(Received for publication, August 15, 1995; and in revised form, November 21, 1995)
From the
To define the molecular basis of human chemoattractant receptor
regulation, rat basophilic leukemia RBL-2H3 cells, which are
thrombin-responsive, were transfected to stably express epitope-tagged
receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g.
N-formyl-methionyl-leucyl-phenylalanine (fMLP)), and
platelet-activating factor (PAF). Here we demonstrate that both
thrombin and a synthetic peptide ligand for the thrombin receptor
(sequence SFLLRN) caused phosphorylation and heterologous
desensitization of the receptors for C5a, IL-8, and PAF but not that
for formylpeptides as measured by agonist-stimulated
[S]guanosine 5`-3-O-(thio)triphosphate
binding to membranes. Consistent with the PAF receptor phosphorylation,
both thrombin and thrombin receptor peptide inhibited phosphoinositide
hydrolysis, Ca
mobilization, and degranulation
stimulated by PAF. Unexpectedly, despite heterologous desensitization
at the level of receptor/G protein activation, there was enhancement
(``priming'') by thrombin of subsequent activities stimulated
by C5a and IL-8 as well as fMLP. The priming effect of thrombin was
blocked by its inhibitor, hirudin. However, two other activators of the
thrombin receptor, the peptide SFLLRN and trypsin, stimulated
Ca
mobilization in RBL-2H3 cells but did not cause
priming. In addition, SFLLRN and the thrombin receptor antagonist
peptide FLLRN both inhibited thrombin-induced Ca
mobilization but not priming. Furthermore, the proteolytically
active
-thrombin, which does not stimulate the tethered ligand
thrombin receptor and caused little or no Ca
mobilization in RBL-2H3 cells, effectively primed the response to
fMLP. These data demonstrate that heterologous receptor phosphorylation
and attenuation of G protein activation are not, by themselves,
sufficient for the inhibition of biological responses mediated by C5a
and IL-8. Moreover, thrombin appears to utilize mechanism(s)
independent of its tethered ligand receptor to selectively prime
phospholipase C-mediated biological responses of the C5a, IL-8, and
formylpeptide receptors but not PAF. Because C5a, IL-8, and
formylpeptide activate phospholipase C
, whereas PAF
stimulates a different phospholipase C, the striking selectivity of
thrombin's priming may be mediated via its ability to enhance
receptor-mediated activation of phospholipase C
.
Phagocytic leukocytes respond to multiple inflammatory signals
and play a key role in immunological reactions. Among the well defined
stimulants for leukocytes are chemoattractants: a cleavage product of
the fifth component of complement (C5a), interleukin-8 (IL-8), ()platelet-activating factor (PAF), and N-formylated peptides (e.g. fMLP)(1, 2, 3) . These chemoattractants
mediate their biological responses via the activation of cell surface
receptors that are coupled to phospholipase C via G proteins. Leukocyte
responsiveness to chemoattractants can be ``primed'' or
desensitized by prior stimulation(1, 4, 5) .
Priming or enhanced responsiveness to subsequent stimuli may be caused
by low concentrations of substance P, chemoattractants, phorbol esters,
Ca
ionophores, and cytokines, including
granulocyte-macrophage colony-stimulating factor and tumor necrosis
factor-
(1, 6, 7) . The molecular
mechanism by which these agents prime responses in leukocytes have not
yet been determined. Desensitization of cellular responses to a
stimulus can either be homologous or heterologous(8) . The
former is specific for a given ligand/receptor, whereas the latter
involves multiple ligands/receptors. There is convincing evidence that
receptor phosphorylation is one mechanism by which many G
protein-coupled receptors undergo both homologous and heterologous
desensitization. Recent evidence indicates an additional mechanism for
chemoattractant receptor desensitization, a process resulting in
reduced phosphoinositide hydrolysis via a decreased activity of
phospholipase C (9) .
