Fig. 7). In contrast, the inverse chimera (p130-(95–
164)/PLC?1-(71–170)-GFP) showed clear membrane localiza-
tion, although this was significantly less than that of the
original PLC?1PH-GFP (Fig. 7). In both cases the expressed
chimeras showed some intracellular “precipitates” in a number
of cells, especially in those expressing high amounts of the
protein. In our experience this indicates limited solubility or
folding problems of the proteins. Similar phenomena were ob-
served with other fluorescent proteins of limited solubility, for
example with the PLC?1PH-BFP protein, but in the latter case
the membrane localization of the protein could still be observed
(not shown).
These data suggested that the C-terminal half of PLC?1PH
contains additional determinant(s) for membrane localization
and also showed that the InsP3-binding region of p130PH can
substitute to some extent for the corresponding part of
PLC?1PH to support membrane localization. Therefore, addi-
tional chimeras were created in which the C-terminal helices
that follow the ?7 strands were exchanged between the two PH
domains. These experiments showed that a p130PH-GFP con-
taining the C-terminal helix of PLC?1PH still failed to localize
to the membrane. Surprisingly, the PLC?1PH domain with the
C-terminal helix of p130PH showed no membrane association
(not shown). Because the PLC?1PH domain used in these ex-
periments was 35 amino acids longer at its C terminus than
p130PH, we examined whether this extra stretch of amino
acids makes a difference in membrane targeting. Truncation of
PLC?1PH to the length corresponding to that of our p130PH
construct (PLC?1PH-(1–135), which still contains the full PH
domain), showed membrane localization comparable with that
of the longer form (not shown). From these data we concluded
that the C-terminal helix contributes to intra- or intermolecu-
lar interaction(s) that contribute to membrane localization.
Next we created a p130PH-GFP in which the short loop be-
tween the ?6 and ?7 strands of PLC?1PH was inserted in place
of the corresponding region in the p130PH wild-type sequence.
This loop is in a position that could come in contact with the
inositide head group or some other component of the membrane
(Fig. 6). As shown in Fig. 7, this protein chimera showed clearly
FIG. 5. Inhibition of agonist-induced Ca2?signaling by expressed InsP3-binding modules fused to GFP. COS-7 cells were transfected
with the indicated GFP fusion constructs, and their cytosolic Ca2?responses were analyzed by ratiometric Ca2?imaging using fura2. The lag time
of the Ca2?response was defined as the time it took for a cell to reach half-maximum of its Ca2?peak after addition of 50 ?M ATP. Upper panel
shows the average Ca2?signals calculated from the number of recordings obtained from transfected cells expressing either p130PH-GFP or GFP.
Lower panel contains the average lag times calculated for the cells expressing the indicated fusion protein. The number of cells recorded within
the individual groups and their average fluorescence are also indicated.
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detectable membrane localization, although it was less pro-
nounced than that of (p130-(95–164)/PLC?1-(71–170)-GFP).
Localization of the chimeras to the plasma membrane was
also assessed by FRET analysis. As shown previously, CFP-
and YFP-tagged versions of the PH domains could transfer
energy when found in molecular proximity at the membrane
(28). We used this approach to assess the extent of membrane
localization of the various chimeras. For this, COS-7 cells ex-
pressing both the CFP- and YFP-tagged PH domains were
examined in suspension using a fluorescence spectrophotome-
ter with excitation of 425 nm and emissions recorded at 475
and 525 nm to form the 525:475 ratio. In addition to FRET
taking place in the plasma membrane, the absolute value of
this ratio depends on several factors, including the relative
expression levels of YFP-PH and CFP-PH and FRET that oc-
curs in compartments other than the plasma membrane (e.g. in
the nucleus). Because ionomycin induces a PLC-mediated
breakdown of PI(4,5)P2in the plasma membrane and elimi-
nates membrane localization, the amplitude of the ionomycin-
induced decrease in the 525:475 ratio was taken as an index of
FRET due to localization of the probes to membrane PI(4,5)P2
(see Ref. 28 for details). These experiments showed that all of
the domains that were able to localize to the plasma membrane
(but none of those unable to bind) showed the characteristic
decrease in 525:475 ratio in response to PLC activation, indi-
cating their binding to the plasma membrane PI(4,5)P2pools
prior to ionomycin addition (Fig. 7). The magnitude of changes
upon Ca2?-induced translocation showed a good correlation
with the extent of PH domain localization reflected by the
confocal images. It is noteworthy that the rate of re-association
of the domains with the membranes during PI(4,5)P2resynthe-
sis also mirrored the apparent affinity differences by which the
various PH domains bound to the membrane.
