(Received for publication, August 25, 1995)
From the
To define the molecular mechanisms of cross-regulation among
chemoattractant receptors, we stably co-expressed, in a rat basophilic
leukemia (RBL-2H3) cell line, epitope-tagged receptors for the
chemoattractants formylmethionylleucylphenylalanine (fMLP), a peptide
of the fifth component of the complement system (C5a), and
interleukin-8 (IL-8). All the expressed receptors underwent homologous
phosphorylation and desensitization upon agonist stimulation. When
co-expressed, epitope-tagged C5a receptor (ET-C5aR) and epitope-tagged
IL-8 receptor (ET-IL-8RA) were cross-phosphorylated by activation of
the other. Activation of epitope-tagged fMLP receptor (ET-FR) also
cross-phosphorylated ET-C5aR and ET-IL-8RA, but ET-FR was totally
resistant to cross-phosphorylation. Similarly, C5a and IL-8 stimulation
of [S]guanosine
5`-3-O-(thio)triphosphate (GTP
S) binding and
Ca
mobilization were cross-desensitized by each other
and by fMLP. Stimulation of [
S]GTP
S binding
by fMLP was also not cross-desensitized by C5a or IL-8, however,
Ca
mobilization was, suggesting a site of inhibition
distal to G protein activation. Consistent with this desensitization of
Ca
mobilization, inositol 1,4,5-trisphosphate release
in RBL-2H3 cells expressing both ET-C5aR and ET-FR revealed that fMLP
and C5a cross-desensitized each other's ability to stimulate
phosphoinositide hydrolysis. Taken together, these results indicate
that receptor cross-phosphorylation correlates directly with
desensitization at the level of G protein activation. The ET-FR was
resistant to this process. Of note, cross-desensitization of ET-FR at
the level of phosphoinositide hydrolysis and Ca
mobilization was demonstrated in the absence of receptor
phosphorylation. This suggests a new form of chemoattractant
cross-regulation at a site distal to receptor/G protein coupling,
involving the activity of phospholipase C.
Leukocytes migrate to sites of inflammation where they
participate in host-defensive and/or tissue-destructive activities via
activation of chemoattractant receptors. Upon stimulation by
proinflammatory agents such as a peptide component of the fifth
complement system (C5a), ()formylpeptides (fMLP),
interleukin-8 (IL-8), platelet-activating factor, or leukotriene
B
chemoattractant receptors couple to guanine nucleotide
binding regulatory proteins (G proteins) to induce cellular
responses(1) . Prolonged stimulation of these receptors results
in desensitization. Originally, two types of desensitization were
described: homologous and heterologous(2) . Homologous
desensitization is specific for a receptor and its agonist.
Heterologous desensitization refers to a process whereby activation of
one type of receptor results in the desensitization of different
receptors. Homologous desensitization occurs as a result of
phosphorylation of the active form of a receptor by a receptor kinase,
whereas heterologous desensitization affects active and inactive
receptor forms by kinases activated by second
messengers(2, 3) .
Cross-desensitization studies of
chemoattractant receptors using Ca mobilization as a
measurement of receptor activation have led us to the description of a
novel type of desensitization whose specificity falls between
heterologous and homologous desensitization(4, 21) .
