© 1997 American Heart Association, Inc.
Articles |
Signal Transduction Through Trimeric G Proteins in Megakaryoblastic Cell Lines
From the Department of Hematology, University Hospital Utrecht, Netherlands.
Correspondence to Dr J.W.N. Akkerman, University Hospital Utrecht, Department of Hematology (G03.647), PO Box 85.500, 3508 GA Utrecht, The Netherlands. E-mail J.W.N.Akkerman{at}lab.azu.nl
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Abstract |
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Abstract The biogenesis of trimeric G proteins was investigated by measurement of the expression of
-subunits in the
megakaryoblastic cell lines MEG-01, DAMI, and CHRF-288-11,
representing stages of increasing maturation, and compared
with platelets. Megakaryoblasts and platelets contained
approximately equal amounts of Gi-1/2,
Gi-3, Gq, and G12 protein.
Maturation was accompanied by (1) downregulation of mRNA for
Gs and disappearance of iloprost-induced
Ca2+ mobilization, (2) upregulation of the long form of
Gs protein (Gs-L) and an increase in
iloprost-induced cAMP formation, and (3) upregulation of
G16 mRNA and G16 protein and appearance
of thromboxane A2-induced signaling
(Ca2+ mobilization and stimulation of
prostaglandin I2–induced cAMP formation).
Gz protein was absent in the megakaryoblasts despite
weak expression of Gz mRNA in DAMI and relatively high
levels of Gz mRNA and Gz protein in
platelets. These findings reveal major changes in G
protein–mediated signal transduction during megakaryocytopoiesis and
indicate that G16 couples the thromboxane
receptor to phospholipase Cß.
Key Words: megakaryocytes • platelets • signal transduction • G protein
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Introduction |
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Platelet stimulation by PAF, TxA2, and
-thrombin is mediated
through transmembrane receptors coupled to trimeric G proteins. These
molecular switches transduce signals to effector enzymes such as
PLCß,1 PLA2, and AC and to ion
channels for Ca2+, K+, and
Na+/H+ exchange.2 3 The
identification of G proteins that take part in signal transduction by
different platelet-activating agents has been difficult. The IP,
EP2, and DP receptors (receptors for I-, E2-
and D-type PGs, eg, PGI2, PGE2, and
PGD2, respectively4 ) and ß-adrenergic
receptors couple to the stimulatory G protein of AC, Gs.
The EP1 and EP3 receptors, the
2A-adrenergic receptor, and the seven-transmembrane
thrombin receptor couple to the inhibitory G protein of AC
(Gi), whereas coupling to the PAF receptor is
controversial.5 The thrombin receptor is also coupled to
Gq, initiating phosphoinositide hydrolysis
via PLCß6 ; the TxA2 receptor
also might signal through this G protein.7 8 Another G
protein identified in platelets is Gz,9
which is phosphorylated by PKC,10 but its
function remains unresolved.11 Additional regulatory
properties are mediated via the ß
-subunits, which may inhibit
AC12 and activate
PLCß.13
Because human platelets lack DNA and have no detectable protein
synthesis, their signal-transducing properties evolve in the maturing
megakaryocyte. Little is known about the biogenesis of signaling
elements in the megakaryocyte, but megakaryoblastic cell lines have
provided insight into the development of the activation
apparatus. MEG-01 cells, representing an early
maturation stage, show thrombin-induced Ca2+ mobilization
and possess receptors for TxA2 and
PGI2.14 15 MEG-01 and also the more mature
DAMI cell line respond to PGE1 with increases in both cAMP
and cytosolic Ca2+,14 a property not seen in
platelets. CHRF-288-11 is the most mature megakaryoblastic cell
line,16 containing secretion granules and signal
transduction for -thrombin, PAF, and TxA2 receptors to
PLC.17
An illustration of differential expression of G proteins during
development is found in cell lines used as a model for the human B-cell
differentiation, where G16 is downregulated during
transition from the pre-B to the B stage.18 Upregulation
of Gi and Gs is observed during
maturation of human thymocytes,19 whereas differentiation
of the murine erythroleukemia cell line RED-1 leads to loss of
Gi-3 and a fall in PTX-sensitive Ca2+
mobilization.20
Assuming a similar asynchronous expression of trimeric G proteins in
megakaryocytes, we set out to explore the transcription of G
subunits during megakaryocytopoiesis in an attempt to clarify the role
of individual G proteins. To compare G-protein expression with signal
processing, the studies were carried out with megakaryoblastic cell
lines that approximate different maturation stages of normal
megakaryocytopoiesis.