Thrombin, a serine protease generated
at sites of vascular injury, is a key enzyme in the coagulation cascade
but may also be important in regulating inflammatory and proliferative
responses(10, 11) . Thrombin activates a variety of
cells including platelets, leukocytes, fibroblasts, and endothelial
cells at least in part via its interaction with cell surface
receptors(12, 13, 14, 15) . One such
receptor is a member of the seven transmembrane domain receptor
superfamily whose activation proceeds via a novel
mechanism(16) . Thrombin binds to and cleaves its
receptor's extracellular amino-terminal extension, thereby
unmasking an amino-terminal peptide, whose binding site resides in the
first six amino acids (sequence SFLLRN)(16, 17) . This
tethered ligand binds to the thrombin receptor and induces its
activation. Thrombin also binds to the platelet membrane glycoprotein
Ib (GPIb) to activate Ca mobilization and platelet
aggregation(18, 19) . However, thrombin-induced
chemotaxis in monocytes and growth factor-like effect in fibroblasts
appear to be mediated via the activation of as yet undefined thrombin
receptors(14, 20, 21) .
To better define
the molecular basis of chemoattractant receptor regulation, we used
thrombin-responsive rat basophilic leukemia RBL-2H3 cells to stably
express epitope-tagged receptors for chemoattractants formylpeptide,
C5a, IL-8, and PAF(4, 5, 9) . Using RBL-2H3
cells expressing fMLP and C5a receptors, we previously showed that
thrombin causes phosphorylation and heterologous desensitization of the
C5a but not formylpeptide receptor at the level of receptor-mediated G
protein activation(4) . The present study was undertaken to
more precisely define the effects of thrombin on responses stimulated
by fMLP, C5a, and other chemoattractant receptors. Here we describe a
novel finding that thrombin, utilizing a mechanism unrelated to the
activation of its tethered ligand G protein coupled receptor,
selectively primes phosphoinositide hydrolysis, Ca mobilization, and exocytosis stimulated via the activation of
receptors for formylpeptide, C5a, and IL-8 but not PAF.
For the assay of
[H]phosphatidylinositol 4,5-bisphosphate
([
H]PIP
), RBL-2H3 cells (0.5
10
) were labeled overnight with
[
H]inositol (10 µCi/ml). The lipids were
extracted and separated by chromatography on silica gel 60 F
that had been treated with a solution of 2 mM EDTA and
1% potassium oxalate as described(24) . Unlabeled PIP
(20 µg) were added to all extracts, which were then
evaporated to dryness under nitrogen. The residues were dissolved in
chloroform:methanol (2:1), and mixture was applied to the plates. The
chromatographs were developed with the following solvent system:
chloroform:methanol:4 N NH
OH (9:7:2, v/v/v). The
plates were exposed to iodine vapor to visualize the PIP
.
The spots were scraped from the plate, and the amount of
[
H]PIP
was determined by
scintillation counting.
Figure 1:
Phosphorylation of stably expressed
chemoattractant receptors. P-Labeled RBL-2H3 cells
expressing ET-FR (A), ET-C5aR (B), ET-IL-8R (C), or ET-PAFR (D) were left unstimulated (lanes
1) or stimulated with their respective ligands (lanes 2)
fMLP (1 µM), C5a (100 nM), IL-8 (100
nM), and PAF (100 nM), with PMA (100 nM) (lanes 3), with thrombin (1 unit/ml) (lanes 4), or
with TRP (100 µM) (lanes 5). The reactions were
stopped 3 min after stimulation by adding excess ice-cold
phosphate-buffered saline. The samples were washed, lysed, and
immunoprecipitated with 12CA5 antibody. The proteins were resolved on a
10% SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography. The data shown are from one of three similar
experiments.
Figure 2:
Effects of thrombin and TRP on
chemoattractant receptor-stimulated [S]GTP
S
binding to membranes. RBL-2H3 cells expressing ET-C5aR and ET-PAFR were
left untreated (CON) or treated with their respective ligands
C5a or PAF, PMA, thrombin (THR), or TRP as in Fig. 1.
Membranes were prepared and assayed for agonist-stimulated
[
S]GTP
S. The values are presented as
percentages of net [
S]GTP
S bound, which is
defined as the maximum amount of agonist-stimulated response from
untreated membranes. For ET-C5aR the basal and C5a-stimulated responses
(untreated) were 7097 ± 216 and 15472 ± 47 cpm,
respectively. For ET-PAFR, the basal and PAF-stimulated (untreated)
responses were 6446 ± 350 and 12330 ± 48 cpm,
respectively. The basal [
S]GTP
S bound to
membranes treated with ligand, PMA, thrombin, and TRP were similar to
basal responses in the absence of pretreatment. The data are the means
± S.E. of three experiments performed in
triplicate.
