Hematology/Oncology Section, Department of Medicine, Veterans Administration Medical Center and University of Minnesota, Minneapolis, Minnesota 55417
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ABSTRACT |
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This study evaluated the mechanism of epinephrine
potentiation of platelet secretion induced by thromboxane
A2 (TXA2). Dog platelets that do not secrete in
response to TXA2 alone (TXA2) were compared
with dog platelets that do secrete (TXA2+) and with human
platelets. TXA2
platelets had impaired TXA2
receptor (TP receptor)-G protein coupling, indicated by 1)
impaired stimulated GTPase activity, 2) elevated basal
guanosine 5'-O-(3-thiotriphosphate) binding, and
3) elevated G
q palmitate turnover that was
corrected by preexposure to epinephrine. Kinetic agonist binding
studies revealed biphasic dog and human platelet TP receptor
association and dissociation. TXA2
and TP
receptor-desensitized TXA2+ dog and human platelets had
altered ligand binding parameters compared with untreated
TXA2+ or human platelets. These parameters were reversed,
along with impaired secretion, by epinephrine. Basal phosphorylation of
TXA2
platelet TP receptors was elevated 60% and was
normalized by epinephrine. Epinephrine potentiates platelet secretion
stimulated by TXA2 by reducing basal TP receptor
phosphorylation and facilitating TP receptor-G protein coupling in
TXA2
platelets and, probably, in normal platelets as well.
G proteins; dogs; platelet activation
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INTRODUCTION |
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EPINEPHRINE BINDS to
2-adrenergic receptors (
2-AR) and
stimulates variable human platelet aggregation and secretion, and it
potentiates other agonist-induced platelet activation in human and dog
platelets (2, 21, 22, 26, 41). The mechanism of
epinephrine-induced platelet activation has been the subject of
numerous studies and considerable debate (see Ref. 41 for review). Previous investigations indicated that epinephrine did not
function as a single platelet agonist but, rather, that it enhanced the
activation initiated by other agonists (2, 26, 41). Banga
et al. (2) found that epinephrine promoted human platelet
aggregation and secretion by increasing thromboxane A2 (TXA2) formation, via activation of phospholipase
A2, and by facilitating TXA2-mediated
signaling. The mechanism responsible for the latter effect was not
defined, but it was suggested that epinephrine potentiated platelet
activation either by enhancing the binding of agonists to
TXA2 receptors (TP receptors) or by the coupling of TP
receptors to phosphoinositide-specific phospholipase C (PLC) (2). This hypothesis was of interest to us because of our
previous observation that dog platelets, which showed little or no
response to TXA2 alone, had impaired signal transduction
via G proteins from TP receptors to PLC (18), but they
aggregated and secreted their granular contents if exposed to
epinephrine before TXA2 (6, 18, 21, 22).
Most dogs, whether random or purpose bred, have blood platelets
that form TXA2, but they have very impaired secretion and aggregation in response to TXA2 (5, 6, 18, 21,
22). These TXA2-insensitive (TXA2) dog
platelets also have impaired responses to TXA2 analogs such
as U-46619 or I-BOP
{[1S-[1
,2
(Z),3
(1E,3S*),4
]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo (2.2.1)hept-2-yl]-5-heptenoic
acid} (18). However, some mixed breeds, and a few
purpose-bred dogs, have TXA2-sensitive (TXA2+) platelets (6, 18, 21) that secrete and aggregate in
response to TXA2 or TXA2 analogs as do human
platelets. Each dog's platelet response to TXA2 is
consistent and genetically determined (20).
TP receptors belong to the family of G protein-coupled receptors
(GPCRs) that activate effectors via G proteins (16).
