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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3412-3422
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Distinct localization and function of
1,4,5IP3 receptor subtypes and the
1,3,4,5IP4 receptor GAP1IP4BP in
highly purified human platelet membranes
Samer S. El-Daher,
Yatin Patel,
Ashia Siddiqua,
Sheila Hassock,
Scott Edmunds,
Benjamin Maddison,
Geeta Patel,
David Goulding,
Florea Lupu,
Richard J. H. Wojcikiewicz, and
Kalwant S. Authi
From the Platelet Section, Thrombosis Research Institute, London,
UK, and the Department of Pharmacology, State University of New York
Health Science Center, Syracuse, NY.
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Abstract |
Platelet activation is associated with an increase of cytosolic
Ca++ levels. The 1,4,5IP3
receptors [1,4,5IP3R] are known to mediate
Ca++ release from intracellular stores of many cell
types. Currently there are at least 3 distinct subtypes of
1,4,5IP3R type I, type II, and type IIIwith
suggestions of distinct roles in Ca++ elevation.
Specific receptors for 1,3,4,5IP4 belonging to
the GAP1 family have also been described though their involvement with
Ca++ regulation is controversial. In this study we
report that platelets contain all 3 subtypes of
1,4,5IP3R but in different amounts. Type I and
type II receptors are predominant. In studies using highly purified
platelet plasma (PM) and intracellular membranes (IM) we report a
distinct localization of these receptors. The PM fractions were found
to contain the type III 1,4,5IP3R and
GAP1IP4BP in contrast to IM, which contained type I
1,4,5IP3R. The type II receptor exhibited a
dual distribution. In studies examining the labeling of surface
proteins with biotin in intact platelets only the type III
1,4,5IP3R was significantly labeled. Immunogold
studies of ultracryosections of human platelets showed significantly
more labeling of the PM with the type III receptor antibodies than with
type I receptor antibodies. Ca++ flux studies were
carried out with the PM to demonstrate in vitro function of inositol
phosphate receptors. Ca++ release activities were
present with both 1,4,5IP3 and
1,3,4,5IP4 (EC50 = 1.3 and 0.8 µmol/L, respectively). Discrimination of the
Ca++-releasing activities was demonstrated with cyclic
adenosine monophosphate (cAMP)-dependent protein kinase (cAMP-PK)
specifically inhibiting 1,4,5IP3 but not
1,3,4,5IP4-induced Ca++ flux.
In experiments with both PM and intact platelets, the
1,4,5IP3Rs but not GAP1IP4BP were
found to be substrates of cAMP-PK and cGMP-PK. Thus the Ca++ flux property of
1,3,4,5IP4 is insensitive to cAMP-PK. These
studies suggest distinct roles for the
1,4,5IP3R subtypes in Ca++
movements, with the type III receptor and GAP1IP4BP
associated with cation entry in human platelets and the type I receptor
involved with Ca++ release from intracellular stores.
(Blood. 2000;95:3412-3422)
© 2000 by The American Society of Hematology.
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Introduction |
Platelet activation represents an important event in
hemostasis and thrombosis. The activation of platelets by all
stimulatory agonists involves the elevation of cytosolic
Ca++ levels (for a review, see Authi1).
Stimulatory agonists acting on their surface receptors lead, through a
number of steps, to the hydrolysis of phosphatidylinositol
4,5-bisphosphate, resulting in the formation of inositol
1,4,5-trisphosphate (1,4,5IP3) and
1,2-diacylglycerol. Elevated cytosolic Ca++ levels arise as
a consequence of release of the cation from intracellular stores and
entry from the extracellular medium. Although Ca++ release
from intracellular stores by the action of
1,4,5IP3 is relatively well understood,
Ca++ entry mechanisms are not.2,3
1,4,5IP3 can be further metabolized to inositol
tetrakisphosphate (1,3,4,5IP4) before being
degraded to 1,3,4IP3 and lower inositol
phosphates. Considerable evidence suggests that Ca++ entry
can occur as a result of Ca++ release from intracellular
stores (termed store-regulated Ca++ entry or capacitative
Ca++ entry2), but the link between the stores
and the plasma membrane (PM) Ca++ entry channel is poorly
defined. Possible mechanisms include the involvement of a soluble
factor,4 small G proteins,5 tyrosine
phosphorylation mechanisms,6 or a coupling action possibly
involving an association of the 1,4,5IP3
receptor (1,4,5IP3R) with the PM
channel.7 In platelets, alternative routes of
Ca++ entry include activation of a receptor-operated
Ca++ entry channel as described for the platelet agonist
ADP8; a second-messenger-operated mechanism such as an
action of 1,4,5IP3 on an
1,4,5IP3R at the PM9 that could be
mediated in a similar manner to that described in endothelial cells by
1,3,4,5IP410; and a possible action
of 1,3,4,5IP4 at the level of the
PM.11 Specific receptors for
1,3,4,5IP4, such as GAP1IP4BP,
GAP1m, and centaurin,12-14 have been described.
GAP1IP4BP and GAP1m are closely related
GTPase-activating proteins that exhibit activities toward the small
G-protein Ras and have been implicated in Ca++
movements.15
There are at least 3 distinct genes that code for
1,4,5IP3Rs, namely type I, type II, and type
III, and at the protein level further heterogeneity arises because of
alternative splicing events.16,17 The 3 subtypes share
approximately 60% to 75% sequence similarity, which is highest at the
ligand-binding domain and the putative channel domain. The possibility
that subtypes may serve distinct functions involved with
Ca++ movements arises from differing properties, modes of
regulation, and distinct localization within cells. Binding affinities
for 1,4,5IP3 differ between the subtypes, with
the highest expressed by the type II receptor, then by type I, and then
by type III.18 Modes of regulation of
1,4,5IP3R include interactions with
Ca++, calmodulin, adenine nucleotides, FK binding proteins,
and protein kinases.17,19 Consensus sites for
phosphorylation by cAMP- and cGMP-dependent protein kinases (cAMP-PK,
cGMP-PK) are present for all 3 receptor subtypes and by tyrosine
kinases on types I and II,18 though not all the functional
consequences of phosphorylation are understood. In addition, with
platelets there is still debate as to whether
1,4,5IP3R is phosphorylated at
all.1,20,21
Platelets have been shown to contain the type I and type II, and
possibly the type III,
1,4,5IP3Rs.20,22,23 The type I
receptor appears to be present predominantly in the intracellular
membranes (IM). There is an uncertain location for the type II
receptor, and a distinct 1,4,5IP3R-like protein
is present exclusively in the PM fraction prepared using free-flow
electrophoresis (FFE).22,23 The purpose of this study was
to determine the localization and functional regulation of
1,4,5IP3R isoforms and GAP1IP4BP
using a range of isoform-specific antibodies, a combination of highly
purified PM and IM preparations, and whole-cell techniques. We report
the expression of all 3 types of 1,4,5IP3R
isoforms in different amounts at distinct locations, in vitro Ca++ flux activities for both the
1,4,5IP3R and the GAP1IP4BP that
co-localize at the PM, and the discrimination of their
activities in vitro by cAMP-PK. These results imply involvement of a
specific population of 1,4,5IP3R (type III and
type II) and GAP1IP4BP with cation influx that localize to
the PM and another population (type I and type II)
1,4,5IP3R that localizes in the IM to be
involved in Ca++ release.