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Methods |
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Materials
FCS, horse serum, and Superscript II reverse transcriptase were from Gibco BRL. SuperTaq DNA polymerase was from HT Biotechnology, and Pfu polymerase was from Stratagene. Human
-thrombin, the cAMP derivative dbcAMP, PTX, and
CTX were from Sigma. PAF, fura 2-AM, DNAse-free RNAseA, and
Pwo DNA polymerase were from Boehringer Mannheim.
The prostacyclin analogue iloprost was a gift from Schering, Berlin,
Germany. The mastoparan analogue Mas-7, the PLC inhibitor
U73122, and its inactive analog U73343 were purchased from Biomol. The
thromboxane A2 analogue U46619, the IP receptor
agonist carbaprostacyclin, and the EP1 receptor agonist
17-phenyl trinor PGE2 were from Cayman Chemical Co. SQ29548
was from Bristol-Meyers Squibb. Nitrocellulose filters were from
Schleicher & Schuel, and Immobilon-P PVDF membranes (PVDF filters) were
from Millipore. The cAMP-[125I] kit and
[-32P]dCTP were purchased from Amersham Life Science,
and [3H]SQ29548 was from New England Nuclear. All other
chemicals were of analytical grade.
Antibodies
The antibodies against Gcommon (GA/1),
Gi-1/2 (AS/7), Gi-3 (EC/2),
Gs (RM/1), and Gq/11 (QL) were rabbit
polyclonal antibodies to C-terminal peptides of the different
-subunits and were purchased from NEN DuPont.
Anti-G12 (N-terminal 2 to 21) and anti-Gz
(N-terminal 3 to 18) were rabbit polyclonal antibodies from Santa Cruz
Biotechnology and Calbiochem, respectively. The rabbit polyclonal
anti-G16 (AS 339, directed against amino acids 361 to
374 of the C-terminus21 ) was a kind gift of Dr K. Spicher,
Free University of Berlin, Germany. Mouse monoclonal antibody 6F6
(anti-CD42b) recognizes free and complexed GPIb and was provided by Dr
H.K. Nieuwenhuis, Department of Hematology, University Hospital,
Utrecht, Netherlands. A FITC-conjugated antibody to GPIIIa
(F803, anti-CD61, Dako) was used with a negative control of the same
isotype (IgG1-FITC, X949, Dako). FITC-conjugated goat anti-mouse
(gm-FITC) was from the Central Laboratory for Blood Transfusion,
Amsterdam, Netherlands. SWARPOs were from Dako.
Cell Cultures
MEG-01 cells,22 kindly provided by Dr H. Saito,
Nagoya University School of Medicine, Nagoya, Japan, were grown in
plastic culture flasks in RPMI 1640 medium supplemented with 20% FCS.
DAMI cells,23 a gift of Dr R.I. Handin, Brigham and
Women's Hospital, Boston, Mass, were grown in IMDM supplemented with
10% horse serum. CHRF-288-11 cells16 were a gift of Dr M.
Lieberman, Department of Molecular Genetics, Biochemistry, and
Microbiology, University of Cincinnati (Ohio) and were grown in
Fischer's medium supplemented with 20% horse serum. All cells were
cultured in the presence of 2 mmol/L
L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin
at 37°C in a humidified atmosphere with 5% CO2 and were
subcultured twice a week to maintain a concentration of
1x106 cells/mL. For some experiments, cells were
incubated with PTX (200 ng/mL) for 14 hours or CTX (0.5
µg/mL) for 5 hours at 37°C in serum-free RPMI supplemented
with 0.2% BSA.