Agonist binding to platelet TP receptors activates G13,
which results in platelet shape change (24), and
G
q, which in turn activates PLC
-isoforms (PLC-
)
(3, 19, 17, 34). Activation of PLC-
liberates two
important intracellular messengers:
D-myo-inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol (DG). The subsequent elevation of
the cytosolic ionized calcium concentration by IP3 and
activation of protein kinase C by DG lead to platelet secretion. Previous investigation of the mechanism responsible for
TXA2
platelets implicated defective signaling from TP
receptors via G proteins to PLC-
(18). Subsequent study
found no mutation in G
q in TXA2
platelets
(19). Therefore, we sought evidence of an alternative mechanism.
An important characteristic of dog TXA2 platelets is the
reversibility of their functional defect by epinephrine. Exposure of
TXA2
platelets to epinephrine before exposure to
TXA2 or TXA2 analogs results in aggregation and
secretion comparable to that observed in TXA2+ platelets
(6, 18, 21, 22). Therefore, we studied the effects of
epinephrine on several aspects of dog TXA2
platelet TP
receptor signal transduction including Gq function, PLC-
activation, TP receptor kinetic agonist binding, and phosphorylation. Because we observed that human platelets with homologously desensitized TP receptors had their defective activation of PLC-
corrected by
exposure to epinephrine in a manner similar to that of dog TXA2
platelets, we also studied the mechanism of
epinephrine correction of human platelets. These studies indicated that
epinephrine corrects the signaling defect of dog TXA2
platelets and desensitized human platelets by facilitating
Gq-TP receptor coupling. In TXA2
platelets
the corrective effect of epinephrine appears to be mediated by
dephosphorylation of hyperphosphorylated TP receptors.
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MATERIALS AND METHODS |
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Materials
[125I]BOP and [127I]BOP were obtained from Cayman Chemical (Ann Arbor, MI). U-46619 (9,11-dideoxy-9Antisera
Antisera N345 and N432 were commercially produced (HRP, Dover, PA) in rabbits by injection of synthetic peptides coupled to keyhole limpet hemocyanin (Pierce conjugation protocol). The dilution of whole antisera used is designated for each experiment. Antiserum N345 recognizes an internal sequence in GSubjects
Dogs with well-characterized TXA2Platelet Preparation
Platelets were prepared as described previously (18) and were resuspended with HEPES citrate buffer for binding assays. For receptor phosphorylation and IP3 generation studies, the resuspension buffer was HEPES Tyrode buffer (18). In [14C]5-HT secretion studies, the resuspension buffer was modified Lindon's buffer (18). Platelet membranes were prepared by the method of Baldassare et al. (1).GTPase Activity
Platelet membrane GTPase activity was assayed by standard methods (18).Binding of [35S]GTPS to Platelet Membranes
[3H]Palmitate Labeling
Palmitate exchange in platelets was determined by the method of Hallak et al. (15). Platelets (3 × 109) suspended in 2 ml of acylation buffer (140 mM NaCl, 2.5 mM KCl, 0.1 mM MgCl2, 10 mM NaHCO3, 0.5 mM NaH2PO4, 5.5 mM glucose, and 10 mM HEPES, pH 7.4, containing 3.6 mg/ml fatty acid-free BSA, 1 U/ml apyrase, and 0.3 µM PGE1). [3H]palmitate (1 mCi) was dried under nitrogen and dissolved in 1 ml of acylation buffer. Both palmitate and cells were incubated at 37°C for 3 min before the reaction was initiated. For stimulated exchange reactions, 30 µl (1:200 dilution) of stock I-BOP were added to the palmitate tube (final concentration 12.5 nM I-BOP). The reaction was initiated by combining the cells with the palmitate, and 315-µl aliquots were withdrawn at 2, 5, 9, 15, 24, and 60 min, diluted into 3.75 ml of HEPES citrate containing 25 µl of 0.1 M EDTA, and centrifuged at 1,700 rpm for 10 min. The supernatant was removed, and 90 µl of SDS disruption buffer (50 mM NaPO4, pH 8.0, 0.5% SDS, and 2 mM EDTA) were added to the pellet. The cells were vortexed, heated at 90°C for 5 min, and cooled, and 30 µl of 4× RIPA (200 mM NaPO4, pH 7.2, 4% deoxycholate, 4% Triton, 0.6 M NaCl, 2% SDS, and 8 mM EDTA) plus inhibitors (1% aprotinin, 200 µg/ml leupeptin) were added. Each sample (~150 µl) was clarified, with 2 µl of preimmune sera and 25 µl of protein A-Sepharose, for 3 h before reaction with GIP3 Formation and Platelet [14C]5-HT Secretion