DISCUSSION
There is increasing interest to understand the principles that
govern regulation of many cellular functions by the highly
compartmentalized changes in the levels of inositol phospho-
lipids. An important aspect of this research is the character-
ization of structural features of the protein modules that inter-
act with the inositide head group of inositides in the
membrane. Several recent studies have analyzed the structural
basis of the specific recognition of 3-phosphorylated inositides
by PH domains and have pointed to the significance of the short
variable loops between the ?1–?2 and ?3–?4 strands of such
domains (46, 47). Similarly, the structural features determin-
ing the binding of FYVE domains (19, 20, 48) and PX domains
to PI(3)P have been recently revealed (21, 49). To test specific-
ity and compare relative affinities, it is convenient to analyze
the binding properties of PH and other protein domains using
soluble inositol phosphates and inositol lipids in vitro. How-
ever, it is equally important to test these interactions within
the intact cell, because it is not possible to mimic in the test
tube the exact conditions that prevail at cellular membranes.
Our interest in characterizing PH domains is originated from
the need to understand and evaluate their usefulness in imag-
ing inositide dynamics in living cells (31). The interpretation of
the results of such imaging studies clearly depends on the
molecular recognition properties of the protein domains. Sev-
eral data suggest that various inositide binding domains inter-
act only with a certain subset of the inositide pools (31). The
restricted recognition of PI(4,5)P2by the PLC?1PH-GFP only
within the plasma membrane prompted us to investigate other
related PH domains for their PI(4,5)P2recognition properties.
The PH domain of the p130 protein was one of our choices due
to its similarity to PLC?1PH. The p130 protein has been orig-
FIG. 6. Sequence homology between
the PH domains of PLC?1and p130
and ribbon diagram showing the
crystalstructure
shows the ribbon diagram based on the
crystal structure of PLC?1PH (23) gener-
ated by Molscript (62). The two different
shades of green represent the two halves
of the PH domain where the chimeric con-
structs were joined, and the red indicates
the regions that were also exchanged be-
tween the two proteins. B shows the ho-
mology between the two primary se-
quences. Green arrows show the locations
of the ?-sheets. Red and blue letters indi-
cate identical residues and conservative
substitutions, respectively, and the basic
residues coordinating the phosphates of
InsP3are highlighted with yellow.
ofPLC?1PH.
A
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FIG. 7. Cellular localization of various chimeras constructed from the PH domains of PLC?1and p130. A is a schematic diagram
showing the various constructs and their plasma membrane localization scores based on confocal microscopy (in NIH 3T3 cells) and FRET analysis
(COS-7 cells) of transfected cells. B shows representative confocal images of NIH 3T3 cells transfected with selected constructs and the FRET
recordings obtained in COS-7 cell expressing the same proteins. Hydrolysis of PI(4,5)P2was initiated by the addition of 10 ?M ionomycin in the
presence of 1.8 mM external Ca2?(labeled as high Ca2?) and was terminated by the addition of 1,2-bis(2-aminophenoxy)ethane-N,N,N?,N?-
tetraacetic acid (BAPTA). PI(4,5)P2hydrolysis is reflected in the redistribution of fluorescence from the membrane to the cytosol, whereas
resynthesis of the lipid is paralleled by the relocalization of the fluorescent proteins to the membrane. Redistribution of the proteins due to
Ca2?-induced hydrolysis of membrane PI(4,5)P2was also assessed by FRET analysis (see “Experimental Procedures” and Ref. 28 for details).
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inally described as an InsP3-binding protein that was purified
on an InsP3affinity matrix (42). Subsequent cloning of the
protein revealed that it is a PLC homologue lacking PLC en-
zymatic activity and showing the greatest similarity to the
yeast protein PLC1, the Drosophila NorpA, and mammalian
PLC? (50). Similarly to PLC?1, p130 possesses a PH domain
that is responsible for the ability of the protein to bind InsP3.
Interestingly, despite its InsP3binding, the GFP-tagged p130
protein was found not to localize to membranes (51), and al-
though wild-type p130 was reported to be associated with mem-
branes when expressed in COS-7 cells, this association was not
mediated by the PH domain (52). However, consistent with its
InsP3binding, the ability of p130 to bind the second messenger
InsP3has been demonstrated in intact cells because its over-
expression altered both Ca2?signaling and InsP3metabolism
(51). The physiological function of this protein is still unknown,
but it has been speculated that it might be important as a
buffer of InsP3(51).
The present study clearly showed that InsP3binding even
with high affinity, such as that of the type I InsP3receptor, is
not sufficient to localize a protein to the membranes of intact
cells through simple binding to the head group of PI(4,5P)2.