This type of desensitization was defined as cross-inhibition of
Ca
mobilization among a particular class of
chemoattractant receptors, i.e. those for peptide but not for
lipid chemotactic factors(4, 21) . Other studies have
shown that phosphorylation of the cytoplasmic domains of G
protein-coupled receptors, followed by their uncoupling from G
proteins, can be responsible for
desensitization(2, 5) . However, experiments in human
neutrophils indicated that component(s) distal from receptor/G-protein
may also be involved in chemoattractant receptor
cross-desensitization(4) . To better define the multiple types
of receptor desensitization, we developed a model system, a rat
basophilic leukemia cell line (RBL-2H3), in which chemoattractant
receptors can be expressed and induced to elicit cellular responses
similar to those in neutrophils. Using this model, we recently showed
that agonist-stimulation of the chemoattractant receptors for fMLP,
C5a, IL-8, and platelet-activating factor expressed in RBL-2H3 cells
resulted in phosphorylation and desensitization of these
receptors(6, 7) . (
)In the present work, we
sought to better define the mechanism(s) of cross-desensitization of
chemoattractant receptors. For that purpose, chemoattractant receptors
were co-expressed in RBL-2H3 cells and studied for their ability to
undergo and/or mediate cross-phosphorylation and correlate this with
consequent GTP
S binding, generation of inositol trisphosphates,
and mobilization of intracellular calcium. The results presented here
demonstrate that receptor phosphorylation and modification of a
downstream component(s) of the chemoattractants signaling cascade
participate in different forms of chemoattractant receptor
cross-regulation.
Figure 1:
Immunoprecipitation of epitope-tagged
chemoattractant receptors expressed in RBL-2H3 cells. A, P-labeled double-transfected RBL-2H3 cells (2.5
10
/60-mm plate) expressing epitope-tagged receptors for FR
and C5aR (ET-FCR), IL-8RA and C5aR (ET-ICR), or IL-8RA and FR (ET-IFR)
were incubated for 5 min with (lanes 2, 3, 5, 6, 8, and 9) or without (lanes 1, 4, and 7) stimulants. Cells were
lysed, immunoprecipitated with 12CA5, and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography. This
experiment was repeated five times with similar results. B,
RBL-2H3 cells expressing either ET-FR and IL-8RA (lanes
10-12) or ET-IL-8RA and FR (lanes 13-15) were
stimulated in the presence or absence of either fMLP or IL-8, and
receptor phosphorylation was assessed as described
above.
We also determined whether ligand cross-reactivity
could result in an apparent receptor cross-phosphorylation. Single
transfected RBL-2H3 cells expressing either ET-C5aR or ET-IL-8RA were P labeled, treated with C5a (0.1 µM), IL-8
(0.1 µM), or fMLP (1 µM), and
immunoprecipitated. Only homologous phosphorylation was observed for
each receptor (i.e. ET-C5aR only by C5a and ET-IL-8RA only by
IL-8) (data not shown), indicating that ligand cross-reactivity does
not occur in these receptors.
Figure 2:
Homologous and cross-desensitization of
peptide chemoattractant receptors stimulated
[S]GTP
S binding. Double-transfected RBL-2H3
cells were treated with fMLP (1 µM), C5a (100
nM), or IL-8 (100 nM) for 5 min. Membranes were
prepared and assayed for agonist-stimulated
[
S]GTP
S binding. The data shown are the
means of three different experiments performed in triplicate. The
values are represented as percentage of maximum stimulation, which is
defined as the maximal increase above basal of
[
S]GTP
S bound to control membranes
(untreated cells) after 10 min of reaction. Basal activities were
0.2-0.3 pmol of [
S]GTP
S bound/mg
of protein). Maximum stimulation was 0.23 ± 0.008 (fMLP) and
0.19 ± 0.010 (C5a) pmol of
S-GTP
S bound/mg of
protein for untreated ET-FCR cells (panel A), 0.2 ±
0.011 (C5a) and 0.21 ± 0.02 (IL-8) pmol of
[
S]GTP
S bound/mg of protein for untreated
ET-ICR cells (panel B), and 0.19 ± 0.008 (fMLP) and
0.22 ± 0.0132 (IL-8) pmol of [
S]GTP
S
bound/mg of protein for untreated ET-IFR cells (panel C).
Specific activity was
380-450 cpm/fmol of
GTP
S.
Figure 3:
Cross-desensitization of chemoattractant
receptors mediated Ca mobilization.
Double-transfected RBL-2H3 cells (3
10
cells/assay)
were loaded with indo-1 and stimulated with fMLP (100 nM), C5a
(10 nM), or IL-8 (10 nM). Cells were rechallenged 3
min later with the same concentration of ligand. Traces are
representative of three experiments.