IP3 formation was assayed by RIA (Amersham), and [14C]5-HT secretion was measured as previously described (18).[125I]BOP Binding
Aliquots of washed platelets (0.02-1 × 109 platelets/ml) in HEPES citrate buffer (pH 7.4) were incubated with [125I]BOP (1-2 µCi) in combination with unlabeled drug over a 100-fold concentration range (0.3-30 nM) at room temperature (~20°C). At multiple time points (association: 0.2-30 min; dissociation after 30-min incubation: 1-45 min), the binding reaction was terminated by the removal of cell aliquots that were immediately added to 10 ml of ice-cold HEPES buffer, followed by rapid filtration through Whatman GF/C glass fiber filters under reduced pressure. The filters were then washed twice with 10 ml of ice-cold buffer and counted. The entire filtration was complete in <15 s. Nonspecific binding was determined in the presence of 1 µM I-BOP and was generally <15% of the total binding. In experiments with U-46619 or epinephrine pretreatment, cells were incubated with U-46619 (1.43 µM [3H]U-46619) or epinephrine (1-2 µM) for 30 min at room temperature, washed twice, and resuspended in HEPES citrate buffer before agonist (1-2 nM [125I]BOP) binding. The residual U-46619 concentration never exceeded 14 nM.Phosphorylation of TP Receptors
The method of Carlson et al. (4) was employed. Briefly, platelets (2.5 × 109 platelets/ml) were labeled with 0.8 mCi/ml H3[32P]O4 for 90 min at 30°C, washed by dilution with HEPES citrate buffer, spun, and resuspended in HEPES Tyrode buffer to 1.8 × 109 platelets/ml. Phosphorylation reactions were conducted at room temperature with 660 µl of labeled cells to which were added (at time 0) buffer, U-46619 (1.43 µM), epinephrine (1 µM), or epinephrine plus U-46619. To inhibit TXA2 formation, indomethacin (5 µM) was added to some samples 3 min before the beginning of the reaction. After 1 and 5 min, 300-µl samples were removed, 30 µl of 0.1 M EDTA were added to each sample, and the samples were spun at 2,000 rpm for 5 min. Supernatants were removed, 90 µl of stopping buffer plus inhibitors (50 mM NaPO4, pH 8.0, 0.5% SDS, 2 mM EDTA, 1 mM DTT, 5 mM NaF, 10 mM Na4P2O7, 2 mM Na3VO4, 1% aprotinin, 0.5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) were added to the pellets, and the pellets were vortexed until dissolved and then heated to 90°C for 3 min. To 90 µl of sample, 30 µl of 4× RIPA plus inhibitors were added, and the mixture was allowed to cool on ice for 30 min. Preimmune sera (1:50) was added, allowed to react for 1 h at 4°C, cleared with 20 µl of protein A-Sepharose for 2 h at 4°C, and centrifuged. Sample supernatant (100 µl) was removed, and TP receptor antisera N432 (1:10 dilution) was added before overnight incubation at 4°C. Protein A-Sepharose (100 µl) was used to capture the immunoprecipitate, and the material was collected by centrifugation. Immunoprecipitates were washed twice, resuspended in 40 µl of buffer plus 20 µl of 3× SDS sample buffer, and boiled for 4 min. Samples were run on 12% mini-gels, transblotted, and exposed to X-OMAT film atMiscellaneous
The protein concentration was determined by dye binding using the Coomassie blue dye reagent (Bio-Rad, Hercules, CA) with ovalbumin as the standard.Calculations
Analysis of the binding association and dissociation parameters under nonequilibrium conditions was done with the use of the computer program KINETIC (BioSoft). Statistical analysis was performed with the program Statworks using the Student's t-test. Data are expressed as means ± SE unless otherwise specified. ![]() |
RESULTS |
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Biochemical Studies
To define the mechanism of the corrective effect of epinephrine on TXA2TP receptor-stimulated GTPase activity.