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Materials and methods |
Materials
All chemical reagents were obtained from Sigma Chemical (Dorset, UK)
unless otherwise stated. Electrophoresis reagents were obtained from
National Diagnostics (Hull, UK) and nitrocellulose membranes were from Schleicher and Schuell (Dassel,
Germany). Sp-5,6-DCl-cBiMPS (BiMPS) and 8pCPT-cGMP
(8pCPT) were obtained from Biolog Life Science Institute (Bremen,
Germany). 2,4,5IP3 was a gift from Prof Robin
Irvine (Cambridge, UK). Regarding antibodies, CT1 was raised to the
C-terminal 19-amino acid sequence of the rat type I
1,4,5IP3R, CT2 to the C-terminal peptide
GFLGSNTPHENHHMPPH of the rat type II receptor, and CT3 raised to the
C-terminal peptide RLGFVDVGNCMSR of the rat type III
1,4,5IP3R. CT1, CT2, and CT3 were raised in the
laboratory of R.J.H.W. and have been previously characterized (for full
details, see Wojcikiewicz24). Two additional polyclonal
antibodies were raised to 1,4,5IP3R in the
laboratory of KSA and were affinity purified as were the CT antibodies.
R26 was raised to the last 12 C-terminal amino acids of the human type
I receptor and is thus similar to CT1, but R45 was raised to the
sequence TASPLGMPHGAA, which resides between the predicted
transmembrane region M5 and M6 of the human type III receptor and is
thus unique. These are characterized in this study and are used
interchangeably. The monoclonal antibody 18A10 recognizes the
C-terminal part of the type I 1,4,5IP3R and was
a kind gift from K. Mikoshiba (Japan). An antibody to the C-terminus of
GAP1IP4BP was also raised using the sequence
(C)VQSYIRQQSETSTHSI and affinity purified. This reagent was found not
to significantly recognize GAP1M (R. F. Irvine,
personal communication).
Purification of platelet plasma and intracellular membranes by
free-flow electrophoresis
Platelet PM and IM were prepared as described in detail in previous
publications.23,25,26 Briefly, platelets were separated from human blood and treated with neuraminidase (0.05 U/mL) for 20 minutes at 37°C. After further washing, platelets were sonicated in
0.34 mol/L sorbitol and 10 mmol/L HEPES, pH 7.4, with a cocktail of
inhibitors including aprotinin (0.3 U/mL), pepstatin A (5 µg/mL), phenylmethylsulfonyl fluoride (PMSF; 0.2 mmol/L), dithiothreitol (DTT;
1 mmol/L), soybean trypsin inhibitor (1 mg/mL), and E64 (2 µmol/L). Platelet sonicates were centrifuged at 40 000g for 90 minutes on a linear (1 to 3.5 mol/L) sorbitol density gradient to
obtain a mixed membrane fraction (free of granular contamination). After centrifugation of the mixed membrane (100 000g for 60 minutes), they were separated into PM and IM by FFE using an Octopus
apparatus (Dr Weber GmbH, Ismaning, Germany) running at 750 V 100 mA.
Two discrete peaks comprising PM (less electronegative) and IM (more electronegative) were obtained. Tops of peaks were pooled, centrifuged (100 000g for 60 minutes), and resuspended in 0.34 mol/L
sorbitol, 10 mmol/L HEPES, pH 7.2, for studies involving Western
blotting and protein phosphorylation.
Western blotting
Samples of purified membranes (50 µg PM and 50 µg IM) were applied to SDS-PAGE gradient gels (5% to 15%) following
the method of Laemmli27 and were subjected to
electrophoresis. Separated proteins were transferred to nitrocellulose
paper by semi-dry blotting using a current density of 0.8 mA/cm2 for 1 hour, and the nitrocellulose was then blocked
for 3 hours (or overnight) in blocking medium (5% dried milk, 1%
normal goat serum, 20 mmol/L Tris, pH 7.4, 500 mmol/L NaCl, 0.1%
Tween). Filters were washed 3 times in the above buffer without dried
milk and normal goat serum, followed by incubation with the
corresponding primary antibody (CT1, CT2, CT3, R26, R45, etc) in
blocking medium for 1 hour. After washing, the membranes were incubated
with an appropriate second antibody (eg, goat antirabbit) conjugated to horseradish peroxidase for 1 hour, followed by detection using ECL
reagents (Amersham, Little Chalfont, UK). The blocking
medium for biotinylation studies (see below) contained 5% bovine serum albumin (BSA), 20 mmol/L NaHPO4, 130 mmol/L NaCl, and 2 mmol/L EDTA, pH 7.5.
Phosphorylation of purified membranes by cyclic nucleotide analogues
and kinases and immunoprecipitation of receptors
Phosphorylation of purified PM was carried out as described
previously.23 The reaction mixture consisted of 50 mmol/L
HEPES buffer (pH 7.4), 10 mmol/L MgCl2, 1 mmol/L DTT, 0.2 mmol/L EGTA, 500 µg plasma membrane protein, 10 µmol/L adenosine
triphosphate (ATP) containing 1 µCi ( -32P
ATP), and, where indicated, cAMP-PK CAT 2 µg protein (70 U)/reaction (see lanes) and ± 1 µmol/L cAMP-PK CAT inhibitor peptide (see El-Daher et al23 and Cheng et al28; also legend
to Table 2 for peptide composition). The reaction was
initiated by the addition of ATP and cAMP-PK CAT, mixing, and
incubation at 30°C for 5 minutes and was terminated by the addition
of cold solubilization buffer (1% NP40 [or 1% Triton X100 where
stated], 0.2% LDS, 10 mmol/L NaF, 20 mmol/L sodium pyrophosphate, 5 mmol/L EDTA, 75 mmol/L NaCl, 1 mmol/L PMSF, 100 µmol/L
sodium vanadate, 0.2 mmol/L leupeptin, 20 mmol/L Tris, pH 7.8). After
30 minutes at 4°C, the samples were centrifuged at 13 000g
for 5 minutes. The supernatant was treated with one-tenth volume 20%
Protein-A Sepharose (PAS; Pharmacia, St Albans, UK) for
30 minutes at 4°C to preclear the sample and centrifuged, and the
supernatant was added to 200 µL appropriate antibody covalently
attached to PAS and incubated for 4 hours at 4°C. (In some
experiments, antibodies alone were added for 2 hours followed by PAS
and incubated with mixing overnight at 4°C). The immunoadsorbent
was spun down, washed 4 times with washing buffer (0.2% NP40 [or
0.2% Triton], 0.1% BSA, 0.01% sodium azide in phosphate-buffered
saline [PBS]) and once in washing buffer without BSA. SDS-Laemmli
buffer was then added to the immunoadsorbent, and this was followed by
SDS-PAGE and autoradiography to visualize the protein bands.