It was
determined whether a depletion of the intracellular calcium pool caused
by the first ligand could account for the attenuation of Ca mobilization in response to a second stimuli. Treatment of ET-FCR
cells with 2 µM thapsigargin before stimulation (301
± 21 nM), 3 min after the first ligand (362 ± 13
nM) and after the second ligand (388 ± 17 nM),
followed by 10 µM ionomycin (621 ± 76 nM)
showed no significant change in the intracellular Ca
pool. These results indicate that the cross-desensitization of
receptor-mediated Ca
mobilization was not due to an
impairment of the intracellullar Ca
storage.
Figure 4:
Measurement of IP
concentration in control and desensitized RBL-2H3 cells. RBL-2H3 cells
(2.5
10
cells) expressing epitope-tagged FR and
C5aR were treated with 1 µM fMLP (fMLP-treated), 100
nM C5a (C5a-treated), or in the absence of stimulants
(untreated cells) for 10 min at 37 °C in serum-free medium. Cells
were then rechallenged for 10 s with fMLP, C5a, or buffer, and IP
was extracted as described under ``Experimental
Procedures.'' The IP
concentration in each extract was
determined using the IP
H assay system from
Amersham. Data are means ± S.E. of four separate determinations
performed in duplicate.
Despite a large body of evidence indicating that
chemoattractant-mediated inflammatory responses are regulated by
desensitization, little is known about the molecular events governing
this process. Wilde et al.(10) reported that
C5a-stimulated GTPase activity was desensitized in membranes from
neutrophils pretreated with fMLP. These results were confirmed by our
previous work, which further indicated that exposure of neutrophils to
fMLP cross-desensitized C5a, IL-8, platelet-activating factor, and
leukotriene B mediated-GTPS binding in membranes. In contrast,
receptors for formylpeptide were resistant to this type of
cross-desensitization due presumably to the absence of the necessary
phosphorylation site (see below)(4) . Heterologous
phosphorylation of chemoattractant receptors by second
messenger-activated kinases (such as PKC) followed by their uncoupling
from G protein has been thought to be the molecular mechanism
responsible for chemoattractant
cross-desensitization(4, 6) . However, Tardif et
al.(11) reported that fMLP stimulation of HL-60 cells did
not induce C5aR phosphorylation. The data presented in the work
reported here clearly indicate that fMLP stimulation resulted in
phosphorylation of both C5aR and IL-8RA in double-transfected RBL-2H3
cells (Fig. 1), and both C5a and IL-8-mediated GTP
S binding
were desensitized under such conditions. The failure of Tardif et
al.(11) to find such cross-phosphorylation in HL-60 cells
is not understood.
The extent of fMLP-mediated phosphorylation of
ET-C5aR and ET-IL-8RA mirrored the ones previously obtained upon
exposure of these receptors to the protein kinase C activator, phorbol
12-myristate 13-acetate(6, 7) . These
results suggest that fMLP cross-desensitization of ET-C5aR and
ET-IL8RA, as well as C5a of ET-IL8RA and IL-8 of ET-C5aR, may be
mediated by receptor phosphorylation by PKC. Indeed, the PKC inhibitor
staurosporine inhibited fMLP-mediated phosphorylation of ET-C5aR in the
ET-FCR cell line (data not shown). fMLP has been shown to increase PKC
activity in neutrophils and several other cell
lines(12, 13, 14) . Molecular cloning has
revealed that the receptor for fMLP lacks sequence motif for PKC
phosphorylation (RXXSXRX). This likely
explains its resistance to PKC-mediated phosphorylation(15) .
Neither C5a nor IL-8 pretreatment resulted in cross-desensitization of
fMLP-mediated GTP
S binding, which correlated with its resistance
to cross-phosphorylation. Taken together, these results are in
agreement with the current concept that receptor phosphorylation leads
to desensitization and indicate that PKC-mediated phosphorylation
results in one form of chemoattractant receptor cross-desensitization
at the level of receptor/G protein activation.