Figure 3:
Effects of thrombin and TRP on
Ca mobilization stimulated by fMLP, C5a, IL-8, and
PAF. RBL-2H3 cells expressing ET-FR, ET-C5aR, ET-IL-8R, and ET-PAFR
were loaded with indo-1 and were left untreated (Control) or
stimulated with thrombin (1 unit/ml) or TRP (100 µM) and 3
min later rechallenged with submaximal concentrations of fMLP, (10
nM), C5a (1 nM), IL-8 (3 nM), or PAF (0.2
nM), and chemoattractant-stimulated Ca
mobilization was determined. The values presented are the peak
Ca
mobilization stimulated by chemoattractants after
the basal (
150 nM) has been subtracted. The data are the
means ± S.E. of three experiments.
Figure 4:
Effects of thrombin and TRP on
dose-response of fMLP-stimulated generation of
[H]inositol phosphates and release of
-hexosaminidase. [
H]Inositol-labeled RBL-2H3
cells expressing ET-FR were left untreated (control) or
treated with thrombin (1 unit/ml) or TRP (100 µM) for 3
min in the presence of LiCl (20 mM) and stimulated with
different concentrations of fMLP. The reactions were quenched 10 min
after stimulation. The generation of total
[
H]inositol phosphates (A) and release
of
-hexosaminidase (B) were determined as described under
``Experimental Procedures.'' The basal responses for A (300 ± 14, 800 ± 3, and 450 ± 20 cpm) and for B (2.3 ± 0.1, 4.8 ± 0.5, and 3.5 ± 0.3%)
in the presence of buffer, thrombin, and TRP, respectively, were
subtracted from the values shown. The data shown are the means ±
S.E. of one of four experiments performed in
triplicate.
The effects of thrombin and
TRP on the generation of [H]inositol phosphates
and secretion of
-hexosaminidase stimulated by C5a, IL-8, and PAF
were also determined. Cells were preincubated with thrombin (1 unit/ml)
or TRP (100 µM) for 3 min and then stimulated with
concentrations of chemoattractants that alone caused a 2-3-fold
increase in the generation of [
H]inositol
phosphates and release of about 20-30% of total cellular
-hexosaminidase. As shown in Fig. 5, thrombin but not TRP
primed both phosphoinositide hydrolysis and secretion stimulated by C5a
and IL-8. In contrast, both thrombin and TRP inhibited these responses
to PAF.
Figure 5:
Effects of thrombin and TRP on C5a-,
IL-8-, and PAF-mediated generation of [H]inositol
phosphates and release of
-hexosaminidase in RBL-2H3 cells.
[
H]Inositol-labeled RBL-2H3 cells expressing
ET-C5aR, ET-IL-8R, or ET-PAFR were left untreated (control) or
were treated with thrombin (1 unit/ml) or TRP (100 µM) for
3 min in the presence of LiCl (20 mM) and stimulated with C5a
(10 nM), IL-8 (100 nM), or PAF (0.3 nM). The
reactions were quenched 10 min after stimulation. The generation of
total [
H]inositol phosphates (A) and
release of
-hexosaminidase (B) were determined from the
same samples. The basal responses were similar to those in Fig. 4and were subtracted from the values shown. The data are
the means ± S.E. of one of three experiments performed in
triplicate.
The fMLP-induced generation of
[H]inositol phosphates was used to further
characterize the priming phenomenon. The priming effect of thrombin was
not dependent on its continuous presence because replacement of
thrombin-containing buffer 3 min after its addition with fresh buffer
without thrombin resulted in the same magnitude of priming as when
thrombin was not removed by washing (Fig. 6A). The
priming effect of thrombin reached a maximum by 3-5 min, remained
elevated for about 15 min, and returned to basal by 60 min (Fig. 6B). When cells were preincubated for 5 min with
thrombin in the presence of Ca
(1 mM) or
EGTA (100 µM; no added Ca
), washed, and
stimulated with fMLP in the presence of Ca
(1
mM), the extent of priming was the same (data not shown).
