From the
We have previously characterized the stably transfected,
clonally selected human placental cell line, 3ASubE P-3, which
overexpresses the type B interleukin-8 receptor (IL-8RB) and responds
to the chemokine melanoma growth stimulatory activity (MGSA) with
enhanced phosphorylation of this receptor. In work described here, we
demonstrate that the MGSA-enhanced phosphorylation of this receptor is
mediated via a process involving pertussis toxin-sensitive G proteins.
Furthermore, treatment of the 3ASubE P-3 cells with either
12- O-tetradecanoylphorbol 13-acetate (TPA) or
1,2-dioctanoyl- sn-glycerol (diC
Chemokines are a group of inflammatory proteins which share
several conserved amino acid residues
(1) . They are subdivided
into two families: the C- X-C (or
Various chemokine
receptors have been identified and their corresponding cDNAs have been
cloned. Two distinct human IL-8 receptors (IL-8R), type A and B (also
referred to as type or class I and II, respectively), have been cloned
from neutrophil and HL60 cDNA libraries, respectively
(2, 3) . The names for these receptors has been rather
misleading since they bind multiple members of the C- X-C
family. The type A IL-8R (IL-8RA) binds IL-8 with high affinity and
MGSA with low affinity, whereas the type B IL-8R (IL-8RB) binds both
MGSA and IL-8 with high affinity and neutrophil activating protein-2
with low affinity
(2, 4, 5) . Recently, a mouse
homolog of the human IL-8 receptors has been cloned
(6, 7, 8) . A C-C chemokine receptor, which
binds multiple members of the C-C chemokine family and two monocyte
chemotactic protein-1 receptors have also been cloned
(9, 10) . These various chemokine receptors are all
members of the seven transmembrane domain, G protein-coupled receptor
family. More recently, the cDNA for the Duffy blood group antigen,
which binds IL-8, MGSA, monocyte chemotactic protein-1, and RANTES, has
also been isolated
(11, 12, 13) . This is the
first reported receptor that binds members of both the C- X-C
and C-C chemokine families. cDNA analysis of the Duffy antigen, also
referred to as the erythrocyte chemokine receptor, predicts that it has
seven transmembrane domains but shows very little homology to the other
chemokine receptors. Furthermore, this receptor does not appear to
couple to G proteins and has been speculated to serve as a
``ligand sink'' to regulate circulating chemokine levels
(13) .
MGSA and IL-8 elicit a variety of common effects on
neutrophils
(1, 14, 15, 16, 17) .
Presumably, the effects of IL-8 and MGSA on neutrophils are mediated
through the type A and B receptors. These two receptors share a 77%
amino acid conservation with the amino and carboxyl termini being the
most divergent regions between these two receptors
(3) .
Receptor chimera studies have demonstrated that the extracellular
amino-terminal domain plays a significant role in the ligand binding
specificity of these receptors
(18) . Specific residues in the
third extracellular loop of the IL-8Rs also play an important role in
ligand binding
(19) . These properties distinguish the IL-8Rs
from the monoamine-binding G protein-coupled receptors, such as the
MGSA and IL-8, along with
their effects on neutrophils, are capable of eliciting both
proliferative and non-proliferative responses in a variety of
non-hematopoietic cell types
(21, 22, 23, 24, 25, 26, 27, 28) .
The receptors through which MGSA and IL-8 transduce their signals in
non-hematopoietic cells have not been characterized. Recently, however,
the IL-8RA and -B mRNAs have been detected by reverse transcriptase
polymerse chain reaction in a variety of MGSA- and IL-8-responsive
non-hematopoietic cell types, including primary human keratinocytes
(27, 29) , melanocyte and melanoma cell lines
(29, 30) . The expression of these receptors suggests
that they may mediate some of the effect(s) of MGSA and IL-8 on
non-hematopoietic cells. To assess the mechanism of signal transduction
through the IL-8RB, and determine which factors may modulate this
signaling in non-hematopoietic cell types, we have previously
established a clonally selected, stably transfected human placental
cell line, termed 3ASubE P-3, which overexpresses the IL-8RB
(29) . This receptor is basally phosphorylated in the 3ASubE P-3
cells and this phosphorylation is markedly enhanced upon treatment with
MGSA
(29) . Thus the IL-8RB behaves similarly to several members
of the G protein-coupled receptor family in that ligand binding results
in receptor phosphorylation. Several members of this receptor family
are also subject to modulation by the protein kinase C (PKC) and
cAMP-dependent protein kinase A pathways
(31) . In this report,
we have examined the effect of PKC activation on the phosphorylation
state of the IL-8RB. We demonstrate that activation of PKC with either
the tumor promoter 12- O-tetradecanoylphorbol 13-acetate (TPA)
or the diacylglycerol analogue 1,2-dioctanoyl- sn-glycerol
(diC
Differentiated U937 cells were washed extensively
with phosphate-free MEM then incubated in phosphate-free MEM containing
10% dialyzed fetal bovine serum (phosphate-free) and 1 mCi/ml
[
The detection of the IL-8RA and B mRNAs, by reverse
transcriptase-polymerase chain reaction, in a variety of MGSA- or
IL-8-responsive non-hematopoietic cell types suggests that these
receptors may play a role in eliciting the MGSA or IL-8 responses
(29, 30) . To investigate the mechanism of signal
transduction through the IL-8RB and determine what factors may modulate
signaling through this receptor in non-hematopoietic cell types, we
have previously established a stably transfected cell line, termed
3ASubE P-3, which overexpresses this receptor
(29) . In this
report we have demonstrated that activation of PKC enhanced the
phosphorylation of the IL-8RB on serine residue(s) in the 3ASubE P-3
cells. This response was not unique to the 3ASubE cell line since PKC
activation in two other stably transfected non-hematopoietic cell lines
which overexpress the IL-8RB also resulted in enhanced phosphorylation
of this receptor. Thus the phosphorylation of the IL-8RB which is
mediated by PKC may be a generalized phenomena throughout
non-hematopoietic cell types. Receptor phosphorylation in response to
PKC activation has been reported for several members of the G
protein-coupled receptor family, including the
Similar to the stable transfectants, MGSA treatment also enhanced
the phosphorylation of the IL-8RB in U937 cells, a promonocytic cell
type which expresses the endogenous IL-8RB. In contrast to our
observations in the non-hematopoietic cell types, we were unable to
detect TPA- or diC
Recently it has been
reported that stimulation of granulocytes with TPA inhibits the IL-8
induced intracellular calcium mobilization
(40) . The response
to TPA was rapid, requiring only a 5-min preincubation with TPA to
prevent the IL-8 effect on calcium. It was proposed that TPA treatment
of the granulocytes may elicit its effect through receptor
phosphorylation, internalization, or shedding. Receptor phosphorylation
and internalization, in response to PKC activation, has been reported
for several members of the seven-transmembrane domain, G
protein-coupled receptor family, including the
The effects of PKC activation on
MGSA binding and receptor protein levels in the 3ASubE P-3 and U937
cells appears to be in striking contrast to effects described for the
adrenergic receptors. PKC activation is associated with phosphorylation
and desensitization of the
One caveat
regarding the effect of PKC activation on the IL-8RB protein level in
U937 cells is that a single application of TPA induces the
differentiation of U937 cells to macrophages (reviewed in Ref. 51).