Labeling of intact platelets with [32Pi] and
activation of cyclic nucleotide kinases
Freshly prepared platelets were resuspended in a wash buffer
consisting of 36 mmol/L citric acid, 103 mmol/L NaCl, 5 mmol/L KCl, 5 mmol/L glucose, pH 6.5, and 60 nmol/L prostacyclin (PGI2). They were then incubated for 90 minutes at 37°C with 0.25 mCi carrier-free [32P] Pi/mL and washed twice before
resuspension in a HEPES tyrode medium containing 1 mmol/L EGTA (and no
Ca++) at 3 × 109 cells/mL. Incubations
with either BiMPS or 8pCPT were carried out using 300-µL suspensions
at 37°C with mixing for 5 minutes, and reactions were stopped by
the addition of Triton solubilization buffer. After 30 minutes at
4°C, the mixtures were centrifuged at 14 000g for 5 minutes at 4°C, and the supernatants were used for
immunoprecipitation with respective antibodies as described earlier,
followed by analysis of phosphorylated proteins by SDS-PAGE and
transfer to nitrocellulose and autoradiography.
Labeling of surface proteins in intact platelets with
biotin
Labeling of surface proteins with biotin was carried out as
described by Clemetson.29 Platelets were resuspended at
5 × 109 cells/mL in 20 mmol/L NaHPO4,
130 mmol/L NaCl, 2 mmol/L EDTA, pH 8.0. N-hydroxysuccinimidobiotin was added at 0.02 mg/mL and left for
1 hour at room temperature with gentle rocking. Platelets were then
washed 5 times in the same buffer at pH 6.5. The washed platelets were
solubilized, and proteins were immunoprecipitated with the appropriate
antibody and then probed to detect for biotin on Western blots using
streptavidin-HRP (1:2000 dilution, 1 hour) and ECL reagents.
Immunogold labeling on ultracryosections
Ultracryosectioning was carried out using the methods of
Tokuyasu30 and van Genderen et al.31 Briefly,
washed platelets were fixed by the addition of equal volume 4%
formaldehyde solution in PBS. After 30 minutes, the cells were
centrifuged at 1200g for 15 minutes, and the pellet placed in
5% sucrose solution and then onto small specimen pins and snap frozen
in liquid nitrogen. Thereafter, the samples were quickly transferred to
a Dupont MT6000 ultramicrotome (Stevenage, UK) adapted
for ultracryomicrotomy and sectioned using a dry glass knife. The
sections were retrieved from the knife using a small drop of 2.3 mol/L
sucrose solution in PBS and transferred onto formar/carbon-coated
specimen grids. Immunolabeling with polyclonal antibodies was carried
out by floating grids section-side down on small drops as follows: (1)
blocking of nonspecific sites with 10% fetal calf serum in PBS for 30 minutes; (2) incubation with primary antibody for 1 hour; (3) wash with PBS 3 × 5 minutes; (4) incubation with Protein A-gold in 1%
BSA/PBS; (5) wash with PBS 3 × 5 minutes and with water
5 × 1 minute. For monoclonal antibodies, an additional step
involving the use of a rabbit antimouse antibody as a bridge (1 mg/mL,
1:200 dilution, 1 hour) and washing is included in step 4 before
Protein A-gold treatment. The sections were then treated with 0.3%
uranyl acetate in methylcellulose for 10 minutes, blotted with Whatman
filter paper (Maidstone, UK), air dried, and examined under a Philips 201 transmission electron microscope. Illustrations
shown are typical of 2 separate preparations with at least 30 cells
examined with each antibody.
Ca++ flux determinations in purified platelet PM
Platelet PM were prepared as above using protease inhibitors in the
sonication medium as follows: PMSF, 0.25 mmol/L; pepstatin, 5 µg/mL;
aprotinin, 0.3 U/mL; DTT, 0.5 mmol/L; soybean trypsin inhibitor, 0.5 mg/mL. PM (40 µg protein in 4µL) resuspended in NaCl-Tris buffer
(150 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.4) was incubated at 37°C
for 30 minutes for Na+ loading and then diluted 60-fold
into exchange buffer containing 150 mmol/L KCl, 10 mmol/L Mopes-Tris
and 45Ca++ (2 µCi/300 µL) and
further incubated for up to 15 minutes to allow
Na+/Ca++ exchange. Reactions were stopped by
the addition of 200 µL ice-cold stop buffer (containing 5 mmol/L
Mopes-Tris, pH 7.4, 150 mmol/L KCl, and 5 mmol/L LaCl3) and
rapid filtration through a 0.45-µm filter under vacuum. The filter
was washed twice with ice-cold stop buffer, and, after semi-drying, the
radioactivity was counted by liquid scintillation spectrometry.
Addition of various concentrations of inositol phosphates or ionomycin
was made at 10 minutes incubation of membranes in exchange buffer and
reactions stopped at 15 minutes (or as indicated).
All Western blots and autoradiographs shown represent typical results
obtained with at least 3 different platelet or membrane preparations.
| |
Results |
Studies initially examined the expression of
1,4,5IP3R in platelet-mixed membranes using
specific antibodies. CT1, CT2, and CT3 represent affinity-purified
antisera specific for the type I, type II, and type III
1,4,5IP3R, respectively, and have been
previously well characterized.24 The specificities of the
newly described antibodies R26 (to the type I receptor) and R45 (to the
type III receptor) are demonstrated in Figure
1A, where immunoprecipitated type I (from
SHSY-5Y cells), mainly type II (from AR4-2J cells), and
mainly type III (from RINm5F cells)
1,4,5IP3R24 are examined with these
antibodies. R26 shows strong recognition of the type I receptor with
identical characteristics as previously described for
CT1,24 and R45 shows specific recognition of the
immunoprecipitated type III receptor. R26 does recognize a band in the
immunoprecipitated type II and type III receptor lanes that reflect the
presence of contaminating type I receptor in the preparations of the
type II and type III from AR4-2J and RINm5F cells,
respectively,24 and, we believe, not a recognition of the
type II and type III receptors by R26. Further, R26 recognized very
well the 1,4,5IP3 receptor from rat cerebellum
but very poorly, if at all, membrane preparations from AR4-2J cells
(rich in the type II receptor) or RINm5F cells (rich in the type III
receptors; results not shown). Figure 1B shows the relative expression
of the type I, type II, and type III receptors using CT1, CT2, and CT3
antibodies at equal antibody dilutions and with equal loading of
platelet-mixed membranes. There is good expression of the type I and
type II receptors and much reduced levels of the type III receptor.