However, these experiments also show that even binding to the
lipid PI(4,5)P2does not necessarily lead to membrane associa-
tion. The small difference found between the PI(4,5)P2-binding
properties of isolated p130PH and PLC?1PH in vitro does not
easily explain the striking difference between the abilities of
the two domains to localize to the plasma membrane. The PH
domain of PLC?4was found to associate with cellular mem-
branes with binding to PI(4,5)P2containing liposomes similar
to that of p130PH-GFP.2Our data obtained with chimeras
constructed from the PH domains of PLC?1and p130 showed
that the regions primarily responsible for InsP3binding (local-
ized within the N-terminal half of the PH domain) are to some
extent interchangeable between the two PH domains, consist-
ent with their almost identical InsP3binding affinities. It is the
C-terminal half of the PLC?1PH domain that seems to contain
additional feature(s) that are needed for membrane localiza-
tion. Within this half of the molecule, we were able to identify
the short loop between the ?6–?7 strands of PLC?1PH as a
sequence that aides membrane localization. However, the sur-
prising loss of membrane localization of the PLC?1PH when
containing the C-terminal helix of p130 also indicates impor-
tant contributions from the helical tail. Further attempts to
pinpoint single amino acids that determine membrane localiza-
tion failed to produce conclusive results.
It is not clear at present how the C-terminal helix could
affect membrane localization. However, it has been reported
that binding of PLC?1PH to PI(4,5)P2-containing lipid vesicles
(but not to soluble Ins(1,4,5)P3) was increased by Ca2?when
the C-terminally adjacent EF-hand motifs were added to the
PH domain (53). This indicates a conformational change that
affects lipid binding and that is initiated via the C-terminal
sequences. It has also been reported that a point mutation
within the PH domain of PLC?1greatly increased the catalytic
activity of the enzyme (54), further suggesting a reciprocal
communication between the various domains of the PLC?1
protein. It is also possible that a putative interacting protein
contributes to the membrane localization of the PLC?1PH do-
main. Further studies will be needed to clarify these questions
and to identify a possible protein-binding partner. Several PH
domains have been shown to bind ?? subunits of heterotrimeric
G-proteins (16, 18) including several PLC isoforms and even
PLC?1PH itself (55, 56). In the few cases where their sites of
interaction were mapped, the determinants to bind inositides
and those with ?? subunits did not completely overlap, the
latter mostly being found toward the C-terminal end of the PH
domain (56, 57). Intriguingly, a recent detailed study (58) lo-
calized some key residues important for ?? binding of GRK2 to
the C-terminal helix of its PH domain. Therefore, ?? subunits
are possible candidates to interact with PLC?1PH to aid its
membrane localization. However, overexpression of a construct
containing the ??-binding region of ?-adrenergic receptor ki-
nase (59) to sequester ??-subunits did not significantly affect
the membrane localization of PLC?1PH-GFP.3
In a recent study, the p130PH-GFP fusion protein (but not
the full-length p130-GFP) was found to localize to the mem-
branes in Madin-Darby canine kidney cells (51). The p130PH
domain used in those studies was fused to the C terminus of
GFP as opposed to our initial construct in which it was in the
N terminus of GFP. This prompted us to create the same
GFP-p130PH protein that was used in the studies of Takeuchi
et al. (51) and to use additional cells types, including Madin-
Darby canine kidney cells, to test the distribution of the pro-
tein. None of the p130PH domain constructs showed membrane
localization in our studies in any of the cells tested, and we
have no explanation for the discrepancy between our findings
and those of Takeuchi et al. (51). However, it is important to
note that the lack of membrane localization in our experiments
was observed with the same construct that showed InsP3as
well as PI(4,5)P2binding comparable with those observed with
PLC?1PH-GFP.
Finally, our results also demonstrate that it is possible to use
isolated protein domains to manipulate the level of inositol
phosphates within intact cells with clearly detectable conse-
quences on Ca2?signaling. Such an approach has been re-
ported recently (60) and will be very useful for the analysis of
InsP3-mediated signaling and to give estimates on the free
InsP3levels that are present in intact cells. These data also
point to the importance of InsP3buffering inside the cells and
may explain why the estimated InsP3concentrations (based on
total mass measurements) have been found so high (even in
resting cells) that they would saturate the InsP3receptor (61).
Therefore, these novel tools to manipulate InsP3changes could
add to our understanding on the physiology of this second
messenger.
In summary, using the PH domains of PLC?1 and the p130
protein, as well as the InsP3binding domain of the type I InsP3
receptor, we demonstrate that binding to the inositol phos-
phate head group of PI(4,5)P2by the PLC?1PH domain is
necessary but may not be sufficient to localize this protein to
cellular membranes. Additional regions within the C-terminal
half of the PH domain, namely the loop between the ?6 and ?7
sheets and the C-terminal ?-helix, are needed for this function.
Exploration of the structural features that determine PH do-
main interaction with InsP3, PI(4,5)P2, and with the intact cell
membrane could yield valuable information on the regulation
of the PLC? enzymes and help to facilitate the development of
strategies for selective inhibition of various inositide-based
regulatory processes.
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