Thus one could argue that the effect of TPA on the IL-8RB is a
nonspecific effect mediated by the differentiation process. In an
attempt to discern a direct effect of PKC on the IL-8RB protein in U937
cells, we restricted our studies to 2 h, typically U937 differentiation
occurs upon exposure to TPA for 3-5 days. Also the effect of two
natural PKC activators, diC
Reconstitution experiments have demonstrated
that the IL-8RA and -B couple to both pertussis toxin-sensitive G
proteins (G
We thank Vinh Lam and Rita Perry-Davis for technical
assistance and Bart Lutterbach for assistance with the phosphoamino
acid analysis. Finally we thank Lee Limbird, Steve Hanks, and Rebecca
Shattuck for critically reading this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
), two different
activators of protein kinase C (PKC), results in a
concentration-dependent increase in the phosphorylation of the IL-8RB.
Inhibition of PKC, by treatment with staurosporin (50 nM for 2
h), or down-regulation of PKC, by prolonged treatment with TPA (400
nM for 40 h) suppresses the TPA-enhanced receptor
phosphorylation, but has no effect on the MGSA-enhanced receptor
phosphorylation. These data suggest that the isoforms of PKC that are
sensitive to these manipulations may not play a role in mediating the
MGSA-enhanced phosphorylation of the IL-8RB. TPA treatment also results
in a time-dependent decrease in
I-MGSA binding to the
3ASubE P-3 cells. A 30-min treatment with 400 nM TPA results
in approximately a 50% decrease in binding, whereas a 2-h treatment
essentially eliminates specific binding of
I-MGSA to
these cells. The TPA-induced decrease in
I-MGSA binding
is accompanied by enhanced degradation of the IL-8RB, as indicated by
Western blot analysis and pulse-chase experiments, suggesting a
potential role for PKC as a negative regulator of the IL-8RB. MGSA
treatment (50 nM for 2 h) also stimulates receptor degradation
in the 3ASubE P-3 cells, indicating that this receptor is
down-regulated in response to prolonged exposure to its ligand. In
similar studies conducted on the promonocytic cell line, U937, MGSA
treatment of the U937 cells resulted in receptor phosphorylation,
whereas PKC activation failed to significantly modulate the
phosphorylation state of the IL-8RB. Treatment of the U937 cells with
MGSA, TPA, or diC
resulted in a loss of receptor protein
present in these cell types. These data imply that MGSA signaling
through the IL-8RB is similar in both the non-hematopoietic and
hematopoietic cell types, whereas activation of PKC by TPA or diC
elicits different responses in these two distinct cell types.
) family, which possess
an intervening amino acid between the first two conserved cysteine
residues, and the C-C (or
) family, in which the first two
conserved cysteine residues are adjacent to one another. Members of the
C- X-C family are generally chemotactic for neutrophils and to
a lesser extent lymphocytes, basophils, and eosinophils. The
C- X-C chemokines include melanoma growth stimulatory activity
(MGSA)
(
)
(also referred to as GRO), interleukin-8
(IL-8), neutrophil activating protein-2,
-interferon inducible
protein-10, and platelet factor-4. The C-C family members, which are
generally chemotactic for monocytes, include macrophage inflammatory
protein-1
, macrophage inflammatory protein-1
, monocyte
chemotactic protein-1, and RANTES
(1) .
-adrenergic receptors, since ligand binding specificity is
typically dictated by the transmembrane domains for the other G
protein-coupled receptors
(20) .
) enhanced the phosphorylation of the IL-8RB on serine
residues. PKC activation also decreased MGSA binding to the 3ASubE P-3
cells and this was accompanied by a decreased amount of receptor
protein present in the cells. PKC does not appear to play a significant
role in the MGSA-enhanced phosphorylation of the IL-8RB since neither
inhibition nor down-regulation of PKC prevented the MGSA response in
these cells. Studies conducted in the promonocytic cell line, U937,
revealed that MGSA treatment had effects on receptor phosphorylation
and degradation similar to those observed in the 3ASubE P-3 cells.
Activation of PKC, however, did not significantly alter the receptor
phosphorylation state in the U937 cells although it did stimulate a
dramatic decrease in the receptor protein levels. These data
demonstrate that receptor responses to ligand binding in hematopoietic
cells is similar to that observed in non-hematopoietic cells yet there
are differences in regulation in response to activation of PKC by TPA
or diC
in these two cell types.
Cell Culture
The human placental cell line,
3ASubE, had previously been stably transfected with the pRC/CMV
mammalian expression vector (Invitrogen) containing the IL-8RB cDNA.
Stable transfectants, which were clonally selected, have previously
been characterized and demonstrated to bind MGSA
(29) . One
clonally selected stable transfectant termed 3ASubE P-3 was used in
these studies. The parental 3ASubE cell line was maintained in 5% fetal
bovine serum/MEM, whereas the 3ASubE P-3 clone was maintained in 5%
fetal bovine serum, 400 µg/ml G418/MEM. U937 cells, a human
promonocytic cell line
(32) , was routinely cultured in the
presence of 10% fetal bovine serum/RPMI and differentiated with 1
µM retinoic acid for 5 days prior to experimentation. All
cell types were incubated at 37 °C and 5% CO.