Flow Cytometric Analysis
Expression of GPIb and GPIIIa was evaluated by FACS
analysis. GPIb content was measured by labeling with mouse
monoclonal antibody 6F6 followed by gm-FITC. As a negative control,
the same gm-FITC antibody was used without primary antibody. GPIIIa
was measured by incubation with FITC-labeled antibody F803; as a
negative control, a nonspecific antibody of the same isotype, X949, was
used. Briefly, cells were incubated with 6F6 or F803 in
HEPES–Tyrode's solution (pH 7.2) supplemented with 1.0% (wt/vol)
BSA, for 20 minutes at 4°C. The cells incubated with the 6F6 antibody
were washed and incubated with gm-FITC for 30 minutes at 4°C.
Finally, cells were washed in PBS (pH 7.4) with 0.2% (wt/vol) BSA and
analyzed on a FACScan (Becton Dickinson).
Platelets
Platelet concentrates (Red Cross blood bank,
Utrecht, Netherlands) were made leukocyte-poor by filtration
through a PALL50 leukocyte removal filter (PALL Biomedical Ltd),
reducing the leukocyte/platelet ratio from 1:3000 to
<1:106. The platelets were washed twice by
centrifugation (20 minutes, 700g at 20°C)
in PBS supplemented with 1/10 vol ACD (2.5 g trisodium citrate,
1.5 g citric acid, 2.0 g D-glucose in 100 mL
distilled water [pH 6.5]).
Western Blotting
Cell lines and platelets were collected by
centrifugation (10 minutes, 100g at 20°C)
and lysed in Laemmli's electrophoresis sample buffer (0.001%
bromphenol blue, 2% wt/vol SDS, 5% ß-mercaptoethanol, and 10%
glycerol in 62.5 mmol/L Tris [pH 6.8]). The samples were
boiled for 5 minutes and stored at -20°C; 40 µg protein was
subjected to SDS-PAGE (10% gels) and electroblotted onto PVDF filters.
The blots were blocked in PBS (pH 7.4) containing 5% (wt/vol) fat-free
dry milk (Protifar, Nutricia) and 0.05% Tween 20 for 1 hour at room
temperature.
The primary antibody incubation was performed overnight at a 1:1000 dilution in PBS supplemented with 0.5% (wt/vol) Protifar and 0.05% Tween 20. The membranes were then washed three times and incubated with SWARPOs in the same buffer. Bands were visualized by chemiluminescence with the Renaissance Western blot chemiluminescence reagent of NEN-DuPont and DuPont NEF-496 Reflection autoradiography films.
RNA Preparation
Total RNA was extracted as described by Davis et
al.24 In short, megakaryoblastic cells and platelets
were pelleted, lysed in guanidine isothiocyanate buffer (4 mol/L
guanidine isothiocyanate, 25 mmol/L sodium citrate [pH
7.0], 0.5% sarkosyl, and 0.1 mol/L ß-mercaptoethanol), and
layered on a CsCl cushion (5.7 mol/L CsCl, 25 mmol/L
sodium citrate). After centrifugation (Beckmann SW41 Ti
rotor, 32 000 rpm, 20 hours at 20°C), the pellet was resuspended in
300 µL 0.3 mol/L sodium acetate, extracted with
phenol/chloroform (1/1, vol/vol), precipitated with 2.5
vol 96% ethanol, and resuspended in H2O.
Northern Blot
Northern blot analysis was carried out as
described.24 25 In brief, 30 µg of total RNA was
migrated on a 1% agarose/formaldehyde gel in MOPS buffer (20
mmol/L 4-morpholinopropanesulfonic acid, 5 mmol/L
sodium acetate, and 1 mmol/L EDTA [pH 7]), blotted onto
nitrocellulose filter, and hybridized overnight at 42°C in
hybridization buffer N,24 with 2x106 cpm of
denatured probe/mL. The blots were washed for 10 minutes in
2XSSC buffer at 65°C, followed by a wash in
0.5XSSC with 0.1% SDS for 30 minutes at 65°C.
Full-length DNA probes were made by random-primer labeling essentially as described by Feinberg and Vogelstein.26 27 A Promega Prime-a-gene kit was used with 32P-labeled dCTP. The 32P signal was measured on a Phosphor Imager (Molecular Dynamics) and quantified by reprobing of the blots with a probe for rat GAPDH.