Interestingly,
receptor cross-phosphorylation cannot explain the cross-desensitization
of formylpeptide receptor-mediated Ca mobilization by
other chemoattractants since the formylpeptide receptors are totally
resistant to the heterologous phosphorylation. Thus, the formylpeptide
receptors provide an important tool to determine the downstream site(s)
for chemoattractant receptor cross-desensitization. The chemoattractant
receptors studied here are coupled to phospholipase C and mediate
intracellular signals via stimulation of phosphatidylinositol
hydrolysis and production of IP
and
diacylglycerol(1) . It has been shown that IP
plays
a pivotal role in stimulating intracellular Ca
mobilization(16) . Thus, cross-desensitization of
fMLP-mediated Ca
mobilization could be at the level
of PIP
hydrolysis or IP
activity. Indeed,
cAMP-mediated phosphorylation of the receptor for intracellular
generated IP
markedly decreases its ability to stimulate
Ca
release(17) . Therefore, the possibility
existed that cross-desensitization of fMLP-induced intracellular
Ca
mobilization reflected either a decrease in the
level of intracellular IP
produced or desensitization of
the receptor for IP
. As shown in fig. 4, fMLP stimulated
IP
production was decreased by
90% in cells pretreated
with C5a. These results indicate that the cross-desensitization of
Ca
mobilization in response to fMLP by the other
chemoattractants is likely due to a decrease in the level of
IP
production. There are several possible explanations for
diminished IP
production. A depletion of the pool of
PIP
prior hydrolysis or stimulation of
phosphatidylinositol-3 kinase activity is one explanation. Against this
hypothesis is that pretreatment of neutrophils with C5a decreased
fMLP-induced IP
production with no significant change in
the level of PIP
(18) . Moreover, in neutrophils
that have been cross-desensitized by fMLP, purinergic receptor ability
to stimulate PLC and Ca
release is normal, indicating
adequate IP
receptor and PIP
level(4) .
A second explanation for diminished IP
production is a
decrease in the catalytic activity of the phospholipase C either by
modification of the enzyme or its activating components. Both
and
subunits of G protein have been shown to activate PLC in
reconstituted systems(19) . Chemoattractant receptors couple to
G
and mediate PLC
activation via
G
subunits(1, 19) .
subunits are known
to be isoprenylated and methylated(20) . It has recently been
shown that demethylation of the
subunit, which does not
affect receptor-mediated GTP
S binding to G protein, caused a
10-fold decrease in
-mediated activation of PLC and, thus,
production of IP
(8) . Therefore, it is possible
that C5a and IL-8-mediated cross-desensitization of FR-induced
Ca
mobilization may be due to either a demethylation
or other modification of
subunits, rendering them less
effective in activating PLC. Modification of PLC itself could also
result in its diminished activity. In any case, the
cross-desensitization of formylpeptide receptor as presented is likely
due to a modification in its ability to activate PLC. C5a- and
IL-8-induced Ca
mobilization and IP
production are also inhibited in cells pretreated with fMLP.
Since all three chemoattractant receptors studied here apparently
utilize the same signal transduction pathways, the downstream effect
observed with the fMLP receptor likely plays a role in the attenuation
of C5a- and IL-8-induced responses in addition to the impairment of
receptor/G protein coupling due to receptor phosphorylation.
In summary, we have developed a system to stably co-express two G protein-coupled receptors and study their cross-regulation. The results presented herein indicate that chemoattractant receptor-mediated inflammatory response are regulated at multiple sites. One is at the level of receptor phosphorylation affecting receptor/G protein coupling. The second is at a site distal to that, presumably involving the activity of phospholipase C. Cross-desensitization at different levels of the signaling cascades may be used by the receptors to control each other's activity at sites of inflammation where multiple chemoattractants are present. It will be important to determine if receptor cross-desensitization at the level of PLC occurs more generally than the subgroup of chemoattractant receptors studied here.