In Vivo Phosphorylation of the IL-8RB in 3ASubE P-3 and
U937 Cells
In vivo phosphorylation studies were
conducted as described previously
(29) . Briefly, confluent
cultures of 3ASubE P-3 cells (35-mm plates) were washed and incubated
in serum-free DMEM for 40 h at 37 °C. The medium was replaced with
phosphate-free MEM and the cells were incubated for an additional 3 h
at 37 °C. After phosphate starvation, cells were incubated in
phosphate-free MEM containing 250 µCi/ml
[P]orthophosphate (9000 Ci/mmol; Amersham) for 3
h at 37 °C. MGSA, TPA, or the appropriate vehicle was added
directly to the [
P]orthophosphate-containing
medium after the 3-h incubation. Prior to use, MGSA, which is stored in
10% acetonitrile, 0.1% trifluoroacetic acid, was lyophilized in the
presence of 25 µg of bovine serum albumin, resuspended in the
appropriate media and used directly. An equal volume of 10%
acetonitrile, 0.1% trifluoroacetic acid was treated in an identical
manner and served as the acetonitrile vehicle control. The TPA stock
was a 1000-fold concentrate in ethanol (EtOH). Inhibitors, when used,
were added to the cells during the 3-h incubation with
[
P]orthophosphate. Staurosporin (50
nM), or vehicle (dimethyl formamide), was added to the cells
for the final 2 h of the 3-h incubation with
[
P]orthophosphate, whereas pertussis toxin (1
µg/ml) or vehicle was added to the cells at the beginning of the
3-h incubation. Pertussis toxin was dialyzed against Tris-buffered
saline to remove the phosphate present in its storage buffer prior to
use in this assay.
P]orthophosphate, at a density of 3
10
cells/ml, for 3 h at 37 °C. Cells were treated with
MGSA, TPA, or the corresponding vehicle controls, for the indicated
period of time.
Immunoprecipitation of the IL-8RB Receptor
Cells
were lysed in either RIPA buffer containing 0.1% SDS, 0.5% sodium
deoxycholate, 1% Triton X-100, 10 mM Tris, pH 7.4, 150
mM NaCl, 1 mM EDTA, 20 mM NaFl, 10 µg/ml
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml
leupeptin, or they were lysed in a Triton X-100 buffer containing 1%
Triton X-100, 10 mM Tris, pH 7.4, 150 mM NaCl, 1
mM EDTA with the above protease inhibitors. Lysates were
centrifuged for 15 min at 4 °C in an Eppendorf Microfuge, the
supernatant was removed, and either trichloroacetic acid-precipitable
counts were determined or protein concentrations were estimated (BCA,
Pierce). Lysates containing either an equal number of trichloroacetic
acid-precipitable counts (5 10
cpm) or an equal
amount of protein (200 µg) were incubated with 5 µg of
affinity-purified anti-amino-terminal peptide polyclonal antibodies
which have previously been demonstrated to be specific for IL-8RB
(29) . The lysates were rocked at 4 °C for 2 h, followed by
precipitation with 30 µl of 1:1 dilution protein A/G-agarose
(Pierce) for 1 h at 4 °C. Thereafter, immunoprecipitates were
washed three times with ice-cold RIPA buffer and pelleted. Pellets were
denatured in 40 µl of 2
Laemmli sample buffer containing 5%
SDS and 10% (v/v)
-mercaptoethanol, electrophoresed through a 9%
SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed to
autoradiographic film (Hyperfilm, Amersham).
I-MGSA Binding
Assay
I-MGSA binding assays were conducted as
described previously
(29) . MGSA (1 µg), generously provided
by R+D Systems, was iodinated using the chloramine-T method.
Routinely, a specific activity of 100 µCi/µg was obtained,
assuming a 100% recovery. Cells were seeded at 2
10
cells/well in 24-well plates. Forty-eight hours later the binding
assay was performed. Prior to the assay, cells were treated with 400
nM TPA, or ethanol as the vehicle control, for 0, 15, 30, 60,
or 120 min at 37 °C. Cells were washed once with ice-cold binding
buffer (0.1 mg/ml ovalbumin, 30 mM Hepes/DMEM).
I-MGSA (20-30,000 cpm/well) was added to the cells
and they were rocked at 4 °C for 4 h. Cells were washed 3 times
with ice-cold binding buffer, the bound
I-MGSA was eluted
with 0.1 N NaOH, 1% SDS, and counted in a
-counter
(Beckman, Gamma 5500). Nonspecific binding was defined as the amount of
I-MGSA bound to the 3ASubE P-3 cells in the presence of
50 ng/ml unlabeled MGSA, which has previously been demonstrated to
eliminate specific binding of
I-MGSA to these cells
(29) .
Western Blot Analysis of the IL-8RB
Whole cell
lysates (equal protein per lane) or IL-8RB immunoprecipitates were
electrophoresed through an 9% SDS-polyacrylamide gel, transblotted onto
either a nitrocellulose or polyvinylidene difluoride (PVDF) membrane
(Bio-Rad) and subjected to Western blot analysis using either 2
µg/ml of the rabbit anti-peptide antibodies described above or 0.25
µg/ml mouse monoclonal anti-IL-8RB (a kind gift from Richard Horuk
and Steven Peiper). Goat anti-rabbit, or rabbit anti-mouse, IgG whole
molecule conjugated with alkaline phosphatase (Sigma) was used at a
1:2000 dilution. Blots were developed typically for 10-15 min at
pH 9.5 using bromochloroindoyl phosphate (Sigma) and nitro blue
tetrazolium (Sigma). Where indicated, the Triton X-100 insoluble
pellet, which remains after the microcentrifugation, was washed twice
with the Triton X-100 buffer then solubilized in 2 Laemmli
sample buffer described above and subjected to Western blot analysis.
Phosphoamino Acid Analysis
Phosphoamino acid
analysis was performed as described by Boyle et al.(33) . Briefly, the IL-8RB was immunoprecipitated from
[P]orthophosphate-labeled 3ASubE P-3 cells that
had been treated with 40 nM TPA for 10 min. The
immunoprecipitates were electrophoresed through a 9% SDS-polyacrylamide
gel, transblotted onto a PVDF membrane and subjected to
autoradiography. After autoradiography, the band corresponding to the
IL-8RB was excised from the membrane, incubated in the presence of 6
M HCl for 1 h at 110 °C, lyophilized, then electrophoresed
through a cellulose thin layer plate in two dimensions (first dimension
was in pH 1.9 buffer, the second dimension was in pH 3.5 buffer).
Phosphoserine, phosphothreonine, and phosphotyrosine standards were
included in the loading buffer. After electrophoresis, the plates were
stained with ninhydrin to detect the position of the phosphoamino acid
standards then exposed to autoradiographic film. [
S]Methionine/Cysteine Labeling of
the 3ASubE P-3 Cells-3ASubE P-3 and the parental 3ASubE cell
lines were grown to confluence in 35-mm plates. Cells were rinsed twice
with phosphate-buffered saline then incubated with cysteine/methionine
free-MEM (Life Technologies, Inc.) for 1 h at 37 °C, the culture
medium was then replaced with cysteine/methionine-free media containing
100 µCi/ml [
S]cysteine/methionine
(Tran
S-label, >1000 Ci/mmol; ICN). Cells were labeled
for 6 h at 37 °C. Cells were rinsed and fresh media containing
unlabeled cysteine/methionine was added to the cells. Cells were either
untreated, or treated with MGSA (50 nM) or TPA (400
nM) for 2 h. Triton X-100 extracts were prepared and the
IL-8RB was immunoprecipitated from an equal number of trichloroacetic
acid-precipitable counts (2
10
cpm),
electrophoresed through a 9% SDS-polyacrylamide gel, dried, and exposed
to autoradiographic film.