Basal GTPase activities of TXA2 and TXA2+
platelet membranes were not significantly different from each other
[42.7 ± 2.1 (n = 13) vs. 46.1 ± 2.6 pmol · min
· mg
1
(n = 10)]. The addition of U-46619 to
TXA2+ membranes significantly increased GTPase activity
(6.9 ± 1.2 pmol · min
· mg
1), but
TXA2
membrane GTPase activity increased only slightly (2.3 ± 1.0 pmol · min
· mg
1) (Fig.
1, whole cells + buffer,
top).
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TP receptor-stimulated GTPS binding.
To further evaluate the effect of TP receptor agonist binding on G
protein activation, we studied GTP
S binding to platelet membranes in
the absence and presence of the TXA2 analog I-BOP (Fig.
2). I-BOP increased mean
[35S]GTP
S binding to TXA2+ membranes at 30 min (P < 0.03, n = 5). I-BOP did not
increase binding to TXA2
membranes; however, the mean
basal binding of TXA2
compared with TXA2+
platelet membranes was significantly elevated at 30 min
(P < 0.02, n = 5). Exposure of
TXA2
platelets to epinephrine before membrane preparation decreased the basal association of [35S]GTP
S with
TXA2
membranes, and subsequent addition of I-BOP to these
membranes resulted in a significant increase in mean [35S]GTP
S binding at 30 min (P < 0.05, n = 2), comparable to that of TXA2+
membranes exposed to I-BOP alone. The similarity of the net
I-BOP-induced increase in GTP
S binding of TXA2
platelets that had been preexposed to epinephrine to that of
TXA2+ platelets without epinephrine preexposure is
illustrated in Fig. 2C.
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TP receptor-stimulated palmitate exchange.
G protein -subunits are palmitoylated at their
NH2-terminal cysteines (44), and they undergo
increased palmitate exchange on receptor activation. To determine
whether impaired agonist-stimulated GTPase activity in
TXA2
platelets was attributable to a failure to activate
G
q, [3H]palmitate exchange
labeling of G
q subunits of TXA2+
and TXA2
platelets was carried out. The results (Fig.
3) closely parallel those obtained from
the study of GTP
S binding. In TXA2+ platelets, I-BOP
stimulated an increased level of palmitate exchange (P < 0.04, n = 2), but in TXA2
platelets,
no increase was observed. Exposure of TXA2
platelets to
epinephrine before the addition of palmitate significantly decreased
basal palmitate turnover compared with control cells (P < 0.05, n = 3), and the addition of I-BOP to these
platelets resulted in significantly increased palmitate turnover
(P < 0.03, n = 3). The similarity of
the net increase in palmitate exchange of TXA2
platelets
that had been preexposed to epinephrine to that of TXA2+
platelets without epinephrine preexposure is illustrated in Fig.
3C.
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Effector activation subsequent to receptor agonist binding.
The results of the biochemical studies reported above indicated that TP
receptor-stimulated G protein activation was impaired in
TXA2 platelets and that it was corrected by epinephrine.
To evaluate the functional consequences of epinephrine exposure, we
observed its effect on activation of PLC-
by assaying
IP3 formation in platelets with intact cyclooxygenase activity.
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TP Receptor Kinetic Agonist Binding
To further elucidate the mechanism responsible for TXA2[125I]BOP association kinetics.
Studies of the time course of [125I]BOP binding to
TXA2+ and TXA2 platelets and human platelets,
performed at six different concentrations (range: 0.3-30 nM; see
MATERIALS AND METHODS), yielded data that were resolved by
the computer program KINETIC to two exponential phases of binding (Fig.