Figure 6:
Characteristics of thrombin-induced
priming. A, [H]inositol-labeled RBL-2H3
cells expressing ET-FR were treated with buffer or thrombin (1 unit/ml
for 3 min) containing LiCl (20 mM) and stimulated with fMLP (1
µM) in the continuous presence of thrombin (+).
Alternatively, cells were preincubated with thrombin, washed, and
exposed to a fresh medium without thrombin but containing LiCl (20
mM) and stimulated with fMLP(-). B, cells were
exposed to thrombin for different time periods in the absence of LiCl,
washed, and exposed to fMLP in a buffer containing LiCl. The reactions
were quenched 10 min after the addition of fMLP and generation of total
[
H]inositol phosphates were determined. The data
shown are the means ± S.E. of one of four experiments performed
in triplicate.
To test whether the effect of thrombin correlated with an increase
in substrate availability for phospholipase C, RBL-H3 cells were
labeled with [H]inositol and exposed to thrombin
(1 unit/ml) or TRP (100 µM) for 5 min and then the amount
of [
H]PIP
was determined. The levels
of incorporation of [
H]inositol in to PIP
in response to buffer, thrombin, and TRP were 4309 ± 95,
4329 ± 210, and 4286 ± 105, respectively.
Hirudin binds to the anion binding exosite and the
enzymatic cleavage pocket of thrombin and prevents thrombin from
binding and cleaving its receptor(25) . Hirudin completely
blocked both Ca mobilization and priming of the fMLP
response by thrombin but had no effect on the fMLP response itself (Table 2). Thrombin inactivated by diisopropylphosphofluoridate
has no catalytic activity but still binds to the seven transmembrane
domain thrombin receptor via the anion binding exosite(26) .
Diisopropylphosphofluoridate-inactivated
-thrombin did not
stimulate Ca
mobilization in RBL-2H3 cells and had no
effect on the fMLP response (Table 2).
-Thrombin retains its
catalytic activity, but its binding site for the tethered ligand
thrombin receptor is disrupted(26) .
-Thrombin itself
caused only a small increase in Ca
mobilization but
primed the response to fMLP by about 3-fold. Trypsin, a serine
protease, stimulated a substantial mobilization of intracellular
Ca
in RBL-2H3 cells but did not prime the response to
fMLP. Another serine protease, elastase, which did not stimulate
Ca
mobilization, had no effect on the fMLP response (Table 2). Other proteases, such as chymotrypsin and cathepsin G
stimulated little or no Ca
mobilization but primed
the response to fMLP by approximately 1.5-fold. This compares with a
4-fold priming of the fMLP response by thrombin.
Regulation of receptor action has been the subject of intense
investigation with one focus being the mechanisms of desensitization.
There is abundant evidence for a role of receptor phosphorylation in
homologous and heterologous desensitization(8) . Indeed, the
inability of the formylpeptide receptor to undergo heterologous
desensitization was attributed to the absence of a phosphorylation site
for protein kinase C on any of its predicted intracellular
loops(4, 27) . The data presented herein demonstrate
that both thrombin and TRP stimulated phosphorylation of the
chemoattractant receptors for C5a, IL-8, and PAF but not for fMLP.
Furthermore, phosphorylation of susceptible receptors was correlated
with heterologous desensitization at the level of receptor/G protein
coupling. Unexpectedly, although both thrombin and TRP decreased G
protein activation by C5a, thrombin primed subsequent phosphoinositide
hydrolysis, Ca mobilization, and degranulation
stimulated by all the peptide chemoattractants (fMLP, C5a, and IL-8).
The specificity of thrombin for the priming of peptide
chemoattractant-mediated responses is demonstrated by the finding that
thrombin inhibited these responses to PAF and had no effect on
Ca
mobilization stimulated by the IgE receptors.
These data suggest a surprising complexity in the regulation of
receptor actions. Whereas signals can lead to receptor phosphorylation
and desensitization at an early stage in the peptide chemoattractant
signal transduction pathway, the same or other signals may result in
markedly enhanced downstream activities.