PKC Activation Enhances Phosphorylation of the
IL-8RB
Since several members of the G protein-coupled receptor
family are targets for phosphorylation by PKC, direct activation
studies were conducted to determine if the IL-8RB is also
phosphorylated in response to PKC activation. The 3ASubE P-3 cell line
is a clonally selected, stably transfected human placental cell line
which expresses the IL-8RB, whereas the parental 3ASubE cell line does
not express this receptor. Previously we have demonstrated that the
IL-8RB expressed in the 3ASubE P-3 cells migrates at approximately 45
kDa on an SDS-polyacrylamide gel
(29) . 3ASubE P-3 cells were
labeled with [P]orthophosphate, then treated in
the presence of increasing concentrations of TPA for 10 min. The IL-8RB
was immunoprecipitated with affinity purified anti-amino-terminal
peptide polyclonal antibodies which recognize the IL-8RB
(29) .
The immunoprecipitates were electrophoresed, then analyzed by
autoradiography and Western blot analysis. TPA treatment of the 3ASubE
P-3 cells enhanced the phosphorylation of the IL-8RB
(Fig. 1 A). Under these conditions, 40 and 400
nM TPA were equally effective in stimulating the maximal level
of receptor phosphorylation. Treatment of the 3ASubE P-3 cells with
4
-phorbol 12-myristate 13-acetate, the inactive isomer of TPA, had
no effect on the phosphorylation of the IL-8RB (data not shown).
Typically in our immunoprecipitations of
[
P]orthophosphate-labeled 3ASubE P-3 cells, two
labeled proteins appear which migrate at approximately 97 kDa and
between the 45- and 66-kDa molecular mass markers. We have previously
demonstrated that these bands are nonspecifically immunoprecipitated
since they can be eliminated by double immunoprecipitations
(29) . Western blot analysis indicated that approximately equal
amounts of the IL-8RB were immunoprecipitated from cells treated with
the various concentrations of TPA (Fig. 1 B).
Phosphoamino acid analysis revealed that TPA enhanced the
phosphorylation of serine residue(s) on the IL-8RB (Fig. 2).
Phosphorylation of the IL-8RB in response to PKC activation was not
unique to stable transfectants of the 3ASubE cell line. Similar
observations were noted in two other stably transfected
non-hematopoietic cell lines, namely the 293 human embryonic kidney
epithelial cell line and the Melan-A mouse melanocyte cell line (data
not shown).
Figure 1:
TPA treatment enhances the
phosphorylation of the IL-8RB in 3ASubE P-3 cells. 3ASubE P-3 cells
were labeled with [P]orthophosphate as described
under ``Experimental Procedures'' and treated with an ethanol
vehicle (0 nM) or increasing concentrations of TPA
(4-400 nM) for 10 min at 37 °C. Whole cell lysates
were prepared and the IL-8RB was immunoprecipitated from an equal
number of trichloroacetic acid-precipitable counts (5
10
cpm), using affinity purified anti-amino-terminal peptide
polyclonal antibodies which recognize the IL-8RB. Immunoprecipitates
were electrophoresed through a 9% SDS-polyacrylamide gel, transblotted
onto a nitrocellulose membrane, and exposed to autoradiographic film
for approximately 16 h ( A). After development, the
nitrocellulose membrane was analyzed by Western blot analysis, using
the above mentioned anti-amino-terminal peptide antibodies. The Western
blot was developed using an alkaline phosphatase detection system
( B). Molecular size standards, in kilodaltons, are shown on
the left. The position of the IL-8RB is marked by an
arrowhead.
Figure 2:
TPA treatment enhances the phosphorylation
of the IL-8RB on serine residues. The IL-8RB was immunoprecipitated
from 3ASubE P-3 cells which had been labeled with
[P]orthophosphate and treated with 40
nM TPA for 10 min. The immunoprecipitates were electrophoresed
through a 9% SDS-polyacrylamide gel, transblotted onto a PVDF membrane,
and exposed to autoradiography. The band corresponding to the IL-8RB
was excised from the membrane and subjected to phosphoamino acid
analysis, as described under ``Experimental Procedures.'' The
position of the phosphoamino acid standards have been traced onto the
autoradiogram.
The effect of the diacylglycerol analogue
diC, a natural activator of PKC, on the phosphorylation
state of the IL-8RB in the 3ASubE P-3 cells was also investigated.
[
P]Orthophosphate-labeled 3ASubE P-3 cells were
treated in the presence of 20 or 200 µM diC
for 10 min. For a comparison, 3ASubE P-3 cells were also treated
with 40 nM TPA. Whole cell lysates were prepared and the
IL-8RB was immunoprecipitated and analyzed by autoradiography and
Western blot analysis. As shown in Fig. 3, diC
enhanced the phosphorylation of the IL-8RB in the 3ASubE P-3
cells. Under the conditions used for these experiments, 200
µM diC
was more potent than 20 µM
diC
in enhancing receptor phosphorylation. Western blot
analysis revealed that approximately equal amounts of the IL-8RB were
immunoprecipitated from the treated and untreated samples (data not
shown).
Figure 3:
DiC treatment enhances the
phosphorylation of the IL-8RB in 3ASubE P-3 cells.
[
P]Orthophosphate-labeled 3ASubE P-3 cells were
treated with either an ethanol vehicle control (not treated,
NT), TPA (40 nM), or diC
(20 or 200
µM) for 10 min at 37 °C. Whole cell lysates were
prepared and the IL-8RB was immunoprecipitated from an equal number of
trichloroacetic acid-precipitable counts (5
10
cpm)
as described under ``Experimental Procedures.''
Immunoprecipitates were electrophoresed through a 9% SDS-polyacrylamide
gel, transblotted onto a nitrocellulose membrane, and exposed to
autoradiographic film for approximately 16 h. The position of the
IL-8RB is marked by an arrowhead.