Amplification of G mRNA by RT-PCR
First-strand cDNA was synthesized from total RNA with oligo-dT
primers using Superscript II reverse transcriptase and was
used for amplification of Gs, G16, and
Gz. The oligonucleotides were designed
to be specific for the different -subunits and were located in the
5' coding region and the 3' noncoding region. The sequences of the
primers were as follows. Gs: 5' primer,
5'-GGAATTCCATATGGGATGTCTCGGGAA-3'; 3' primer,
5'-CCGCTCGAGGCCCTATGGTGGGTGATTATTA-3'; G16:5'
primer,
5'-GGAATTCCATATGAGCGCTTGGCGTCACCCGCAGTTCGGTGGTATGGCCCGCTCGCTGACCTGGCGCTGCT-3';
3' primer, 5'-GAAGATCTGGCGTTCCTTCTCCTGTCCACTAGAGTGCG-3';
Gz: 5' primer,
5'-GGAATTCCATATGGGATGTCGGCAAAGCTCAGAGG-3'; 3' primer,
5'-GGATGATCAAAAGTGAAGGGGCAGGTTGGG-3'. The PCR was performed for 30
cycles using Pwo polymerase for Gs,
SuperTaq for G16, and Pfu
polymerase for Gz. The conditions for denaturation
between cycles, annealing, and extension were, respectively, 1 minute
at 95°C, 1 minute at 52°C, 2 minutes 72°C for Gs;
1 minute at 95°C, 1 minute at 60°C, 2 minutes at 72°C for
G16; and 1 minute at 95°C, 1 minute at 53°C, 2
minutes at 72°C for Gz. All PCRs were performed with a
hot start at 95°C; SuperTaq and Pfu were used
with 5% DMSO.
Ca2+ Mobilization
MEG-01, DAMI, and CHRF-288-11 cells were cultured overnight in
serum-free RPMI, pelleted (5 minutes, 200g at 20°C), and
resuspended in Ca2+-free HEPES–Tyrode's buffer (in
mmol/L: NaCl 145, KCl 5, Na2HPO4 0.5,
MgSO4 1, HEPES 10, and glucose 5, and 2 g/L BSA [pH
6.5]; buffer A). Cells were incubated at 37°C for 1 hour with 3
µmol/L fura 2-AM, pelleted, resuspended in
albumin-free buffer A, and stored at room temperature. Five
minutes before the start of each measurement, the suspensions were
diluted to 3x105 cells/mL in albumin-free buffer A
(pH 7.2) and prewarmed at 37°C. Cells were incubated with 1
mmol/L CaCl2 for 1 minute to load intracellular
Ca2+ stores. Subsequently, 1 mmol/L EGTA (final
concentration) was added to trap extracellular Ca2+
immediately before the addition of agonist, making the measurements
specific for Ca2+ mobilization. Measurements were performed
at 37°C under mild stirring (50 rpm) in a Hitachi F-4500
fluorescence spectrophotometer with a multiwavelength timescan
program. Fura 2 fluorescence was measured at 340 nm (F1) and
380 nm (F2) excitation, chosen on both sides of the isosbestic point,
and 510 nm emission. [Ca2+]i was calculated
from the ratio (R=F1/F2) of fluorescence intensities obtained
from the formula of Grynkiewicz et al28 as modified by
Gillis and Gailly29 :
[Ca2+]i=Kdx(R-Rmin)/(Rmax-R)xF2min/F2max.
Fmax was measured at saturated Ca2+ concentration, and Fmin in the absence of calcium ions. Rmax was determined by addition of Triton X-100 (final concentration, 0.1%) in the presence of 1 mmol/L Ca2+, and Rmin by the subsequent addition of 10 mmol/L EDTA and 20 mmol/L Tris base. Calculations were based on a dissociation constant (Kd) of the fura 2–Ca2+ complex of 224 nmol/L.
Determination of cAMP
Samples (300 µL) of control and agonist-treated suspensions
(1x105 cells/mL in HEPES-Tyrode's buffer [pH 7.2]) were
added to 600 µL of ice-cold 96% ethanol and transferred to liquid
nitrogen. Defrosted samples were centrifuged (15 minutes,
14 000g at 4°C), and the pellets were washed with
ice-cold 65% ethanol. The combined supernatants were evaporated under
a stream of nitrogen at 60°C. cAMP was determined according to the
manufacturer's instructions with a cAMP-[125I]
radioimmunoassay.