The data herein indicate
that the priming effect of thrombin requires its proteolytic activity
but is unlikely to be mediated via its tethered ligand receptor. This
contention is based on the following observations. The peptide agonist
of the thrombin receptor, SFLLRN, stimulated Ca mobilization in RBL-2H3 cells but did not prime the response to
fMLP. Desensitization of the thrombin receptor by SFLLRN and treatment
with the thrombin receptor antagonist peptide FLLRN both inhibited
thrombin-induced Ca
mobilization by >70%, but
neither blocked thrombin's ability to prime Ca
mobilization to fMLP.
-Thrombin, which does not bind to the
tethered ligand thrombin receptor but has proteolytic
activity(26) , stimulated little or no Ca
mobilization but effectively primed the response to fMLP. The
priming signal is also unlikely to be derived from peptide
chemoattractant receptors, because they desensitize each other's
inositol 1,4,5-trisphosphate formation and Ca
mobilization(9) . The effect of thrombin is not solely
due to its serine protease activity because trypsin and elastase, which
are serine proteases(28) , did not cause priming. However,
other proteases such as cathepsin G and chymotrypsin primed
fMLP-stimulated Ca
mobilization, albeit to a lesser
extent than thrombin. Whether cathepsin G and chymotrypsin utilize the
same or different mechanisms to cause priming remains to be determined.
In fibroblasts, both thrombin and TRP stimulate a transient
Ca mobilization of similar magnitude, but only
thrombin is mitogenic(14) . The mitogenic signal of thrombin
persist long after (30 min) the transient Ca
mobilization has returned to basal(29) . Based on these
observations, the existence of an additional receptor for
thrombin's mitogenic activity has been
postulated(14, 29) . The identity of this putative
receptor or mechanism is yet to be determined. The data herein showing
that both thrombin and TRP stimulate transient Ca
mobilization but only thrombin provides a sustained priming
signal suggest that the priming and mitogenic responses of thrombin may
be mediated via a similar pathway. In platelets, two glycoproteins
(GPIb and GPV) interact with thrombin, but the biological significance
of these interactions is not
known(18, 19, 30, 31, 32) .
GPIb is a high affinity thrombin receptor in human
platelets(18, 19) . The demonstration that
-thrombin, which does not bind to GPIb in human
platelets(33) , causes priming in RBL-2H3 cells suggests that
GPIb is unlikely to be involved in priming. GPV (molecular mass, 82
kDa) is the only detectable membrane surface protein to be hydrolyzed
by both
- and
-thrombin as well as by
chymotrypsin(30, 31, 32) . Its proteolysis
results in the generation and extracellular release of a 69.5-kDa
soluble fragment. If GPV is expressed in RBL-2H3 cells, it is a
candidate for involvement in priming.
The phenomenology of priming
of chemoattractant receptor-mediated responses have been well
delineated, but the molecular mechanisms are not known. For example,
granulocyte-macrophage colony-stimulating factor enhances
Ca mobilization and superoxide generation stimulated
by both peptide (fMLP and C5a) and lipid (PAF) chemoattractants as well
as by direct activation of G proteins(6) . It has been proposed
that the priming effect of granulocyte-macrophage colony-stimulating
factor may reside in its ability to cause de novo synthesis of
cellular G proteins or activation of phosphoinositide kinase, which
increases the availability of substrates for phospholipase
C(7, 34) . In the studies presented here, the effect
reached a maximum 3-5 min after thrombin addition, and thus de novo protein synthesis is unlikely to be involved. In
addition, thrombin actually decreased G protein activation by the C5a
receptor yet primed its subsequent responses. Furthermore, under
optimal conditions of priming, thrombin had no significant effect on
PIP
levels in RBL-2H3 cells. The striking selectivity of
thrombin for priming responses for peptide chemoattractants (fMLP, C5a,
and IL-8) indicates that the target is distal to the receptor but is a
component of the signal transduction pathway shared by fMLP, C5a, and
IL-8 but not PAF.