Immunodetection by each antibody was specific as co-incubation with the
respective peptide to which the antibody was generated resulted in loss
of the immune signal (results not shown). Figure 1C shows typical immunoprecipitation analysis of the 1,4,5IP3R
types (repeated at least 6 times) using equal quantities of membranes
and antibodies. There is good immunoprecipitation of the type I and
type II receptors and significantly reduced amounts of the type III
receptor, suggesting much lower expression of this subtype. The reduced
immunoprecipitation of the type III receptor from platelets was not
caused by a reduced ability of the antibodies to immunoprecipitate the
protein because both CT3 and R45 were effective at immunoprecipitating
the type III receptor from RINm5F cells that are rich in the type III
receptor (lane III in Figure 1A reflects this). In cross-checking
experiments, each antibody did not co-immunoprecipitate any other
subtype from platelet membranes. For example, CT1 (or R26) only
immunoprecipitated the type I receptor, and other types were not
detected; the same was true for the CT2, CT3 and R45 (results not
shown). The post-antibody lysates were found to be devoid of the
immunoprecipitated receptor, suggesting the antibody amounts used
totally extracted the appropriate receptor. These results suggest that
in platelets these receptors most probably exist as homotetramers.
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| Fig 1.
Specificity of antibodies and presence of
1,4,5IP3R in platelets.
(A) Approximately 18 ng immunopurified type I, mainly type II, and
mainly type III 1,4,5IP3R (see "Results"
and ref. 24) were run in the lanes indicated and probed with antibodies
R26, R45, and CT3 using Western blotting. Arrow indicates position of
IP3R. R45 and CT3 are highly specific for the type III
receptor and R26 for the type I receptor. Cross- reactivity by R26 in
lanes for the types II and III receptors reflects co-immunopurification
of the type I receptor from AR4-2J and RINm5F cells. (B) Western blot
analysis of platelet-mixed membranes with the antibodies CT1 (to type
I), CT2 (to type II), and CT3 (to type III receptor). Arrows reflect
position of IP3 receptors. (C) Equal amounts of platelet
lysates were immunoprecipitated with equal amounts of either CT1, CT2,
or R45 antibodies. Immunoprecipitates were reprobed with the respective
antibodies on Western blots. Each blot is typical of 3 separate
experiments.
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High-voltage FFE enables human platelet PM and IM to be obtained with
high purity. These membrane fractions have been extensively characterized since their first description (see Menashi et
al,32 Crawford et al,33 and Hack and
Crawford34). Full documentation of the analytical and
enzymatic characterization including protein, lipid, and marker enzymes
(eg, NADH cytochrome C reductase for IM, actin and myosin for PM) are
reported.33 We have recently shown the IM to be the site of
the sarco-endoplasmic reticulum Ca++ATPase (SERCA) 2b and
SERCA 3.26 The PM preparations have been shown to be rich
in the surface glycoproteins GPIIb-IIIa and GP1b and the proteins VASP
and Rap1B, which are associated with the inner surface of the
PM.23,34 Western blot analysis of purified platelet
membranes using the 1,4,5IP3R antibodies is
shown in Figure 2A. Immune recognition by
the antibodies CT1 and CT3 is striking. The IM fractions contain the type I receptor, and the PM fractions contain the type III receptor, with little or no cross-contamination. This clear separation of cellular localization may suggest a differentiation of function for the
1,4,5IP3R in platelets. The antibody CT2
recognized a protein in the 250-kd range in both PM and IM fractions
(Figure 2B; some blots showed more intensity in the IM than PM
fractions). This suggests a dual distribution for the type II receptor.
Immunodetection of 1,4,5IP3R with R26 (to the
type I receptor) and R45 (to the type III receptor) showed a similar
distribution to that observed with CT1 and CT3, respectively (results
not shown). With a number of membrane preparations, we have observed
that the type III receptor is more prone to degradation by endogenous
proteases during the lengthy procedure than either the type I or type
II receptors. An example of this phenomenon is illustrated in Figure
2B, which shows a disappearance of the type III receptor parent protein and an increase of smaller products (at approximately 200 and 180 kd)
as the procedure progresses, whereas this effect was observed to a
lesser extent with the type II and type I receptors (the latter not
shown). Preparation of platelet extracts in the presence of the
protease inhibitor cocktail (see "Materials and methods") plus a
10-fold excess of aprotinin (but not PMSF, soybean trypsin inhibitor,
leupeptin, or pepstatin) resulted in greater protection against
proteolysis of the type III receptor, suggesting that perhaps a
kallikrein-type protease (rather than trypsin) was probably responsible. Coupled with the reduced expression, this may explain the
lack of detection of the type III receptor with platelet membranes in
the earlier studies (eg, Quinton and Dean22).
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| Fig 2.
Localization of 1,4,5IP3R types
in platelets.
(A) Western blot analysis of platelet 1,4,5IP3R
isoform distribution in plasma (PM) and intracellular membranes (IM)
prepared using high-voltage FFE. Numbers reflect molecular size markers
in kilodaltons. Marker enzyme NADH cytochrome C reductase activity
IM = 1.5 µmol/min per milligram protein; PM = 0.005 µmol/min
per milligram protein. Actin was only detected in PM, not
in IM. Ref. 33 shows full characterization of membranes. (B)
Degradation of type III 1,4,5IP3R by endogenous
proteases during membrane purification. PM were prepared using FFE, as
outlined in "Materials and methods." Samples loaded; lane 1, platelet lysate; lane 2, mixed membranes (MM) before FFE; lane 3, PM.
Antibodies used in Western blot analysis reflect CT2 for type II and
CT3 for the type III receptor. Numbers reflect positions of molecular
size markers. (C) Intact platelets were surface labeled with biotin
(see "Materials and methods") and
1,4,5IP3R were immunoprecipitated with R26,
CT2, and R45 antibodies. Biotinylated proteins were detected using
streptavidin-HRP.
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The near exclusive localization of the type III receptor to the PM
fraction prompted us to examine this property further; particularly,
was this receptor surface exposed? Intact platelets were labeled with
N-hydrosuccinimidobiotin (see "Materials and methods").
After washing, platelets were solubilized and receptors were
immunoprecipitated with the respective antibodies. A typical experiment
(repeated 3 times) is shown in Figure 2C. Immunoprecipitates of the
type III 1,4,5IP3R (using the antibody R45)
showed avid labeling with biotin. Immunoprecipitates of the type I and
type II receptors (with R26 and CT2) showed little, if any,
biotin associated with these proteins, as was to be expected for IM
proteins. In parallel experiments, all 3 antibodies
immunoprecipitated their respective receptors as in Figure 1B.