5A). The rate constants for
the fast and slow components of binding were determined from linear
regression of the secondary plots of the observed rate constant
(kobs) vs. agonist concentration (Fig.
5B) and were analyzed according to the mass action equation:
kobs = k
+ k+[A], where the dissociation constant
(Kd) is defined as
k
/k+ (dissociation rate
constant/association rate constant) and [A] is the agonist
concentration.
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[125I]BOP dissociation kinetics. [125I]BOP dissociation rates at equilibrium (30 min at room temperature) were measured from the same ligand reaction samples used in the association studies. Biphasic kinetic patterns were observed in all three platelet types, and the rate constants were independent of agonist concentration (Fig. 5C). More than 95% of [125I]BOP-specific binding dissociated by 150 min, indicating that virtually all of the binding was reversible.
TXA2+ platelets (n = 4) exhibited a rapid dissociation of 34% of the total binding with a half-time of 3.7 min (kAnalysis of paired association/dissociation parameters.
To further evaluate TP receptor kinetic binding parameters, we
performed multiple studies with agonist concentrations of 1-2 nM
(to allow comparison of dog and human platelet results with minimal
interference from the abundant human low-affinity receptors), and we
calculated association constants (ka) using the
relationship ka = [ka(obs) k
a]/[L], where [L] is the ligand
concentration. Dissociation parameters were determined
concurrently. An important result of the analysis of these data was
recognition of the fact that the association/dissociation relationships
expected from mass action kinetics were not observed. Instead, we
observed a proportional relationship between rapidly associating and
slowly dissociating TP receptors of human and both dog platelet types (Fig. 6). TXA2
platelets
demonstrated significantly less fast association binding
(Re1) and slow bound ligand dissociation (R02), but total binding was not significantly different from that of TXA2+ or human platelets (Fig. 6).
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Effects of homologous or heterologous agonists on [125I]BOP binding. We evaluated the binding distribution and rate constants for [125I]BOP binding as functions of prior exposure to U-46619 (homologous agonist), epinephrine (heterologous agonist), or both to correlate change in the TP receptor functional state with change in the fast and slow components of binding.
After prior U-46619 incubation of TXA2+ platelets, significant changes were noted in several I-BOP binding parameters (Table 1). The fast rate constant (k1) increased and the proportion bound (Re1) decreased, while the slow constant (k2) decreased and the proportion bound (Re2) increased (Table 1). The amplitude of fast dissociation (R01) increased, and that of slow dissociation (R02) decreased significantly. Total binding (Re1 + Re2) decreased 56 fmol/mg. Human platelet parameters, after exposure to U-46619, were similar to those observed with TXA2+ platelets (Table 1). In TXA2TP Receptor Phosphorylation
Because our kinetic binding studies revealed that homologously desensitized TP receptors of TXA2+ and human platelets had impaired agonist binding similar to that observed in TXA2Basal TP receptor phosphorylation and effect of epinephrine.
Basal phosphorylation of TP receptor protein in TXA2
platelets was significantly higher (160%) than that observed for
TXA2+ platelets (Fig. 7).
Exposure to epinephrine alone increased phosphorylation of
TXA2+ platelet TP receptor protein, but this increase was
indomethacin sensitive, indicating that the increase was attributable
to TXA2. Epinephrine decreased TXA2
platelet
TP receptor phosphorylation to a level comparable to the basal state of
TXA2+ platelets, and this change was not affected by
indomethacin (Fig. 7).
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Agonist-stimulated TP receptor phosphorylation.