Analysis of TxA2 Receptors
MEG-01, DAMI, and CHRF cells were suspended in
HEPES-Tyrode's buffer (1.5x106 cells/mL in buffer A,
without BSA [pH 7.2]). Aliquots of 0.2 mL were incubated with
increasing concentrations (1 to 50 nmol/L) of
[3H]SQ29548 at 25°C for 60 minutes. The incubations
were stopped with 1.5 mL of ice-cold buffer, and the samples were
rapidly filtered through a Whatman GF/C filter. The filters were rinsed
three times with 3 mL of ice-cold buffer. Scintillation fluid was
added, and radioactivity was counted according to standard procedures.
Nonspecific binding was assessed in the presence of 10
µmol/L excess of unlabeled ligand. Data were analyzed
by nonlinear regression (GraphPad Prism) and were displayed as
Scatchard plots.
Statistics
Statistical significances were calculated by a two-tailed paired
or unpaired (where appropriate) Student's t test. Data are
expressed as mean±SD (for n data).
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Results |
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Characterization of Megakaryoblastic Cell Lines
Flow cytometric analysis showed that almost all cells expressed GPIb (Fig 1
); small differences
in relative fluorescence intensity indicated that the number of
expressed GPIb molecules per cell was somewhat higher on MEG-01 and
DAMI than on CHRF. Analysis of GPIIIa expression showed that
only 36±2% of MEG-01 cells expressed this protein, in contrast to
91±2% and 99±1% for DAMI and CHRF, respectively. The relative
fluorescence intensities differed considerably, ranging from
6±1% in MEG-01 and 15±1% in DAMI to 75±2% (n=4) in CHRF. This
indicates that the number of GPIIIa molecules per cell on MEG-01 and
DAMI is much lower than on CHRF. On the basis of these data and
morphological criteria (not shown), these data confirm that MEG-01,
DAMI, and CHRF represent stages of increasing maturation.
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Identification of G Subunits in Megakaryoblastic Cell Lines
and Platelets
Western blots revealed that MEG-01, DAMI, CHRF, and platelets
contain Gi-1/2, Gi-3, Gq,
G12, and Gs (Fig 2). Gs is known to consist
of a short (45-kD) and a long (52-kD) isoform, designated
Gs-S and Gs-L,
respectively.30 MEG-01 and DAMI contained mainly
Gs-S; CHRF and platelets contained both types in
approximately equal amounts, although the total amount of
Gs appeared lower. MEG-01 did not contain
G16 protein, but DAMI, CHRF, and platelets showed
increasing amounts of this protein. The three cell lines appeared
devoid of Gz protein, although this -subunit was
easily detectable in platelets.
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To study the expression of G16 and
Gz in more detail, mRNAs for these subunits were
analyzed and compared with mRNA for Gs as an
example of an early expressed -subunit. Northern blot
analysis (Fig 3) showed
Gs mRNA in MEG-01, DAMI, and CHRF cells, in agreement
with the Western blots. G16 mRNA was absent in MEG-01,
but DAMI and especially CHRF showed expression for this -subunit. In
contrast, Northern blots failed to show mRNA for Gz in
the three cell lines (not shown). Reprobing of the blots with a probe
for GAPDH and quantification of the amount of mRNA on the Phosphor
Imager revealed that MEG-01 contained the highest amount (100%) of
mRNA for Gs, whereas DAMI and CHRF contained 45% to
54% and 61% to 55%, respectively, in two separate experiments. These
data are in agreement with the weaker expression of Gs
on the Western blot. In contrast, G16 mRNA signal was
absent in MEG-01, 20% to 21% in DAMI, and 100% in CHRF.