Inhibition, or Down-regulation, of PKC Fails to Block the
MGSA-enhanced Phosphorylation of the Type B IL-8R
MGSA has
previously been demonstrated to enhance the phosphorylation of the
IL-8RB on serine residue(s)
(29) . Initially, we reported
enhanced receptor phosphorylation in response to 5 nM MGSA
(29) . A more extensive analysis of the concentration dependence
of MGSA on receptor phosphorylation revealed that 50 nM MGSA
maximally increased receptor phosphorylation in the 3ASubE P-3 cells
(Fig. 4). Basal phosphorylation of the IL-8RB is not apparent in
this figure due to the shorter exposure time for this autoradiogam
(approximately a 5-h exposure for Fig. 4compared to a 16-h
exposure used in Fig. 1). Upon longer exposure of the gel shown
in Fig. 4, the basal phosphorylation of the receptor was apparent
(data not shown). Since both MGSA and TPA enhanced the phosphorylation
of the IL-8RB on serine residue(s), we postulated that PKC may serve as
a second messenger for the IL-8RB in response to ligand binding.
Studies including PKC inhibition or down-regulation were subsequently
conducted to further elucidate a role for PKC in this signaling
pathway. 3ASubE P-3 cells were pretreated with either dimethylformamide
(vehicle control) or 50 nM staurosporin for 2 h, after which
the cells were treated with 5 nM MGSA (or an acetonitrile
vehicle control) or 400 nM TPA (or an EtOH vehicle control)
for 10 min. As demonstrated in Fig. 5, both MGSA and TPA enhanced
the phosphorylation of the IL-8RB in cells which had been pretreated
with the dimethylformamide vehicle control. Pretreatment with
staurosporin suppressed the TPA-enhanced phosphorylation of the IL-8RB,
however, it had no apparent effect on the MGSA-enhanced receptor
phosphorylation. A similar response was observed in cells which had
been pretreated with 400 nM TPA for 40 h to down-regulate PKC.
Prolonged TPA treatment suppressed the TPA-enhanced phosphorylation,
whereas it had no effect on the MGSA-enhanced phosphorylation of this
receptor (Fig. 5). Inhibition of PKC by staurosporin, or
down-regulation by prolonged TPA treatment also had no effect on the
MGSA-enhanced receptor phosphorylation with 50 nM MGSA (data
not shown). Interestingly, the basal level of receptor phosphorylation
was greater in the cells which had been pretreated with TPA for 40 h.
Similar results were observed in cells treated with 200 nM TPA
for 24 h (data not shown). Since neither inhibition nor down-regulation
of PKC significantly altered the effect of MGSA on the phosphorylation
state of the IL-8RB, PKC isoforms which are sensitive to these
manipulations may not play a role in the MGSA-enhanced phosphorylation
of this receptor.
Figure 4:
Concentration dependence of the
MGSA-enhanced phosphorylation of the IL-8RB. 3ASubE P-3 cells were
labeled with [P]orthophosphate as described
under ``Experimental Procedures'' and treated with an
acetonitrile vehicle (0 nM) or increasing concentrations of
MGSA (0.5-100 nM) for 10 min at 37 °C. Whole cell
lysates were prepared and the IL-8RB was immunoprecipitated as
described under ``Experimental Procedures.''
Immunoprecipitates were electrophoresed through a 9% SDS-polyacrylamide
gel, dried, and exposed to autoradiographic film for approximately 5 h.
The position of the IL-8RB is marked by an
arrowhead.
Figure 5:
PKC inhibition, or down-regulation, does
not block the MGSA-enhanced phosphorylation of the IL-8RB. 3ASubE P-3
cells were labeled with [P]orthophosphate for 3
h at 37 °C. During the final 2 h of this incubation, cells were
treated with dimethylformamide ( DMF, vehicle control) or 50
nM staurosporin. Alternatively, the 3ASubE P-3 cells were
cultured in the presence of 400 nM TPA for 40 h and maintained
in TPA during the [
P]orthophosphate labeling
period. Once labeled, cells were treated with 5 nM MGSA, or an
acetonitrile vehicle control, or with 400 nM TPA, or an
ethanol vehicle control, for 10 min at 37 °C. The IL-8RB was
immunoprecipitated from whole cell lysates, as described under
``Experimental Procedures,'' electrophoresed through a 9%
SDS-polyacrylamide gel, dried, and exposed to autoradiographic film for
approximately 8 h. The position of the IL-8RB is indicated by the
arrowhead.
Pertussis Toxin Treatment Suppresses the MGSA-enhanced
Phosphorylation of the IL-8RB
Previously we have demonstrated
that the IL-8RB expressed in the 3ASubE P-3 cells is coupled, at least
in part, to pertussis toxin-sensitive G proteins
(29) . To
determine whether the MGSA-enhanced receptor phosphorylation required
pertussis toxin-sensitive G proteins, or whether other G proteins
participated in this pathway, 3ASubE P-3 cells were treated or not
treated with pertussis toxin (1 µg/ml) during the 3-h labeling with
[P]orthophosphate. Cells were then treated in
the absence or presence of 5 nM (40 ng/ml) MGSA or 400
nM TPA for 5 min, lysed in RIPA buffer, and the IL-8RB was
immunoprecipitated. As demonstrated in Fig. 6, pertussis toxin
treatment of the 3ASubE P-3 cells suppressed the MGSA-enhanced
phosphorylation of the IL-8RB. These data suggest that pertussis
toxin-sensitive G proteins participate in the MGSA-enhanced
phosphorylation of the IL-8RB. As expected, pertussis toxin had no
effect on the TPA-enhanced phosphorylation of the IL-8RB
(Fig. 6). However, these data demonstrate that the concentration
and duration of the pertussis toxin treatment was not toxic, nor did it
nonspecifically inhibit kinases within the cells.
Figure 6:
Pertussis toxin suppresses the
MGSA-enhanced phosphorylation of the IL-8RB in 3ASubE P-3 cells. 3ASubE
P-3 cells were labeled with [P]orthophosphate in
the presence of either pertussis toxin (1 µg/ml) or its vehicle for
3 h. Cells were subsequently treated with MGSA (5 nM) or TPA
(400 nM) for 5 min at 37 °C. Whole cell lysates were
prepared and the IL-8RB was immunoprecipitated from 5
10
trichloroacetic acid-precipitable counts/min. Immunoprecipitates
were analyzed as described. The position of the IL-8RB is marked by an
arrowhead.
TPA Treatment Concomitantly Reduces both
I-MGSA Binding to 3ASubE P-3 Cells and IL-8RB Protein
Levels
I-MGSA binding assays were conducted to
determine whether IL-8RB phosphorylation in response to TPA was
accompanied by a change in ligand binding to this receptor. 3ASubE P-3
cells were treated with TPA (400 nM) for increasing periods of
time (0-120 min) prior to the initiation of the binding assay. As
shown in Fig. 7 A, there was a time-dependent decrease in
total binding of
I-MGSA to the TPA-treated 3ASubE P-3
cells. Pretreatment of the 3ASubE P-3 cells with TPA for 30 min
resulted in approximately 50% reduction in
I-MGSA
binding, whereas a 2-h pretreatment essentially reduced
I-MGSA binding to background levels. This effect was
transient;
I-MGSA binding to the 3ASubE P-3 cells treated
with 400 nM TPA for 24 h was equivalent to the
I-MGSA binding to untreated cells (data not shown).