The peptide chemoattractant receptors couple to a
Ptx-sensitive G protein, presumably G, whereas PAF
receptors utilize a predominantly Ptx-insensitive G
protein(3, 35) . Cotransfection studies in COS cells
demonstrated that fMLP, C5a, and IL-8 utilize
subunit
(G
) of Gi
2 to activate PLC
2, whereas PAF
utilizes the
subunit of a G
-like G protein to
activate a different PLC(2, 36, 37) . The
finding that Ptx blocks fMLP-induced Ca
mobilization
in both thrombin-treated and untreated cells indicates that priming is
not simply a switch in the coupling of the fMLP receptor from a
Ptx-sensitive to a Ptx-insensitive G protein.
The activity of PLC is
a likely locus for the priming event because phosphoinositide
hydrolysis stimulated by peptide chemoattractants is enhanced, although
the enzyme's substrate level is not. How thrombin primes
chemoattractant receptor-mediated phospholipase C activation can only
be speculated at present. Thrombin primes responses to chemoattractant
receptors that utilize G to activate PLC, so modification of
either G
or PLC by thrombin may lead to enhanced generation
of inositol phosphates. The
subunits of heterotrimeric G proteins
(G
) are members of a family of proteins that are
post-translationally modified at their carboxyl termini by
isoprenylation, proteolytic cleavage, and
carboxymethylation(38) . This modification of G
is
essential for membrane association of G
and activation of
effector molecules such as PLC
2(39) . Indeed,
demethylation of G
, which does not affect receptor-mediated
GTP
S binding to G proteins, is at least 10-fold less effective in
stimulating PLC
2 than its methylated counterpart(40) .
Moreover, fMLP-stimulated carboxymethylation of G
2 is associated
with neutrophil activation(41, 42) . It is therefore
possible that thrombin enhances the carboxymethylation levels of
G
2, resulting in more efficient stimulation of PLC
2. This
would explain the observation that thrombin enhances PLC-mediated
respones to peptide chemoattractant receptors despite the fact that
thrombin partially inhibits their G protein activation, as measured by
GTP
S binding to membranes.
In human platelets, thrombin causes
proteolytic cleavage of the 155-kDa PLC3 in a calpain-dependent
manner to generate a 100-kDa fragment(43) . The demonstration
that this and a 110-kDa truncated form of the enzyme display markedly
enhanced stimulation by G
when compared with the native
PLC
3 (43, 44) suggests that priming by thrombin
could involve proteolytic cleavage of PLC
. In addition, a
synthetic peptide corresponding to amino acids 448-464 of
PLC
2 stimulates the activity of this enzyme(45) . It has
been suggested that the peptide does not stimulate PLC
2 activity per se but enhances the ability of an already active enzyme to
hydrolyze additional substrates. It has not been determined whether
thrombin leads to the formation of such a peptide in RBL-2H3 cells.
There is evidence that PLC
possesses a domain within its structure
that inhibits the catalytic activity of the enzyme(46) . Thus,
processes that enhance the ability of PLC to be activated may do so via
the release of an inhibitory constraint. Therefore, the ability of
thrombin to modify the G
, cause proteolytic cleavage of
PLC
2, remove an inhibitory constraint from the enzyme or enhance
its activation by other mechanisms could explain the observed
specificity for thrombin-induced priming. These possibilities are
amenable to future investigations.
The data herein demonstrate that
thrombin modifies RBL-2H3 cells to selectively enhance the
phospholipase C-mediated responses to peptide chemoattractants (fMLP,
C5a, and IL-8). We speculate that the striking selectivity of thrombin
for priming peptide chemoattractant receptor-mediated responses is due
to its ability to enhance the ligand-stimulated activation of
PLC2. Of note, we have recently characterized a new form of
cross-desensitization among receptors for peptide chemoattractants and
suggested that the mechanism of this type of regulation involves
inhibition of PLC
2 activity(9, 47) . Thus,
modulation of the activity of phospholipases could have a major
regulatory role in cross-receptor signaling. Understanding the
molecular details of how differential regulation of PLC
2 activity
results in the priming and cross-desensitization of peptide
chemoattractant receptors should provide new and important information
on the mechanisms controlling receptor regulation.