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The amplification of megakaryoblastic and platelet mRNAs by RT-PCR
with oligonucleotides specific for G16
was in line with these observations. Although Gs mRNA
was present in all cell types, a concurrently run sample was
negative for G16 mRNA in MEG-01. A weak band of 1.6
kb was detected in DAMI and CHRF, and a strong band was found in
platelets (Fig 4). A similar
amplification of Gz mRNA was negative for MEG-01 and
CHRF, which again accords with the Northern blots. However, a weak band
of 1.4 kb was found in DAMI, despite the negative Northern blot for
this cell line. Again, amplification of platelet RNA led to a clear
band of the same size.
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BstEII.
Signal Transduction
To analyze whether incomplete G expression led to
abnormal signal transduction, MEG-01, DAMI, and CHRF cells were
stimulated with agonists known to affect Ca2+ homeostasis
in platelets (Table 1). -Thrombin
and PAF induced Ca2+ mobilization in the three cell lines,
albeit to different extents. These responses could be completely
blocked by a preincubation with the PLC inhibitor U73122,
whereas the inactive analogue U73343 had no effect. Preincubation with
PTX abolished the -thrombin–induced Ca2+ mobilization
by 40%, indicating that the thrombin receptor was coupled to PLC
via a G protein of the Gi class and via a different G
protein. In contrast, the PAF-induced Ca2+ mobilization was
insensitive to PTX. Interestingly, the TxA2 analogue U46619
failed to mobilize Ca2+ in MEG-01, whereas DAMI and CHRF
responded with increases of 60% and 70%, respectively, compared
with unstimulated cells. As in platelets, these responses were
insensitive to PTX.5
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The PGI2 analogue iloprost also triggered Ca2+ mobilization. The response was particularly evident in MEG-01, lower in DAMI, and virtually absent in CHRF. The rise in cytosolic Ca2+ was abolished by the PLC inhibitor U73122 and could be partially blocked by PTX. Although iloprost shows the highest affinity for IP receptors, binding to EP1 receptors has been reported.31 To better discriminate between these receptors, studies were repeated with carbaprostacyclin, an agonist with optimal specificity for IP receptors, and with 17-phenyl trinor PGE2, an agonist for EP1 receptors. After stimulation of DAMI cells with 17-phenyl trinor PGE2 (1 µmol/L), the rise in cytosolic Ca2+ in response to carbaprostacyclin (1 µmol/L) was 110±16% (n=3) of normal carbaprostacyclin signaling. Also, a primary incubation with carbaprostacyclin failed to change subsequent responses by 17-phenyl trinor PGE2. Thus, both ligands were specific for their respective receptors. The carbaprostacyclin data were similar to those obtained with iloprost, confirming that the IP receptor signals to Ca2+ via PLC, a property that disappears at later maturation stages. In contrast, the Ca2+ responses triggered by 17-phenyl trinor PGE2 did not disappear during maturation.
In platelets, a slight increase in cAMP is sufficient to abolish agonist-induced Ca2+ mobilization.32 As expected, iloprost raised cAMP in the three megakaryoblasts (see below), but this treatment did not change the Ca2+ responses by platelet ligands. Also, the cell-permeable analogue dbcAMP (250 µmol/L, 5 minutes) failed to change these Ca2+ responses, revealing a marked discrepancy with those patterns in platelets (data not shown).
The cAMP accumulation in the megakaryoblasts is listed in Table 2. Unstimulated cells contained 10
pmol cAMP/106 cells. Incubation with CTX increased cAMP
2-fold in all cell lines, reflecting the presence of a functional
Gs protein. Iloprost raised the level of cAMP from 4- to
6-fold in MEG-01 and DAMI to 12-fold in CHRF. Preincubation with the
mastoparan analogue Mas-7 completely inhibited the iloprost-induced
cAMP accumulation, reflecting the presence of a functional
Gi protein. As in platelets, -thrombin, PAF, and the
thromboxane analogue U46619 failed to increase the cAMP
level. Surprisingly, -thrombin amplified the cAMP accumulation by
iloprost, a property not seen in platelets. There was a 2-fold to
3-fold enhancement in MEG-01 and DAMI and a 6-fold increase in CHRF.
PAF failed to affect the iloprost-induced cAMP formation in any of the
cells. Potentiation of cAMP formation was also absent when MEG-01 was
treated with TxA2 analogue, but both DAMI and CHRF showed a
stimulation by TxA2 similar to that seen with
-thrombin.