The elevated basal level of TXA2 platelet TP receptor
phosphorylation did not increase further on exposure to U-46619 alone; however, exposure of TXA2+ and TXA2
platelets
to epinephrine before U-46619 treatment resulted in a significant
increase in TP receptor phosphorylation of both types of platelets
(Fig. 7). The increase in phosphorylation of TXA2
platelets over the basal level observed after exposure to epinephrine
was nearly equal to that of TXA2+ platelets after exposure
to U-46619 alone. TP receptor-linked G
q and
G
13, which are phosphorylated on agonist stimulation
(25, 29, 33, 43), were quantitatively similar in
TXA2
and TXA2+ platelets, and they migrated
at lower molecular weights than TP receptors on the gels we utilized
(data not shown).
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DISCUSSION |
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The current study yielded additional evidence of impaired
receptor-Gq coupling in TXA2 platelets (e.g.,
diminished GTPase stimulation by TP receptor agonists and elevated
basal levels of GTP
S binding and palmitate turnover, with the latter
attributable to G
q). On activation, G protein
-subunits (G
) exchange GDP for GTP, separate from
-subunits
(G
), and some G
undergo increased palmitate turnover
(44). They subsequently hydrolyze GTP to GDP by the
intrinsic GTPase activity of G
. G
q in
TXA2
platelets appears to be cycling at an increased rate
without being effectively linked to TP receptors. These results could
be due to mutant G
q or mutant TP receptors, such as the
-adrenergic mutants previously described (39, 46).
However, G
q is not mutated (19); recent
studies in our laboratory indicated that the TP receptor is not mutated
(unpublished observation); and PLC-
activation in the absence of TP
receptor agonists (expected from a constitutively activated receptor)
is not present in TXA2
platelets (18). In
contrast to the impaired signal transduction via G
q,
TXA2
platelet signaling via G
13 is intact
since G
13 is not deficient, and shape change is normal
(21, 22).
An important observation made in the biochemical studies of dog
platelets is that the basal level of GTP-ase activity, which was higher
than that of human platelets, was reduced in both types of dog
platelets following the addition of epinephrine, but the reduction seen
in TXA2 platelets was approximately three times greater
than that in TXA2+ platelets. This reduction in basal GTPase activity was followed by an agonist-stimulated rise in activity
that was comparable to that in TXA2+ platelets. Thus we
observed increased agonist-receptor-coupled GTPase stimulation, after
epinephrine exposure of TXA2
platelets, that was
indicative of G
q heterotrimer coupling. Subsequent
studies of IP3 formation demonstrated restoration of second
messenger formation. When TXA2
platelets were pretreated
with epinephrine, the agonist-stimulated IP3 rise was
threefold higher than that observed with epinephrine alone, indicating
significantly greater PLC-
stimulation. That degree of stimulation
was more likely the result of G
q stimulation of
PLC-
3 than G
stimulation of PLC-
2
(40). Thus these observations strongly suggest that
epinephrine restored productive TP receptor-Gq interaction
in TXA2
platelets.
Because G protein activation is dependent on heterotrimer association
with receptors (7), and agonist binding to GPCRs is
influenced by receptor-G protein association (8, 32, 37, 46), we studied the kinetic binding characteristics of untreated and homologously desensitized TXA2 and TXA2+
platelet TP receptors in the absence and presence of epinephrine. As a
control, we studied intact human platelets. These studies yielded data
that closely paralleled those obtained in prior studies of agonist
binding to
2-AR (31, 32).
Three basal-state TP receptor binding parameters distinguished
biochemically unresponsive TXA2 platelets from
TXA2+ or human platelets. First, the elevated fast
association rate (k1) correlated with a reduced
proportion of bound ligand (Re1) and an elevated proportion
of ligand bound by slow association (Re2). Second, the
proportion of ligand undergoing fast dissociation (R01) was increased, and that involved with slow dissociation (R02)
was decreased. Third, TXA2
platelet TP receptor binding
parameters were comparable to TXA2+ and human platelet
parameters when TXA2
platelets were exposed to
epinephrine, and secretion followed the addition of agonist. After
homologous desensitization of TXA2+ and human platelets,
fast association rates increased significantly toward those seen with
simple diffusion-controlled binding. The similarity of the association
rate of TXA2
platelets to that of desensitized platelets,
previously shown to manifest receptor-G protein uncoupling
(30), was further evidence that a similar state existed in
TXA2
platelets. The altered binding parameters and
impaired function of desensitized platelets were restored by
epinephrine. Thus fast-affinity TP receptor binding parameters correlated with biochemical efficacy.