Thus, although the type III receptor was present in the least amount,
it was the only one with significant surface exposure. The PM
association of the type II receptor may thus reflect the presence of a
distinct membrane fraction closely associated with the PM through
protein or other linkages.
The cellular distribution of 1,4,5IP3R was also
examined using immunogold electron microscopy. All the antibodies to
the 1,4,5IP3R were tested; however, only
antibodies to the type I and type III receptors showed positive
immunogold labeling. It is not known why CT2 was not successful in this
regard. Figure 3 shows representative labeling patterns obtained for the type I and type III receptors using
2 distinct antibodies for each receptor typeR26 and a monoclonal antibody 18A10 [23] for the type I receptor, R45 and CT3 for the type
III receptor. The type I receptor is observed to have a widespread distribution among membrane structures throughout the cytoplasm, including the dense tubular system and granular membranes, with some
staining near the PM and in some cells with mitochondrial membranes
(Figures 3A and 3B). The type III receptor also showed some staining
with intracellular structures, but noticeably significant staining was
seen in the PM and the open canalicular system that represents
invaginated portions of the PM (Figures 3C and 3D). A statistical
analysis of the distribution of immunogold label obtained for each
polyclonal antibody for either 15 or 16 cells is shown in Table
1. Gold particles associated with PM versus IM for the type I receptor were statistically significant
(P = .0001). Further comparison of the PM association of the
type I receptor versus the PM association of the type III receptor
obtained with either CT3 or R45 was significant (P = .0013
and P = .047, respectively).
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| Fig 3.
Immunogold labeling of type I and type III
1,4,5IP3R in human platelets.
Ultracryosections were prepared and immunostained for type I
1,4,5IP3R using the monoclonal antibody 18A10
(A) and R26 (B) and for the type III receptors using CT3 (C) and R45
(D). Bar represents 1 µm; arrows point to selected gold particles.
dts, dense tubular system; M, mitochondria; ocs, open canalicular
system.
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We have also examined the distribution of the
1,3,4,5IP4R GAP1IP4BP using an
antibody raised to a sequence in the C-terminal portion of the protein.
Previously we had reported a binding site for [3H]
1,3,4,5IP4 in the PM fractions.35
Figure 4 shows that the antibody recognized
GAP1IP4BP as a 97-kd protein present in mixed membranes,
with enrichment in the PM but little staining is seen in the IM
fraction. Immunoprecipitation of GAP1IP4BP with this
antibody from platelet lysates prepared from biotinylated cells did not
reveal the protein to be biotinylated, confirming the protein to be
associated with the inner surface of the PM (results not shown).
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| Fig 4.
Detection of GAP1IP4BP in purified human
platelet membranes.
50 µg platelet-mixed (MM), plasma (PM), and intracellular membranes
(IM) were probed for Western blotting detection with an antibody raised
to a C-terminal peptide of GAP1IP4BP. Numbers reflect
molecular size markers.
|
|
Demonstration of an involvement of inositol phosphate receptors in
Ca++ flux in purified PM in vitro
Studies were carried out to determine the possible involvement of
the 1,4,5IP3R and the GAP1IP4BP
with cation influx by examining 45Ca++ flux in
the purified PM. Membrane vesicles were loaded with
45Ca++ using a Na+/Ca++
exchange activity,9 and the functionality of the
receptors was measured using inositol phosphate-mediated
Ca++ flux. Figure 5A shows the
characteristics of 45Ca++ loading into PM. The
steady-state 45Ca++ was retained within the
vesicle, as measured up to 30 minutes of incubation. This level of
trapped 45Ca++ at 10 minutes represented 0.63 nmol/mg protein. The addition of 10 µmol/L ionomycin at 10 minutes of
incubation led to the rapid release of approximately 80%
45Ca++ retained by the vesicle, indicating that
it represented Ca++ that was trapped and not membrane
bound. Inclusion of ionomycin at the start of the incubation resulted
in no loading of 45Ca++ into the vesicles.
Na+/Ca++ exchange activity was predominantly
associated with PM; IM vesicles contained approximately one tenth the
exchange activity of PM. Additionally, the Ca++ exchange
mechanism was not ATP dependent.
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| Fig 5.
Inositol phosphate-mediated Ca++ flux
activity in platelet PM.
(A) Na+/Ca++ exchange activity of PM and
inositol phosphate-mediated Ca++ flux. The illustration
shows kinetics of Na+/Ca++ exchange and
addition of either 10 µmol/L ionomycin (iono) or 50 µmol/L
1,4,5IP3 (IP3) at 10 minutes. All
points are mean ± SEM of triplicate determinations, with similar
results obtained in at least 2 other membrane preparations. (B)
Dose-response relationship of 1,3,4,5IP4 ( )
and 1,4,5IP3 ( ) induced Ca++
flux in plasma membranes.
|
|
The effect of inositol phosphates on PM Ca++ flux was
tested by addition at equilibrium conditions (10 minutes of
incubation). The addition of 20 µmol/L
1,4,5IP3 led to the release of approximately
40% trapped 45Ca++ within 5 minutes; a similar
response was elicited with 20 µmol/L 1,3,4,5IP4. The time course of
1,3,4,5IP4- or
1,4,5IP3-induced Ca++ release was
found to be maximal at 5 minutes, and there was no further
Ca++ release at 10 or 15 minutes of incubation. Figure 5B
shows the dose-response relationship of inositol phosphate-induced
Ca++ release from PM, with an EC50 = 0.8 µmol/L and 1.3 µmol/L for 1,3,4,5IP4 and
1,4,5IP3, respectively, with concentrations of
10 µmol/L and above giving maximal response. Calcium flux
determinations were carried out on the day of electrophoresis because
we detected a decrease in the sensitivity of the response the next day.
Concentrations of 100 µmol/L of either inositol phosphate did not
cause more than a maximal 50% Ca++ release (variation from
25% to 50% between preparations) under conditions in which ionomycin
released 75% to 85% trapped 45Ca++. This
suggested that approximately half the PM vesicles were of inside-out
orientation. Such vesicles would not be expected to exhibit inositol
phosphate-stimulated Ca++ flux because the ligand would
only have access to its receptor on the cytoplasmic face of the
membrane. The effects of other inositol phosphate isomers, namely
2,4,5IP3, 1,3,4IP3,
1,4IP2, and IP6, were tested in
this model of PM Ca++ flux. With all isomers, addition was
made after 10 minutes, and reactions were stopped after a further 5 minutes. 45Ca++ release activity equipotent
with 1,4,5IP3 was observed with
2,4,5IP3 but not with
1,3,4IP3 (EC50 = 20 µmol/L),
and 1,2IP2 or IP6 was found to be
ineffective tested up to 100 µmol/L maximum.