Figure 7:
TPA treatment of 3ASubE P-3 cells
concomitantly reduces both I-MGSA binding and IL-8RB
protein levels. A,
I-MGSA binding to TPA-treated
3ASubE P-3 cells. 3ASubE P-3 cells were incubated with TPA (400
nM) or ethanol vehicle, in DMEM for 0, 15, 30, 60, or 120 min
prior to the binding assay.
I-MGSA (20-30,000
cpm/well, specific activity of 100 µCi/µg) was incubated with
the 3ASubE P-3 cells in 100 µg/ml ovalbumin, 30 mM
Hepes/DMEM for 4 h at 4 °C. Cells were washed and counts were
eluted with 0.1 N NaOH, 1% SDS. Nonspecific binding was
estimated as the binding of
I-MGSA in the presence of 50
ng/ml unlabeled MGSA. Each point represents the mean of triplicate
determinations (standard deviations were typically less than 10%).
A, inset, TPA time dependence on receptor protein levels.
Triton X-100 extracts were prepared from 3ASubE P-3 cells which had
been treated with 400 nM TPA for increasing periods of time.
Fifty micrograms of protein per lane was electrophoresed through a 9%
SDS-polyacrylamide gel, transblotted onto a PVDF membrane, and
subjected to Western blot analysis. The position of the IL-8RB is
marked by an arrowhead. B, TPA concentration
dependence on receptor protein levels. 3ASubE P-3 cells were treated
with 0, 4, 40, or 400 nM TPA for 2 h prior to extraction with
Triton X-100. Molecular size standards in kilodaltons are shown on the
left. The position of the IL-8RB is marked by an
arrowhead.
Western blot analysis was conducted to determine if the TPA-induced
decrease in I-MGSA binding was accompanied by a decrease
in the level of IL-8RB protein present in the 3ASubE P-3 cells. Triton
X-100 extracts were prepared from 3ASubE P-3 cells which had been
treated for increasing periods of time with 400 nM TPA.
Western blot analysis of the extracts (50 µg of protein/lane)
revealed a time-dependent decrease in the amount of immunoreactive
IL-8RB present in the TPA-treated cells (Fig. 7 A,
inset). A decrease in the level of IL-8RB protein was notable
after a 15-min treatment with 400 nM TPA and it was maximal
after a 2-h treatment. The effect of TPA on receptor protein level was
also concentration-dependent. Treatment of the 3ASubE P-3 cells for 2 h
with either 40 or 400 nM TPA resulted in a significant
decrease in the level of the IL-8RB protein present in the whole cell
lysates (Fig. 7 B). Similar to the transient effect of
TPA treatment on ligand binding to the 3ASubE P-3 cells, the effect of
TPA on receptor protein levels was also transient. Cells treated with
400 nM TPA for 24 h expressed the same level of receptor
protein as untreated cells, as determined by Western blot analysis
(data not shown). DiC
treatment also resulted in a decrease
in the IL-8RB protein level in the 3ASubE P-3 cells. Western blot
analysis of whole cell lysates (25 µg of protein/lane) prepared
from 3ASubE P-3 cells which had been treated with increasing
concentrations of diC
indicated that a 2-h treatment with
500 µM diC
resulted in a decrease in the
receptor protein level (Fig. 8). Greater concentrations of
diC
did not result in an increased reduction of the IL-8RB
protein level in the 3ASubE P-3 cells. Under these conditions, the
effect of diC
was not as potent as the effect observed in
response to 40 nM TPA (Fig. 8).
Figure 8:
DiC treatment reduces the
IL-8RB protein level in 3ASubE P-3 cells. Triton X-100 extracts were
prepared from 3ASubE P-3 cells which had been treated with an ethanol
vehicle control ( NT), TPA (40 nM), or increasing
concentrations of diC
(200-800 µM) for 2
h at 37 °C. Twenty-five micrograms of protein per lane was
electrophoresed through a 9% SDS-polyacrylamide gel, transblotted onto
a PDVF membrane, and subjected to Western blot analysis. The position
of the IL-8RB receptor is marked by an
arrowhead.
A similar experiment
was conducted to determine if MGSA treatment would alter the amount of
IL-8RB protein present in the 3ASubE P-3 cells. Cells were treated with
increasing concentrations of MGSA (0-50 nM) for 2 h,
Triton X-100 extracts of cells were prepared and subjected to Western
blot analysis. For comparison, a Triton X-100 extract of the parental
3ASubE cell line, which does not express the IL-8RB, was included in
this Western blot. Similar to TPA, MGSA treatment also resulted in a
concentration-dependent decrease in the level of IL-8RB protein in the
3ASubE P-3 cells (Fig. 9). This decrease was detectable after a
2-h treatment with 25 nM MGSA. The IL-8RB present in the
lysates prepared from cells treated with the greater concentrations of
MGSA migrated at a slower rate, consistent with hyperphosphorylation of
this receptor. Under these experimental conditions, the amount of
IL-8RB protein present in cells treated with 400 nM TPA was
consistently less than the amount present in cells treated with 50
nM MGSA.
Figure 9:
MGSA treatment reduces the level of the
IL-8RB protein in 3ASubE P-3 cells. Triton X-100 extracts were prepared
from either the parental 3ASubE cell line or the 3ASubE P-3 cells which
had been treated with MGSA (0, 5, 25, or 50 nM) for 2 h at 37
°C. Twenty-five micrograms of protein were electrophoresed through
a 9% SDS-polyacrylamide gel, and transblotted onto a PVDF membrane. The
blot was analyzed as described under ``Experimental
Procedures.'' The position of the IL-8RB is marked by an
arrowhead.
The decrease in immunoreactive IL-8RB protein
present in the Triton X-100 extracts of TPA- or MGSA-treated 3ASubE P-3
cells could result from the mobilization of this receptor to a Triton
X-100 insoluble pool. Such mobilization has previously been reported
for the fMet-Leu-Phe receptor in response to ligand binding in
granulocytes
(34) . Alternatively, the decrease in receptor
protein could be due to an increase in receptor degradation. Two
distinct approaches were taken to address the question of receptor
mobilization versus degradation in response to TPA or MGSA
treatment. First, to assess receptor mobilization we examined both the
Triton X-100 soluble and insoluble fractions of 3ASubE P-3 cells that
had been treated with MGSA (50 nM), TPA (400 nM), or
the appropriate vehicle controls for 2 h. Under these conditions, we
again detected decreased levels of IL-8RB protein in the Triton X-100
extracts from cells treated with MGSA or TPA, as compared to the cells
treated with vehicle, however, no apparent IL-8RB immunoreactivity was
detectable in the Triton X-100 insoluble pellets (Fig. 10). A
Coomassie stain of a polyacrylamide gel of Triton X-100 insoluble
pellets revealed that proteins from the insoluble pellets did migrate
through the gel (data not shown). These data suggest that the decrease
in IL-8RB immunoreactivity in response to MGSA or TPA treatment is not
due to the mobilization of this receptor to a Triton X-100 insoluble
pool.