Although epinephrine treatment of U-46619-desensitized platelets reversed the parameter shifts and restored biochemical responsiveness, the increased fast component of association that resulted was not equal to the sum of the loss with desensitization plus the gain from epinephrine exposure. Rather, it was similar to the increase observed with exposure to epinephrine alone. This suggested that only some of the TP receptors were restored to fast association status after epinephrine exposure or that they were derived from "spare" receptors (10). The apparently nonrecoverable decrease in the total binding capability (Re1 + Re2) following homologous desensitization was not due to internalization, since only ~1% residual [3H]U-46619 was detected, and internalization of TP occurs only after hours of agonist exposure (10, 30, 36, 45).
TP receptors are phosphorylated in the absence of agonists, and agonist
binding to TP receptors stimulates time-dependent and
concentration-dependent increased phosphorylation (13, 14, 35). Agonist-induced receptor phosphorylation plays an important role in TP receptor desensitization, but the influence of basal phosphorylation on receptor binding has not been evaluated. The proximal cause of impaired Gq-TP receptor interaction in
TXA2 platelets appears to be an elevated state of basal
TP receptor phosphorylation. Because epinephrine resulted in a change
in TP receptor binding parameters in both TXA2
and
homologously desensitized TXA2+ and human platelets, and
because it decreased the elevated phosphorylation and the elevated
GTP
S binding and palmitate cycling of TXA2
TP
receptors, our studies suggest that basal TP receptor phosphorylation
is a regulatory mechanism for TP receptor agonist binding. Epinephrine
might influence TP receptor efficacy via G
liberated from
2-AR (38), but the mechanism is unknown.
Agonist binding to GPCRs has been characterized by cyclic
agonist-receptor-G protein ternary complex models (12, 32, 39, 42). The allosteric ternary complex model (28, 39)
of agonist binding to receptors includes spontaneous isomerization
(isomerization constant, J) between a resting (constrained)
state, R, and an activated (relaxed) state, R*,
that favors agonist binding and permits G protein interaction. Agonists
may bind preferentially to the R* receptor conformation, or
to other intermediate conformations (12, 27), resulting in
an increased proportion of receptors in an activated state and
stabilization of the agonist-bound receptor-G protein complex
(12). The increased basal phosphorylation that we observed
in TXA2 platelets could result in a shift of receptor equilibrium toward the R configuration by impairing receptor
conformational change. Conversely, the reduction in phosphorylation
observed after epinephrine exposure could shift the equilibrium toward R*. Other investigators have hypothesized that high- and
low-affinity TP receptors might be the result of different levels of
phosphorylation (35).
Thus phosphorylation may control the binding state of normal TP
receptors by influencing the J constant of receptor
isomerization. Epinephrine may potentiate TP receptor-mediated
activation of normal as well as TXA2 platelets by
reducing the basal state of receptor phosphorylation. The expected
result would be an increase in fast-association ligand binding and G
protein coupling that would be expressed as increased agonist potency.
The data available are not sufficient to confirm this speculation, but
the strong parallels between the kinetic TP receptor binding
characteristics of desensitized TXA2+ and human platelets
and those of TXA2
platelets, plus the effects of
epinephrine on TP receptor binding and function, provide support for
this hypothesis. Basal receptor phosphorylation is a potentially
important control mechanism for GPCRs that merits further study.
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ACKNOWLEDGEMENTS |
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This work was supported by the Merit Review Program of the Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. J. Johnson, Hematology/Oncology (111E), VA Medical Center, 1 Veterans Drive, Minneapolis, MN, 55417 (E-mail: johns337{at}tc.umn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 March 2000; accepted in final form 6 July 2000.
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