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The inability of TxA2 analogue to mobilize Ca2+
or to stimulate iloprost-induced cAMP formation in MEG-01 but not in
DAMI and CHRF pointed to a defect in TxA2-induced signal
transduction. Because all cell lines showed PLC-mediated
Ca2+ responses (Table 1
) and AC-mediated cAMP formation
(Table 2), the impairment was either at the receptor level or caused by
the absence of G16 (Figs 2 through 4). Analysis
of [3H]SQ29548 binding to intact cells revealed that
MEG-01, DAMI, and CHRF all contained TxA2 receptors (Fig 5). MEG-01 showed a binding affinity of
6.9 nmol/L, with a Bmax of 14 800 binding sites per
cell, which is in good agreement with the Kd of
8.2 nmol/L and 12 800 binding sites per cell reported
earlier.15 DAMI cells had a similar
Kd (7.5 nmol/L) but contained 44 000
binding sites per cell. CHRF had a Kd of 5.4
nmol/L and 21 600 binding sites per cell, which is again close
to reported values (Kd=2.1 nmol/L;
Bmax=35 00033 ). These data indicated that
neither receptor affinity nor the number of receptors varied
appreciably between the cell lines.
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), DAMI (
),
and CHRF cells (
). Data represent means of triplicate
samples. Analysis was performed by nonlinear regression of
untransformed data. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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With MEG-01, DAMI, and CHRF cell lines used as models for different stages of megakaryocyte maturation and the platelet taken as the most matured cell, the following stages in the biogenesis of G
subunits can be identified.
There is an early stage represented by MEG-01 that already
contains the proteins for Gi-1/2, Gi-3,
Gq, G12, and Gs. These
cells respond to -thrombin and PAF with Ca2+
mobilization, indicating that the respective receptors are present
and that steps downstream of the -subunits are intact. Indeed, the
fact that the Ca2+ response is abolished by U73122 is
evidence for the presence of PLC. In platelets, -thrombin
mobilizes Ca2+ via Gi-ß/ and a poorly
characterized PTX-insensitive G protein, possibly
Gq.34 MEG-01 cells also accumulate cAMP in
response to iloprost, in line with the presence of Gs
and the CTX-induced cAMP formation in these cells. The
Gs-type is mainly the short isotype,
Gs-S, which in reconstitution experiments had a much
lower activity than the long isotype,
Gs-L.30
The maturation stage represented by DAMI contains the same
-subunits as present in MEG-01 and, in addition,
G16. G16 is a member of the Gq
family and is expressed primarily in hematopoietic
cells.35 A recent publication reported that platelet
RNA contains the message for this G protein.36 The
appearance of G16 is accompanied by Ca2+
mobilization by TxA2. Because MEG-01, DAMI, and CHRF all
contain TxA2 receptors and the sequences that mediate
Ca2+ mobilization by -thrombin, these findings suggest
that G16 couples the TxA2 receptor to PLC. A
second property that accompanies the appearance of G16
is stimulation of iloprost-induced cAMP formation by TxA2,
again pointing to the formation of a receptor-G16
complex that is activated by this agonist.
The late stage of megakaryocyte maturation, represented by
CHRF, shows downregulation of Gs-S and the appearance of
Gs-L, which is accompanied by an increase in
iloprost-induced cAMP formation. At the same time, there is a
downregulation of total Gs, as evident from the weaker
bands in the Western blot and the lower expression of Gs
mRNA on Northern blot. Platelets also contain approximately equal
amounts of Gs-S and Gs-L. Thus, the
extreme responsiveness of platelets to
PGI237 and its analogues might result from the
prevalence of the long isotype, which has a 3- to 10-fold higher
activity than the short isotype.30 A similar shift is seen
in rat platelets during aging.38
Apart from the signaling properties that develop during maturation, a property that disappears is the iloprost-induced Ca2+ mobilization. Whereas MEG-01 showed a 95% increase in cytosolic Ca2+ content, there was only a 30% increase in DAMI. In CHRF, iloprost failed to mobilize Ca2+, a property these cells share with platelets. A similar change is seen in cultures of human CD34+ cells, in which young megakaryoblasts, cultured in the presence of thrombopoietin, show iloprost-induced Ca2+ mobilization and matured megakaryocytes lose this property (van der Vuurst et al, unpublished results, 1997). The Ca2+ mobilization by iloprost in MEG-01 was for the major part inhibited by PTX, suggesting the involvement of a G protein of the Gi family. The PTX-insensitive part might reflect a role for a Gs protein. HEL cells also show PTX-insensitive Ca2+ mobilization in response to iloprost.39 The PLC inhibitor U73122 completely abolished the iloprost-induced Ca2+ mobilization. Similar findings were obtained with carbaprostacyclin, which is a more specific agonist for IP receptors, indicating that these receptors couple to PLC via a member of the Gs class. Ca2+ mobilization through IP receptors disappears during maturation, in contrast to responses evoked by EP1 receptors.