Figure 10:
TPA or MGSA treatment of 3ASubE P-3 cells
does not mobilize the IL-8RB into a Triton X-100 insoluble pool. 3ASubE
P-3 cells were incubated in the presence of 50 nM MGSA (or an
acetonitrile vehicle control), or with 400 nM TPA (or an EtOH
vehicle control) for 2 h at 37 °C. Triton X-100 extracts were
prepared and 25 µg of extract were electrophoresed per lane.
Alternatively, the entire Triton X-100-insoluble pellet from 3ASubE P-3
cells was resuspended in 2 Laemmli buffer containing 5% SDS and
10%
-mercaptoethanol and electrophoresed. Proteins were
transblotted and subjected to Western blot analysis as described. The
migration of the IL-8RB is indicated by the
arrowhead.
Pulse-chase experiments were conducted to assess whether MGSA
or TPA stimulated receptor degradation. The parental cell line, 3ASubE,
along with the 3ASubE P-3 cells were metabolically pulsed with
[S]cysteine/methionine
(Tran
S-label, ICN) for 6 h then chased in the presence of
cold cysteine and methionine in the absence or presence of MGSA (50
nM) or TPA (400 nM) for 2 h. As shown in
Fig. 11
, a decreased level of the IL-8RB was immunoprecipitated
from the MGSA- or TPA-treated cells, as compared to the untreated
3ASubE P-3 cells, suggesting that both MGSA and TPA stimulate receptor
degradation. As expected, the IL-8RB was undetectable in the
S-labeled parental 3ASubE cell line (Fig. 11).
Figure 11:
TPA or MGSA treatment of the 3ASubE P-3
cells results in degradation of the IL-8RB. The parental 3ASubE cell
line, or the IL-8RB expressing 3ASubE P-3 cells were pulse labeled with
[S]cysteine/methionine as described under
``Experimental Procedures.'' Cells were then chased with
unlabeled cysteine and methionine for 2 h in the absence or presence of
MGSA (50 nM) or TPA (400 nM). Triton X-100 extracts
were prepared and the IL-8RB was immunoprecipitated from an equal
number of trichloroacetic acid-precipitable counts (2
10
), electrophoresed through a 9% SDS-polyacrylamide gel,
dried, and subjected to autoradiography. The migration of the IL-8RB is
indicated by the arrowhead.
Differential Effects of MGSA and TPA Treatment on the
IL-8RB Expressed in U937 Cells
Experiments were conducted on the
promonocytic cell line, U937, as an initial attempt to compare the
signaling by MGSA through the IL-8RB, and the effect of PKC activation
on this receptor in non-hematopoietic cell types versus hematopoietic cell types. Previous studies have demonstrated that
U937 cells, differentiated with retinoic acid, express the endogenous
IL-8 receptors
(35) . U937 cells were treated with 1
µM retinoic acid for 5 days prior to experimentation.
Differentiated U937 cells were labeled with
[P]orthophosphate, then treated in the absence
or presence of MGSA (50 nM) for 10 min or TPA (40 nM)
for 1-10 min. Whole cell lysates were prepared, the IL-8RB was
immunoprecipitated, electrophoresed, transferred onto a nitrocellulose
membrane, then analyzed by autoradiography and Western blot analysis.
MGSA treatment of the U937 cells enhanced the phosphorylation of the
IL-8RB (Fig. 12 A). Phosphoamino acid analysis revealed
that, similar to the 3ASubE P-3 cells, MGSA enhanced the
phosphorylation of serine residues on the IL-8RB expressed in the U937
cells (data not shown). There was no evidence for either threonine or
tyrosine phosphorylation on this receptor. Unlike the effect observed
for MGSA, TPA treatment, under these conditions, did not appear to
modulate the phosphorylation state of the IL-8RB expressed in U937
cells (Fig. 12 A). Western blot analysis of the
immunoprecipitates, using a mouse monoclonal anti-IL-8RB antibody,
indicated that approximately equal levels of receptor protein were
immunoprecipitated from the treated and untreated U937 cells
(Fig. 12 B). Greater concentrations of TPA (400
nM) also failed to modulate the IL-8RB phosphorylation status
(data not shown). Similarly, treatment with diC
(200
µM) or 1-oleoyl-2-acetyl- sn-glycerol (200
µM), another diacylglycerol analogue, for 10 min failed to
alter the phosphorylation state of this receptor in the U937 cells
(data not shown).
Figure 12:
MGSA, but not TPA, treatment enhances the
phosphorylation of the IL-8RB in U937 cells. Differentiated U937 cells
were labeled with [P]orthophosphate as described
under ``Experimental Procedures'' and treated with an
acetonitrile vehicle control ( AN), MGSA (50 nM) for
10 min, or TPA (40 nM) for 1-10 min at 37 °C. Whole
cell lysates were prepared and the IL-8RB was immunoprecipitated from
an equal number of trichloroacetic acid-precipitable counts (5
10
cpm). The immunoprecipitates were electrophoresed
through a 9% SDS-polyacrylamide gel, transblotted onto a nitrocellulose
membrane, and exposed to autoradiographic film for approximately 16 h
( A). After development, the nitrocellulose membrane was
analyzed by Western blot analysis, using a mouse monoclonal antibody
against the IL-8RB ( B).
The effect of MGSA treatment and PKC activation on
the level of IL-8RB protein present in the differentiated U937 cells
was also examined. U937 cells were treated with MGSA (50 nM),
TPA (4 or 40 nM), diC (10-100
µM), or the appropriate vehicle control for 2 h at 37
°C. Whole cell lysates were prepared, the IL-8RB was
immunoprecipitated using the rabbit anti-amino-terminal peptide
antibody, and the immunoprecipitates were subjected to Western blot
analysis using the above mentioned mouse monoclonal antibody. Results
from this experiment (Fig. 13) indicated that MGSA treatment
resulted in a modest decrease in the IL-8RB protein level, whereas TPA
or diC
treatment significantly reduced the level of IL-8RB
protein in the U937 cells.