Contamination of PM with a Ca++ flux activity associated
with IM was checked with the use of the Ca++ATPase
inhibitor thapsigargin (Tg). At 3 µmol/L, Tg is sufficient for total
inhibition of SERCA Ca++ ATPases in platelets26
and to release Ca++ by 70% within 5 minutes from platelet
IM loaded with 45Ca++ (results not shown). The
addition of 3µmol/L Tg at 10 minutes of incubation with
45Ca++-loaded PM, followed by
stopping 5 minutes later, resulted in no release of
45Ca++ (101% of controls), indicating little
or no contamination of the PM by SERCA Ca++ pumps. Further
inclusion of Tg with 1,4,5IP3 did not
significantly alter Ca++ flux mediated by
1,4,5IP3 (results not shown).
We have attempted to differentiate the Ca++-releasing
actions of 1,4,5IP3 from those by
1,3,4,5IP4. First, heparin (at 1 mg/mL) was
considered unsuitable for this purpose because it inhibited the
Na+/Ca++ exchange activity by approximately
50%, added at either the start of the incubation or at equilibrium (10 minutes). We have recently shown that PM are rich in both cAMP- and
cGMP-dependent protein kinases.23 Thus, the effects of
protein phosphorylation on Ca++ fluxes in PM was
investigated using exogenous addition of the catalytic subunit of
cAMP-PK (cAMP-PK CAT). Addition of 50 U cAMP-PK CAT to PM at the start
of Na+/Ca++ exchange resulted in a stimulation
of 45Ca++ uptake by at least 3-fold (maximum up
to 7-fold). The kinetics of 45Ca++ loading was
similar, with equilibrium reached within 10 minutes of incubation and
maintained for up to 30 minutes (results not shown). (This increased
45Ca++-exchange activity was probably mediated
by the presence of phosphate ions and DTT used to stabilize the kinase
preparation because it was evident in the absence of added ATP or in
the presence of an inhibitor peptide specific for cAMP-PK; results not
shown.) The effect of cAMP-PK CAT on 45Ca++
flux induced by 1,4,5IP3 and
1,3,4,5IP4 was investigated in the presence or
absence (reflecting controls) of 100 µmol/L ATP. Table
2 shows 2 typical experiments with similar results obtained in 3 other membrane preparations. Stimulation of
Ca++ flux by 1,3,4,5IP4 was found
to be unaffected by cAMP-PK CAT in the presence of ATP, but
45Ca++ flux stimulated by
1,4,5IP3 was markedly inhibited. In 3 other
experiments, 50 U cAMP-PK CAT in the presence of ATP inhibited
1,4,5IP3 induced Ca++ flux by 65%,
78%, and 100% while having no effect or, in some cases, causing a
slight stimulation (18%) of 1,3,4,5IP4
stimulated Ca++ flux. The effect of cAMP-PK CAT was
reversed by a peptide inhibitor specific for this kinase (Table 2). The
possibility that the action of cAMP-PK CAT may be to hasten the
metabolism of 1,4,5IP3 and thus contribute to
its inhibitory effect on Ca++ release was considered
unlikely because the nonmetabolizable analogue,
2,4,5IP3 (50 µmol/L) also caused
Ca++ release from PM that was similarly inhibited by
cAMP-PK CAT (results not shown). In a further series of experiments,
100 µmol/L ATP added in the absence of cAMP-PK CAT did not affect the
dose-response relationship of 1,4,5IP3-induced
Ca++ release, suggesting that the nucleotide alone, which
has been suggested to have a modulatory role on the
1,4,5IP3 receptor, did not account for the
observed effects. The results suggest that in platelets, the
Ca++-releasing function of
1,4,5IP3, but not that of
1,3,4,5IP4, is sensitive to the action of
cAMP-PK.
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|
Table 2.
Effect of cAMP-PK CAT on (1,4,5)IP3 and
(1,3,4,5)IP4 induced Ca2+ flux in highly
purified plasma membranes
|
|
Phosphorylation of 1,4,5IP3R and
1,3,4,5IP4R by cyclic nucleotide kinases in
platelet PM fractions and whole cells
The above studies with cAMP-PK CAT provided a means to discriminate
the actions of 1,4,5IP3 from that of
1,3,4,5IP4 and also implied that the
1,4,5IP3R (but probably not
GAP1IP4BP) were substrates for cAMP-PK. Studies were
carried out to verify this in PM fractions by examining possible
phosphorylation of the receptors using [32P]ATP and
either cAMP-PK CAT or BiMPS, which directly activates endogenous
cAMP-PK (23 and references therein), and with intact platelets labeled with [32Pi] and incubated with either
prostacyclin (PGI2) or BiMPS. The results obtained with PM
preparations and intact cell systems were similar in the extent of
phosphorylation for each 1,4,5IP3R isoform and
GAP1IP4BP. The intact cell experiments reflect information
that is more physiologically important, and, to avoid duplication, only
these are presented here. Figure 6A shows
that incubation of platelets labeled with [32Pi] with
either PGI2 or BiMPS resulted in increased phosphorylation of the type III receptor, as observed after immunoprecipitation with
CT3. Both treatments proved effective to phosphorylate the receptor
with BiMPS marginally better probably because it directly activates
cAMP-PK. Using BiMPS the relative extents of phosphorylation of all 3 1,4,5IP3R isoforms was examined (Figure 6B).
BiMPS treatment led to the stimulated phosphorylation of the type I and
type III receptors avidly, but phosphorylation of the type II receptor
was considerably less; parallel experiments showed comparable
immunoprecipitation of each receptor in control and BiMPS-treated cells
(results not shown). An essentially similar finding was obtained with
PM fractions stimulated with either BiMPS or cAMP-PK CAT in the
presence of [32P]ATP (ie, good phosphorylation of the
type III receptor that was also blocked by inclusion of the cAMP-PK
inhibitor peptide and with markedly poor effect on the type II
receptor; results not shown). Figure 6C shows stimulation of platelet
cGMP-PK using 8pCPT23 and effects on phosphorylation of the
types I and III 1,4,5IP3 receptors and on
GAP1IP4BP. Both cyclic nucleotide kinases showed nearly
equal ability to phosphorylate each of the IP3R examined,
but neither kinase was an effective stimulus to phosphorylate
GAP1IP4BP. In each set of experiments, there was equal
immunoprecipitation of the respective receptor in control and treated
incubations (shown only for GAP1IP4BP).