In all cell lines, stimulation with iloprost raises cAMP. This leads to
the peculiar observation that PGI2 stimulates both
inhibitory and stimulatory G proteins for AC regulation.
The fact that iloprost raises cAMP indicates that the stimulating
signals prevail and that additional mechanisms separate the two
pathways. Equally surprising is the fact that Ca2+
mobilization by -thrombin, PAF, or TxA2 analogue in
megakaryoblasts is insensitive to an increase in cAMP, induced either
via the PGI2 receptor or by direct addition of dbcAMP. In
platelets, cAMP inhibits multiple steps in agonist-induced
Ca2+ mobilization, such as the binding of -thrombin to
its receptor and activation of PLC.32 In many cell types,
cAMP is not an inhibitor or even signals as an activating
factor. Activation of adenosine A1 receptors in
smooth muscle cells led to a Gi-mediated Ca2+
mobilization through PLC, which was insensitive to cAMP.40
A dopamine receptor expressed in human embryonic kidney 293 cells
mediated Ca2+ mobilization, which depended on
cAMP.41
The insensitivity to PTX and the fact that the protein is
phosphorylated during platelet activation makes
Gz a topic of specific interest. None of the
megakaryoblasts contained detectable Gz protein, and
there was only a weak 1.4-kb band in DAMI after RT-PCR. Nevertheless,
these cells showed a normal Ca2+ mobilization on
stimulation with -thrombin and PAF and undisturbed cAMP
production by iloprost, indicating that these pathways can
function without Gz. In platelets, activation of PKC
results in phosphorylation of
Gz10 and suppression of
PGI2-induced cAMP formation,42 but a causal
relationship has never been established.
A property not seen in platelets is the amplification of
iloprost-induced cAMP formation by -thrombin and TxA2
analogue. For TxA2 analogue, this property required the
presence of G16, suggesting that a
TxA2-G16 complex was involved. -Thrombin
enhanced cAMP formation even in MEG-01, which lacks G16,
and might therefore involve a complex between the thrombin receptor and
a Gi subtype or Gq. Preliminary studies show
that the enhancement by both agonists is mediated via protein kinase C
and a second step upstream of PLC (van der Vuurst et al, unpublished
results, 1997). The stimulation in megakaryoblasts is in sharp contrast
with platelets, whereas PKC inhibits this
pathway.35
Most classifications of megakaryocyte maturation presume their
development from an incomplete precursor to a cell that contains most
of the proteins present in platelets, such as membrane
receptors, glycoproteins, and -granule proteins.
Williams and Levine43 separated megakaryocyte development
into four stages, ranging from the early megakaryoblast to the mature
polyploid megakaryocyte capable of platelet formation. The protein
composition in developing megakaryocytes is unknown, and it is equally
uncertain how well megakaryoblastic cell lines resemble the different
maturation stages of megakaryocytopoiesis. The present results
illustrate that maturation might imply that expression increases for
one protein and decreases for another. Whereas the more mature DAMI and
CHRF cells show increased levels of G16 mRNA compared
with the immature MEG-01 cells, the levels of Gs mRNA
are lower. Furthermore, the shift to the more active
Gs-L subtype illustrates that maturation also includes
changes between closely related proteins.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Netherlands Organization for Scientific Research/the Netherlands Heart Foundation (grant 940.50.102/900.526.094).
Received March 22, 1996; accepted December 12, 1996.
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References |
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