Figure 13:
MGSA, TPA, or diC treatment
reduces the level of the IL-8RB protein present in U937 cells. U937
cells were treated in the absence (-) or presence (+) of
MGSA (50 nM), TPA (4 or 40 nM), diC
(10-100 µM), or an EtOH vehicle control (0
µM) for 2 h at 37 °C. Cells were washed and whole cell
lysates were prepared using a Triton X-100 buffer. The protein content
of the lysates were estimated (BCA, Pierce) and 200 µg of protein
was immunoprecipitated using 5 µg of affinity purified
anti-amino-terminal rabbit polyclonal antibody. The immunoprecipitates
were electrophoresed through a 9% SDS-polyacrylamide gel, transblotted,
then subjected to Western blot analysis using the mouse monoclonal
antibody.
- and
-adrenergic receptors
(31) . Since PKC and MGSA enhanced
the phosphorylation of serine residue(s) on the IL-8RB, and
furthermore, since PKC has been implicated in the signaling pathway of
IL-8 in both neutrophils and lymphocytes
(36, 37, 38) , experiments were conducted to
elucidate a role for PKC in the MGSA-enhanced phosphorylation of the
IL-8RB. Under conditions where the TPA-enhanced phosphorylation of the
IL-8RB was suppressed, either by staurosporin or prolonged exposure to
TPA, MGSA still increased the phosphorylation of this receptor. Thus,
PKC may not be involved in the MGSA-enhanced phosphorylation of this
receptor. Alternatively, MGSA may elicit its signal through PKC
isoform(s) not inhibited by staurosporin or down-regulated by TPA under
our experimental conditions, such as PKC
or PKC
(39) .
The sites of receptor phosphorylation mediated by either PKC or MGSA
have not been determined. Based upon the proposed membrane topology of
the IL-8RB, there are 8 candidate serine residues in the cytoplasmic
carboxyl tail of the receptor which may be target(s) for the PKC- or
MGSA-mediated phosphorylation. There are no serine residues in the
other intracellular domains of the IL-8RB. Studies are currently
underway to identify which serine residue(s) are phosphorylated.
-enhanced phosphorylation of the IL-8RB
in U937 cells. One explanation for the difference in receptor
phosphorylation in response to PKC activation in the stable
transfectants versus the U937 cells is that regulation of
receptor phosphorylation by PKC is different in monocytic cells
versus the three different non-hematopoietic stable
transfectants which we have examined. It is conceivable that the effect
of PKC activation on receptor phosphorylation in non-hematopoietic cell
types is mediated through a second kinase which is activated by PKC. If
such a kinase is absent in U937 cells, this may explain the lack of
IL-8RB phosphorylation in response to PKC activation. In vitro kinase assays should be useful to determine whether PKC is capable
of directly phosphorylating the IL-8RB.
- and
-adrenergic receptors
(41, 42) , the angiotensin II
receptor
(43) , and the bombesin receptor
(44) . Although
our experiments with the 3ASubE P-3 and other stably transfected
non-hematopoietic cell lines would be consistent with receptor
phosphorylation playing a role in the desensitization response to PKC
activation of the granulocytes, the U937 experiments described here do
not support this hypothesis. Recently it has been demonstrated that PKC
activation enhances the phosphorylation of G
,
resulting in an attenuated inhibition of the adenylyl cyclase pathway
(45) . Since IL-8 and MGSA mediate their effects on neutrophils
via pertussis toxin-sensitive G proteins, and furthermore, since it has
been demonstrated that both the IL-8RA and B can couple to
G
(46) , the ability of PKC to attenuate
signaling by this route must also be considered as a means of
desensitizing granulocytes to IL-8.
- and
-adrenergic
receptors, however, it has not been reported to alter the agonist
binding characteristics of these receptors
(41, 42) . In
contrast, we have shown that TPA treatment of the 3ASubE P-3 and U937
cells results in a rapid decrease in the amount of receptor protein
present in these cells. Furthermore, down-regulation, or degradation,
of the adrenergic receptors requires agonist occupation of the receptor
(31) . The effect of PKC activation on the IL-8RB protein levels
in the 3ASubE P-3 and U937 cells indicates that receptor occupancy is
not necessary for the degradation of this receptor. Although the
PKC-mediated degradation of the IL-8RB is novel with respect to G
protein-coupled receptors, PKC has previously been demonstrated to
stimulate the shedding of several different cytokine and homing
receptors, including the IL-6, the tumor necrosis factor-
and
-
, and the gp90
receptors
(47, 48, 49, 50) . Whereas it is
unlikely that a seven-transmembrane domain receptor would be shed in
response to PKC activation, the overall effect would be similar for
receptor degradation and shedding; both would render the cell
insensitive to a subsequent cytokine challenge. Thus activation of PKC
via different extracellular signals may initiate a negative feedback
loop for responses to various cytokines, whereby their respective
receptors are either degraded or shed by the cell.
and
1-oleoyl-2-acetyl- sn-glycerol, on the IL-8RB protein level was
investigated. Single application of either of these two PKC activators
fails to induce U937 differentiation
(52, 53) . Our
studies demonstrate that a single 2-h treatment of the U937 cells with
25 µM diC
resulted in a decrease in the IL-8RB
protein. 1-Oleoyl-2-acetyl- sn-glycerol treatment also elicited
a modest decrease in the level of the IL-8RB protein present in the
U937 cells; the magnitude of this effect was comparable to the effect
observed in response to treatment with MGSA (data not shown). Thus
under conditions where one would not anticipate that the U937 cells
would be differentiated, we have observed that treatment with either
PKC activators or MGSA decreased the total amount of IL-8RB protein
present in these cells.
and G
) and pertussis
toxin-insensitive G proteins (G
, G
,
G
)
(46) . Since pertussis toxin treatment
suppresses the MGSA-enhanced phosphorylation of the IL-8RB, it is
inferred that this receptor couples to endogenous G
family member(s) in the 3ASubE P-3 cells and that activation of
the kinase(s) which mediate the MGSA-enhanced phosphorylation of the
IL-8RB is mediated, at least in part, through the pertussis
toxin-sensitive G protein family. Candidate kinase(s) which may play a
role in this phosphorylation include members of the recently described
G protein-coupled receptor kinase family
(54) . Investigations
into the potential role of these kinase(s), in response to MGSA, are
currently underway.
,
1,2-dioctanoyl- sn-glycerol (8:0); IL-8, interleukin-8; IL-8R,
interleukin-8 receptor; PKC, protein kinase C; PVDF, polyvinylidene
difluoride; TPA, 12- O-tetradecanoylphorbol 13-acetate; DMEM,
Dulbecco's modified Eagle's medium.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.