GAP1IP4BP was also not phosphorylated in PM preparations
when stimulated by cAMP-PK CAT in the presence of
[32P]ATP (results not shown). Failure of
GAP1IP4BP to be phosphorylated by either cyclic nucleotide
kinase in whole cells or PM preparations supports the lack of effect by
cAMP-PK CAT on 1,3,4,5IP4-mediated
Ca++ flux in the PM preparations (Table 2). Verification
that both cyclic nucleotide kinases were activated in the negative
experiment involving GAP1IP4BP was carried out by assessing
the phosphorylation status of the phosphoprotein VASP in reaction
lysates after GAP1IP4BP immunoprecipitation. In its
nonphosphorylated state, VASP migrates on SDS-PAGE as a 46-kd band. On
phosphorylation in intact platelets by either cAMP-PK or cGMP-PK, the
protein migrates as a 50-kd species.36 Figure 6D shows that
both BiMPS and 8pCPT induced the phosphorylation of VASP as measured by
the appearance of the upper 50-kd species, suggesting that both cAMP-PK
and cGMP-PK had been activated to similar extents.
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| Fig 6.
Phosphorylation of 1,4,5IP3R and
GAP1IP4BP in intact platelets by cAMP-PK and cGMP-PK.
(A) [32P] labeled platelets were incubated with either
vehicle (CON) or PGI2 (0.5 µmol/L) or BiMPS (500 µmol/L), and type III 1,4,5IP3R
immunoprecipitated with CT3. (B) Experiments were carried out as in
(A), and immunoprecipitations were carried out using CT1, CT2, and CT3.
(C) Experiments were carried out as above with additional stimulation
of cGMP-PK using 8pCPT. Immunoprecipitations were carried out using R26
(for type I receptor), R45 (for type III receptor), and
anti-GAP1IP4BP antibody. Western blotting analysis of
immunoprecipitates of GAP1IP4BP is shown. (D) Lysates from
experiments involving GAP1IP4BP were subjected to analysis
of the phosphorylation status of VASP using Western blot analysis.
Activation of the kinases is associated with an increase of the upper
50-kd form of VASP. In control cells, nonphosphorylated VASP migrates
as a 46-kd protein.
|
|
| |
Discussion |
In this study, we report the distinct localization of inositol
phosphate receptors in human platelets using a number of techniques. We
demonstrate functional activity in vitro for both
1,4,5IP3R and GAP1IP4BP related to
Ca++ flux in a highly purified PM preparation. We also
describe a mechanism to discriminate the Ca++ flux
activities of 1,4,5IP3 and
1,3,4,5IP4 in platelets using cAMP-PK under
conditions in which phosphorylation of
1,4,5IP3R but not of GAP1IP4BP is
demonstrated in both PM and intact cells.
The distinct distribution of 1,4,5IP3R types
described in this study supports earlier observations of the
distribution of the type I and type II receptors reported by Quinton
and Dean,22 our own report of the presence of the type I
receptor in the IM,23 and suggestions of distinct roles of
1,4,5IP3R subtypes in specialized functions
related to Ca++ signals.17-19 Our studies
imply, in platelets, that the main role for the type I receptor is
involvement with Ca++ release from intracellular stores and
for the type III receptor a role in cation influx. The type II
receptor, depending on its location, may be involved in both processes.
A specialized role for the type III receptor is indicated because it
appears to be the only member of this family to have surface exposure,
as measured by biotin labeling of surface proteins in intact cells and
an almost exclusive elution of the protein with PM prepared using FFE.
The PM location of the type III receptor is further supported by
studies using immunogold labeling of platelet ultracryosections in
which R45 and CT3 both show significant labeling to the PM and the open
canalicular system, whereas the type I receptor shows a widespread
distribution among membrane structures in the cytoplasm. The type II
receptor that partially elutes with the PM, but is not surface exposed,
may reside on a membrane system that is tightly linked to the PM, but
this membrane system is substantially free of SERCA contamination.
Since the initial suggestions of a possible function for
1,4,5IP3 at the PM,37,38 a number
of studies have provided evidence for the possible presence of
1,4,5IP3R in the PM (eg, Fujimoto et
al39 and Cunningham et al40) with one
study41 suggesting that between 10% and 20% of the receptors in MDCK cells may be accessible to biotin labeling. It is
only recently that a specific role for the type III receptor in
Ca++ entry has been suggested, namely in lymphocyte cell
lines undergoing apoptosis42 and in Xenopus where
overexpression of the type III receptor led to increases of
Ca++ influx. Overexpression of the type I receptor,
however, led to an increase of the rate of Ca++
elevation.43 The ability of the type III receptor to remain in the open state at higher Ca++
concentrations44 is compatible with this function.
Putney45 has even hypothesized that in some cells the type
III receptor may serve as a capacitative Ca++ entry channel.
The Ca++ flux measurements suggest that the
1,4,5IP3R
and 1,3,4,5IP4R in the PM are functional
in stimulating cation movements. We have previously demonstrated
1,4,5IP3 to release Ca++ from
intracellular membranes with high potency (EC50 = 0.25
µmol/L46). We now show these membranes to contain mainly
the type I and type II 1,4,5IP3Rs. A
Ca++ flux activity for
1,4,5IP3, along with the same for
1,3,4,5IP4, is demonstrated in PM. An important
property of this PM preparation is its insensitivity to thapsigargin in
Ca++ATPase activity26 and also to
Ca++ flux (this study). The system may thus be useful to
provide a better dissection of the inositol phosphate receptors
involved with cation entry from those involved in Ca++
release. We show that the Ca++ flux activity stimulated by
1,3,4,5IP4 in the PM can be distinguished well
by the use of cAMP-PK. This kinase has no observable effect on the
cation flux activity of 1,3,4,5IP4 but markedly
inhibits that by 1,4,5IP3. cAMP-PK is also
found to be ineffective at stimulating the phosphorylation of
GAP1IP4BP, even though the protein has a number of
consensus sequences suitable for this kinase (eg, Ser90 in
the sequence ARDS that is suitable also for cGMP-PK)12 and may reflect an inaccessibility of this site by the kinase. This observation supports a Ca++ flux action of
1,3,4,5IP4 by its specific receptor,
GAP1IP4BP15,47, rather than by a possible effect on
1,4,5IP3R, as indicated by Wilcox et
al.48 A function for 1,3,4,5IP4
related to Ca++ movements is still
controversial49 but has been found in a number of
preparations, including those from lachrymal cells,50-52 platelets,11 NIH/3T3 cells,53 and many others.
The exogenous addition of phosphatidylinositol 3,4,5-trisphosphate
(PIP3), which has the same head group as
1,3,4,5IP4, was found to induce
Ca++ entry and to activate rabbit platelets. It has been
suggested that it also acts through
GAP1IP4BP.54 There is no unified mechanism to
explain 1,3-5IP4-stimulated Ca++
flux, and GAP1IP4BP is not an ion channel. Therefore,
future studies must probe its possible association with cation
channels. It is of particular interest that in overexpression studies
using COS 7 or HeLa cells, GAP1IP4BP appears to locate
solely at the plasma membranes (as with our studies), whereas
GAP1m exhibits a perinuclear localization.55
The relative expression and location of these 2 receptors may thus be
important determinants that stipulate whether
1,3,4,5IP4 exerts an effect on Ca++
at the level of intracellular stores (and be exerted by
GAP1m) or be related to cation entry (and be mediated by
GAP1IP4BP).
The distribution of 1,4,5IP3R in the PM and IM
fractions is of particular interest. Recently, Parekh et
al,56 from studies of 1,4,5IP3
perfusion in RBL-1 cells, have suggested that there are at least 2 types of Ca++ stores; 1 involved in Ca++
release with a high sensitivity to 1,4,5IP3 and
another responsible for Ca++ influx wherein higher levels
of 1,4,5IP3 are required. These reflect well
the properties of our PM and IM system. In addition,
1,4,5IP3 bound to its receptor has been shown
to activate Htrp3 channels overexpressed in HEK 293 cells.57 The latter study provides long-awaited support for
the conformational coupling mechanism for Ca++ entry first
suggested by Irvine.7 The coupling mechanism in HEK 293 cells did not require channel activity by the
1,4,5IP3R because it was equally shown by
1,4,5IP3 bound to a truncated type I receptor
and appeared to be shown by all the 1,4,5IP3R
isoforms. Thus, the localization of isoforms may be particularly important. Although that study suggested no direct contact between the
1,4,5IP3R and Htrp3,57 more recent
data from the same group suggest that direct contact has been
demonstrated (S. Muallem, personal communication). We
suggest that the type II receptor eluting with the platelet PM may
serve to link a PM cation channel to intracellular stores. As far as we
are aware, the presence of trp isoforms in platelets is yet to be
demonstrated. However, as we have reported, a number of megakaryocytic
cell lines express mRNA for Htrp1 and Htrp3,58 and it is
likely that they are present in platelets. The presence of
1,4,5IP3 receptors and GAP1IP4BP at
the PM may shed new light on a number of previous studies on platelets
that suggested a (more) direct role for
1,4,5IP3 in cation entry than mediated by store
depletion. Stopped flow studies of thrombin-stimulated platelets
suggested a component of Ca++ entry that preceded
Ca++ release from intracellular stores.59
1,4,5IP3 infusion in rat megakaryocytes has
been reported to activate Na+ entry by a mechanism
independent of store depletion.60 Exogenous PIP3 added to rabbit platelets stimulates Ca++
entry.54 All these could be accommodated as occurring
through the type III receptor that is surface exposed or through a
possible interaction of the cation channel with the type II receptor or GAP1IP4BP. Figure 7 depicts the
possible organization of inositol phosphate receptors in human
platelets encompassing these suggestions.
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| Fig 7.
Localization of IP3 receptors
(IP3R) and GAP1IP4BP in platelets.
The plasma membrane preparation (PM) contains the type III receptor,
which is surface exposed and is likely to be involved in either
Ca++ or Na+ entry (see text). The type II
receptor and GAP1IP4BP eluting with the plasma membrane
preparation are suggested to link with a plasma membrane cation
channel. The intracellular membranes contain the type I and type II
receptors involved with Ca++ release from intracellular
stores and thapsigargin (TG) sensitive SERCA Ca++ATPases.
|
|
Our studies also clarify an important but controversial aspect of
1,4,5IP3R phosphorylation by cAMP-PK and its
effects on Ca++ fluxes. Depending on the cell type,
elevation of cAMP has been suggested to potentiate or to inhibit
agonist-induced Ca++ elevation1,19 with the
effect of cAMP thought to be mediated by cAMP-PK. However, the effects
of phosphorylation of the 1,4,5IP3R by cAMP-PK
are unclear, with conflicting results presented from studies on
isolated membranes61 and immunopurified
receptors.62 In platelets, though it is well established
that cAMP (and cGMP) elevation leads to inhibition of cytosolic
Ca++ elevation,63, 64 the site of action for
cAMP-PK or cGMP-PK, whether this be at the level of phospholipase
C,65 the 1,4,5IP3R21 or
Ca++ pumps,66,67 and to what extent an effect
at each of these sites is important, is unclear. Recently studies on
intact platelets68 and on rat megakaryocytes69
implied an effect at the level of the
1,4,5IP3R, though no direct examination of the
phosphorylation status of the 1,4,5IP3R
subtypes was carried out. The need for studies on each receptor subtype
was further emphasized by the finding that purified type I
1,4,5IP3R from platelets was found not to be a
substrate for cAMP-PK.20 Rather, in isolated membranes, it
was reported to be phosphorylated by an endogenous kinase and then
further phosphorylated by cAMP-PK.21 Our studies
demonstrate for the first time in intact platelets that all 3 types of
1,4,5IP3Rs are substrates of cAMP-PK to
differing extents. The type I and type III receptors are highly favored
substrates for both kinases, and the type II receptor is less favored.
Our results support the work of Quinton et al21 regarding
cAMP-PK dependent phosphorylation of the
1,4,5IP3R and the inhibition of
Ca++ release. Similar findings were recently reported for
the 3 subtypes of 1,4,5IP3R isolated from 3 distinct cell types, each expressing high levels of 1 subtype but with
the kinase enhancing Ca++ release.70 Our
finding that cGMP-PK acts on platelet
1,4,5IP3Rs extends the work of Komalavilas and
Lincoln71 in which phosphorylation of the type I
1,4,5IP3 receptor by cGMP-PK in smooth muscle
cells was demonstrated. It is unclear why in some cell types the effect
of cAMP-PK phosphorylation on 1,4,5IP3R is
stimulatory to function but in others (such as
platelets) the effect is inhibitory and if this effect requires the
participation of another protein. But clearly these receptors represent
highly potent targets of cross-talk regulation with cyclic nucleotides and 1,4,5IP3 representing second messengers
with strongly opposing actions on platelet Ca++ movements.
In conclusion, our studies report a distinct location for
1,4,5IP3R isoforms and GAP1IP4BP
that most probably relate to their specific roles in the promotion of
Ca++ release and in the propagation of cation entry.
| |
Acknowledgments |
We thank Prof R. F. Irvine (Cambridge, UK) and Dr P. Cullen (Bristol,
UK) for many discussions during the course of this work.
| |
Footnotes |
Submitted May 13, 1999; accepted January 26, 2000.
Supported by grants from the Wellcome Trust, the British Heart
Foundation, and the Thrombosis Research Trust.
Reprints: Kalwant S. Authi, Platelet Section, Thrombosis
Research Institute, Manresa Rd, Chelsea, London SW3 6LR UK; e-mail;
ksauthi{at}tri-london.ac.uk.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction