 |
INTRODUCTION |
The extracellular Ca2+-sensing receptor
(CaR)1 is a G protein-coupled
receptor that is expressed in the parathyroid and kidney and senses
extracellular Ca2+ in the millimolar range. This receptor
acts through at least two G proteins, G
i and
G
q, to regulate multiple intracellular enzymes that
control production of second messengers including cAMP, inositol
trisphosphate (IP3), diacylglycerol (DAG), intracellular Ca2+ (Ca2+i), and arachidonic acid (AA)
metabolites (1). In the parathyroid, the CaR inhibits parathyroid
hormone production and secretion in response to elevated extracellular
Ca2+ (Ca2+o) levels, and in the kidney,
activation of the CaR inhibits NaK2Cl cotransporter activity,
Ca2+ reabsorption, and the action of vasopressin leading to
a Na+, Cl
, Ca2+, and
H2O diuresis. CaR-stimulated production of AA metabolites, possibly products of 12- and 15-lipoxygenase, contributes to inhibition of parathyroid hormone secretion in parathyroid cells (2-4). In cells
from the thick ascending limb of Henle, activation of phospholipase
A2 (PLA2) by the CaR results in the production
of 20-hydroxyeicosatetraenoic acid, a cytochrome P450 metabolite, that
inhibits the apical 70-picosiemens potassium channel activity that
would reduce NaK2Cl transporter activity (5). In these tissues and
others including the brain, pancreas, stomach, colon, and skin, the CaR
may also sense Ca2+ extrusion by adjacent cells and
function in cell-cell communication (6-8).
Phospholipase A2, the rate-limiting enzyme in AA
metabolism, hydrolyzes cellular phospholipids to form lysophospholipids
that lead to the production of platelet-activating factor and
liberation of polyunsaturated fatty acids including AA that are the
precursors for prostaglandins, thromboxanes, leukotrienes, and a
variety of other metabolites (eicosanoids) (1, 9). Eight different groups of PLA2 enzymes have been described. The majority of
these enzymes are extracellular and not hormonally regulated, whereas two groups, group IV and group VI, are intracellular and are subject to
regulation by extracellular signals. The hormone-regulated enzymes are
the 85-kDa Ca2+-sensitive cytosolic PLA2
(cPLA2, a group IV enzyme) and the 80-88-kDa Ca2+-insensitive PLA2 (iPLA2, a
group VI enzyme). Both enzymes are subject to activation by G
protein-dependent signaling systems (10, 11).
cPLA2 is expressed in most tissues and is activated by many
G protein-coupled receptors including those for angiotensin II, ATP,
bradykinin, endothelin, lysophosphatidic acid, and thrombin (1). The AA
products of cPLA2 appear to be involved primarily in
signaling functions. cPLA2 activity is inhibited by the
substrate analogue arachidonyl trifluoromethyl ketone
(AACOCF3) but not bromoenol lactone (BEL) (12). The precise
mechanism of activation of cPLA2 is variable and depends on
the receptor. Activation of cPLA2 requires a rise in
Ca2+i which leads to translocation of the enzyme
from the cytosol to the plasma membrane. In different cell types,
pathways that involve pertussis toxin-sensitive or -insensitive G
proteins, phospholipase C (PLC), protein kinase C (PKC), MAP kinases
(extracellular signal-regulated kinases, ERK), calmodulin (CaM), and
calmodulin-dependent protein kinase (CaMK) have been
described (13). cPLA2 is a substrate for ERKs in many cell
types, and phosphorylation of cPLA2 by ERK appears to be
required for activation of the enzyme (14).
iPLA2 also appears to be ubiquitously expressed and is
activated by receptors for ATP,
2 agonists, vasopressin,
and Fas (15-17). The proposed functions of iPLA2 include
cellular lipid remodeling, generation of substrates for leukotriene
biosynthesis, and generation of second messengers that regulate ion
channel activity (11, 18). iPLA2 activity is inhibited by
both AACOCF3 and BEL (12). iPLA2 is activated
by Ca2+ store depletion and PKC and is inactivated by
association with calmodulin (16).
The form of PLA2 that is activated by the CaR and the
mechanism by which the CaR stimulates PLA2 activity have
not been defined in any cell type. To determine which PLA2,
cPLA2 or iPLA2, is activated by the CaR and
which G protein, second messenger, and kinase pathway(s) are involved,
we expressed the CaR in HEK-293 cells and defined the early components
of the signaling pathway that lead to activation of PLA2.
We find that the CaR activates cPLA2 via a
G
q, PLC, Ca2+i, CaM, and
CaMK-dependent but ERK-independent signaling pathway.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Chemicals were purchased from the Sigma or Fisher
unless specified otherwise. Fura-2-AM was obtained from Molecular
Probes (Eugene, OR). PMA was supplied by LC Laboratories (Woburn, MA). U73122 and BEL were purchased from Biomol (Plymouth Meeting, PA).
PD98059, U0124, U0126, W7, KN-93, and AACOCF3 were obtained from Calbiochem-Novabiochem. G418 sulfate was supplied by Life Technologies, Inc. The myo-[2-3H]inositol
(10-25 Ci/mmol) and [5,6,8,9,11,12,14,15-3H]AA (60-100
Ci/mmol) were purchased from NEN Life Sciences. SuperSignal West Pico
chemiluminescent substrate was obtained from Pierce. AG1-X8 anion resin
(200-400 mesh, formate form) was supplied by Bio-Rad.
Sources and Construction of cDNAs--
The cDNAs
encoding human wild-type and nonfunctional mutant CaR (R796W) in
pcDNA3 were generous gifts from Drs. E. M. Brown, S. C. Hebert, and M. Bai in the Department of Medicine, Brigham and Women's
Hospital, Harvard Medical School, and Vanderbilt University School of
Medicine. To add the HA epitope tag to the C termini of CaR, we
synthesized a 57-bp oligonucleotide coding for the last 6 amino acids
of CaR followed by 9 amino acids of the HA epitope, a stop codon, an
XbaI restriction site, and a 3-bp tail (5' CGC TCT AGA CTA
AGC GTA GTC TGG GAC GTC GTA TGG GTA TGA ATT CAC TAC GTT TTC 3'). The
cDNA was amplified by polymerase chain reaction using an
oligonucleotide that is 5' to a unique BamHI site in the
C-terminal region of receptor (5' ACC TTT ACC TGT CCC CTG AA 3') and
the 57-bp oligonucleotide coding for C terminus of receptor and the HA
epitope. The polymerase chain reaction products were digested with
BamHI and XbaI, purified, and ligated into the
expression vector (pcDNA3) that contained the remainder of the
cDNA for the CaR. The human cPLA2 cDNA was obtained
from Dr. Harry Nick, University of Florida, and subcloned into
pcDNA3 (19). MEKK97R and the HA-tagged
ERKK53R were generous gifts from Dr. Melanie Cobb,
University of Texas, Southwestern Medical School. The HEK-293 cells
that stably express RGS4 were described previously (20).
Cell Culture and Expression of cDNAs--
HEK-293 cells were
obtained from the American Type Culture Collection and cultured in DMEM
supplemented with 10% calf serum, and 25 mM Hepes (pH
7.4). The cDNAs, pcDNA3, CaR-HApcDNA3, or CaR(R796W)-HApcDNA3 and cPLA2-pcDNA3 were
introduced into cells using the calcium phosphate precipitation method.
Studies using transiently transfected cells were performed 48 h
after transfection. In these transfections, the total amount of DNA
transfected was held constant by addition of pBluescript so that the
total amount used was 3 µg. For stable expression of cDNAs, the
cells from each individual well were plated in 100- or 150-mm dishes or
96-well plates and cultured in medium containing 500 µg/ml G418
24 h after transfection. G418-resistant clones were isolated after
3-4 weeks and screened by immunoblotting for the expressed protein.
Cells were used for experiments between passages 5 and 15. Similar
results were obtained with multiple clones.
Antibodies and Immunoblotting--
To verify expression of
proteins, membranes were immunoblotted with the anti-HA antibody 12CA5
(CaR), the anti-Myc antibody 9E10 (Myc-RGS4) (both antisera were from
the UF hybridoma core), and anti-human cPLA2 (Santa Cruz
BioTechnology, Santa Cruz, CA), and to measure ERK activity,
anti-phospho-ERK (Promega, Madison, WI) and anti-ERK (Promega). HEK-293
cell membranes were prepared by homogenization in a buffer containing
10 mM Tris-HCl (pH 7.8), 1 mM EDTA, and
protease inhibitors and brought to a final concentration of 30 mM NaCl and 2 mM MgCl2 and
centrifuged at 250 × g for 2 min. The supernatant was
centrifuged at 23,000 × g for 10 min; the pellet was
resuspended in the same buffer, and the protein concentration was
measured. Samples containing equal amounts of protein were subjected to
SDS-PAGE and processed for immunoblotting. The immunoreaction signals
were detected by enhanced chemiluminescence.
Fluorescent Measurement of Ca2+i--
Cells
were released from dishes with phosphate-buffered saline containing 0.5 mM EDTA and rinsed twice with calcium measurement solution
(CaMS) containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,
10 mM Hepes (pH 7.4), 1 mg/ml bovine serum albumin (BSA),
and 2 mg/ml glucose. The cells were resuspended and incubated in 2 ml
of uptake medium containing 2.5 µM Fura-2-AM in CaMS for 30 min at 37 °C, washed twice with CaMS, and diluted to a
concentration of ~106 cells in 2 ml of CaMS.
Ca2+i was measured fluorometrically in
Fura-2-loaded cells in suspension by a dual excitation microfluorometer
(SLM Fluoromax) at 37 °C with constant stirring. Excitation was at
340 and 380 nm, and emission was at 510 nm. Experiments were started
when a steady fluorescent base line was obtained. Each tracing was calibrated by lysing the cells with digitonin (75 µg/ml) in the presence of 2 mM Ca2+o to determine
Fmax, and then 20 mM EDTA (pH 8.6)
was added to obtain Fmin. The
Ca2+i concentration was calculated from the
expression [Ca2+]i = Kd{(F
Fmin)/(Fmax
F)} where Kd = 224 nM
(21).
Measurement of Arachidonic Acid Release--
The HEK-293 cells
were grown in collagen-coated 24-well plates and were prelabeled with
0.1 µCi/well [3H]AA in serum-free DMEM at 37 °C for
6 h. After removal of the labeling medium, the cells were rinsed
with 1.5 ml of buffer A containing 130 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 0.5 mM CaCl2, 10 mM glucose, and 10 mM Hepes (pH 7.4) and incubated with buffer A containing 2 mg/ml BSA for 30 min in the presence or absence of inhibitor. In the
PKC down-regulation experiments, cells were incubated with or without
100 nM PMA for 20-24 h and prelabeled with
[3H]AA for the 6 h preceding the experiment. For
pertussis toxin treatment, cells were pretreated with or without 100 ng/ml pertussis toxin for 12-15 h and labeled with
[3H]AA during the last 6 h. After equilibration,
cells were incubated at 37 °C for 15 min with 0.5 ml of the same
buffer in the presence or absence of 5 mM CaCl2
or 0.5 mM neomycin. LPS (lipopolysaccharide)-stimulated [3H]AA release was measured in RAW 264.7 cells in a
similar manner except that the cells were grown and labeled in DMEM
(high glucose) with 10% FCS in 24-well trays without collagen coating.
The BEL (10 µM) was added 15 min before the LPS in buffer
A without BSA, and the cells were exposed to LPS (5 µg/ml) for 1 h with BSA present (22). Equal volumes of medium were removed from the
wells and centrifuged at 13,000 rpm for 5 min. The radioactivity in the cell-free medium was quantitated by liquid scintillation spectrometry and normalized for total radioactivity incorporated into cellular phospholipids that were solubilized with 0.2 N NaOH, 0.2%
SDS. [3H]AA release was linear for 60 min.
Measurement of Inositol Trisphosphate Formation--
Cells at
65% confluence in 12-well plates were prelabeled with 5-10 µCi/ml
myo-[3H] inositol in 0.5 ml of inositol-free
DMEM containing 10% calf serum for 48-50 h. For pertussis toxin
treatment, cells were incubated with pertussis toxin 100 ng/ml or
vehicle for the last 12-15 h of the prelabeling period. Before
experiments, cells were equilibrated for 30 min in inositol-free DMEM
containing 20 mM Hepes (pH 7.4) and 20 mM LiCl
with or without 10 µM U73122. Cells were then incubated
at 37 °C for 5 min in 0.4 ml of equilibration medium with or without
5 mM CaCl2. The reactions were terminated by
adding 0.4 ml of 10% perchloric acid to each well, and the plates were stored at 4 °C for 3-5 h. The samples were transferred to
microcentrifuge tubes, neutralized by the addition of 0.32 ml of a
solution containing 2 M KOH, 1 mM EDTA, and 1 M Hepes (pH 7.4), and centrifuged at 12,000 × g for 5 min. The supernatants were applied to AG1-X8 anion
exchange columns, and tritiated inositol-containing compounds were
separated as described (20).
Statistical Analysis--
Concentration-response relationships
(EC50 and Hill coefficient values) were determined using
GraphPad prism software, and significance was calculated using the
Instat two-tailed, unpaired t test statistics program.
 |
RESULTS |
Expression of CaR in Mammalian Cells--
To study regulation of
PLA2 by the CaR, we expressed an HA-tagged wild-type and an
HA-tagged nonfunctional mutant receptor (R796W) in HEK-293 cells (23).
The expressed proteins were detected by immunoblotting with the
monoclonal anti-HA antibody (12CA5) (Fig.
1). Blots with both the transiently and
stably transfected cells revealed bands of the appropriate molecular
mass, ~125 and 140 kDa, and demonstrated similar levels of CaR
protein expression. The two bands of the CaR result from differential
glycosylation (24, 25). Bands were not present in membranes from the
G418-resistant cells.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 1.
Stable and transient expression of the CaR in
HEK-293 cells. Membranes from cells that transiently (upper
panel) or stably (lower panel) express the HA-tagged
wild-type (WT) or mutant (CaRR796W,
Mut) CaR or G418-resistant control cells (V) were
immunoblotted with a monoclonal antibody 12CA5 that recognizes the HA
tag on the receptors.
|
|
Activation of cPLA2 by the CaR--
Extracellular
Ca2+ and neomycin, both ligands for the CaR, stimulated
[3H]AA release from cells that express the CaR in a
dose-dependent manner but not from cells that express the
nonfunctional mutant CaR R796W (Fig. 2).
The EC50 for Ca2+ was 4.2 mM, and
the EC50 for neomycin was 0.15 mM. To determine which form of PLA2, cPLA2 or iPLA2,
is activated by the CaR, we treated cells with AACOCF3, an
inhibitor of both enzymes, and BEL, a specific inhibitor of
iPLA2 (1, 12). The CaR was activated with neomycin to avoid
a possible increase in Ca2+ entry as a result in
increased extracellular calcium. As shown in Fig.
3A, AACOCF3
inhibited the CaR-stimulated [3H]AA release
(IC50 25 µM), but BEL had no effect up to a
concentration of 10 µM, a concentration that inhibits
iPLA2 in other systems (26). In parallel experiments, 10 µM BEL inhibited LPS-stimulated [3H]AA
release in RAW 264.7 cells, an iPLA2-dependent
response, by 92% (26). To confirm that the CaR activates
cPLA2, we transiently coexpressed cDNAs coding for the
CaR and cPLA2, and we measured [3H]AA release
(19). Fig. 3B shows that coexpression of increasing amounts
of the cPLA2 cDNA with the CaR resulted in increased
CaR-stimulated [3H]AA release without an increase in
basal [3H]AA release. Inhibitor studies and increasing
CaR-stimulated PLA2 activity with increasing levels of
expression of cPLA2 demonstrate that the CaR activates
cPLA2.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration dependence of CaR-stimulated
PLA2 activity for Ca2+ or neomycin.
HEK-293 cells that stably express the CaR were prelabeled with
[3H]AA for 6 h and treated with Ca2+
(0.5-10.0 mM, closed circles) or neomycin
(0-2.0 mM, closed squares) at 37 °C for 15 min. Cells that stably express the nonfunctional CaR,
CaRR796W, were treated with Ca2+ (open
circles) or neomycin (open squares).
[3H]AA release was normalized for [3H]AA
incorporated into total cellular lipids. The EC50 for
neomycin was 0.15 mM and the EC50 for
Ca2+ was 4.2 mM.
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Identification of cPLA2 as the
CaR-stimulated PLA2 activity using chemical phospholipase
inhibitors (A) and expression of cPLA2
(B). A, cells that stably express the
CaR were pretreated with AACOCF3 (0-200 µM)
for 20 min or BEL (0-10 µM) for 15 min and stimulated
with 0.5 mM neomycin for 15 min. [3H]AA
release was measured and is expressed as percent of maximum
neomycin-stimulated activity. B, HEK-293 cells were
transiently cotransfected with cDNAs coding for the CaR and vector
(pcDNA3) or increasing amounts of cDNA coding for human
cPLA2. The cells were labeled with [3H]AA,
and CaR-stimulated PLA2 activity was measured as described.
Results are expressed as percentage of [3H]AA released
normalized for the amount released in the presence of 0.5 mM Ca2+. Equal amounts of extracts of cells
transfected with different amounts of cDNA coding for
cPLA2 were immunoblotted with an antibody to
cPLA2, and a representative blot is shown below
the bar diagram in B.
|
|
Role of PLC and G Proteins--
We tested the role of PLC-
in
activation of cPLA2 by first demonstrating inhibition of
CaR-stimulated [3H]AA release by U73122, an inhibitor of
PLC-
. Pretreatment of cells that express the CaR with U73122
eliminated the CaR-stimulated cPLA2 activity demonstrating
that the CaR activates cPLA2 via PLC-
(Fig.
4A). To confirm that U73122
inhibits PLC-
activity, we measured CaR-stimulated IP3
production with and without U73122 (Fig. 4B), and we found
that it was completely inhibited by U73122. PLC-
can be activated by
pertussis toxin-sensitive (G
i-dependent) or
pertussis toxin-insensitive (presumably
G
q-dependent) signaling systems. Treatment
of the cells with pertussis toxin had a minimal inhibitory effect on
[3H]AA release (Fig. 4A), and IP3
production (Fig. 4B) indicating that G proteins of the
i family have a minimal role in stimulation of
cPLA2 and PLC-
by the CaR. To test specifically for a
role for a G
q family member in the activation of PLC-
and consequently cPLA2, we transiently expressed the CaR in
HEK-293 cells that stably express RGS4, an RGS protein that interacts
preferentially with members of the G
q family,
accelerates their GTPase activity, and reduces their activation (20,
27). Fig. 4C shows that RGS4 eliminated CaR-stimulated
PLC-
activity. These results indicate that the CaR acts through a
pertussis toxin-insensitive, G
q-dependent pathway to activate PLC, the products of which stimulate
cPLA2.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
Role of PLC- and
G i and
G q family proteins in
CaR-stimulated cPLA2 and PLC activity. A,
[3H]AA release in cells that stably express the mutant
(Mut) and wild-type (WT) CaR. Cells were
prelabeled with [3H]AA for 12-15 h. Where indicated,
cells were treated with pertussis toxin (PTx) 100 ng/ml for
the 12-15 h before experiments or U73122 for the 30 min before
experiments. Cells were treated with 0.5 mM
Ca2+ or 5.0 mM Ca2+ for 15 min for
AA release. B, IP3 production in cells that
stably express the wild-type or mutant (R796W) CaR. Cells were
pre-labeled with [3H]inositol for 48 h. Where
indicated, cells were treated with 100 ng/ml pertussis toxin for the
12-15 h before experiments or U73122 for the 30 min before
experiments. Cells were treated with 0.5 mM
Ca2+ or 5.0 mM Ca2+ for 5 min, and
[3H]IP3 production was measured by column
chromatography. C, effect of RGS4 on CaR-stimulated PLC
activity. HEK-293 cells that stably express RGS4 or G418-resistant
control cells (V) were transiently transfected with the CaR
(wild-type). Twelve hours after transfection, they were prelabeled with
[3H]inositol for 48 h and exposed to 0.5 or 5.0 mM Ca2+ for 5 min, and
[3H]IP3 production was separated by column
chromatography. Each bar represents the mean of triplicate
samples ± S.D., and this figure is representative of three
experiments.
|
|
Role of Ca2+i--
PLC-
produces both DAG,
which activates PKC, and IP3, which raises
Ca2+i, by releasing Ca2+ from
intracellular stores. A rise in Ca2+i could
activate cPLA2 by mechanisms involving calmodulin, Ca2+, calmodulin-dependent protein kinase,
Ca2+-dependent tyrosine kinases, PKC in
cooperation with DAG, or the ERK pathway. To document and characterize
the CaR-stimulated rise in Ca2+i in the cells that
express the CaR, we measured Ca2+i in cells that
express the wild-type and mutant CaR (CaRR796W) using
Fura-2 fluorescence (Fig. 5). In the
cells that express the wild-type CaR, activation of the CaR with 4 mM extracellular Ca2+
(Ca2+o) leads to a rapid rise in
Ca2+i followed by a slow fall to a plateau level
above base line. This type of Ca2+ signal has two
components, release of Ca2+ from intracellular stores and
Ca2+ entry across the plasma membrane. Subsequent
stimulation of purinergic receptors in these cells with ATP (100 µM) also leads to a rapid rise in
Ca2+i and a plateau phase. Cells that express the
CaR R796W do not respond to 4 mM extracellular
Ca2+ but do respond to ATP. Fig. 5B shows that
the effect of pertussis toxin on the CaR-stimulated
Ca2+i signal, like its effect on cPLA2
and PLC activity, is minimal. Incubation of the cells with EGTA to
chelate Ca2+o and deplete Ca2+i
(Fig. 5C) reduced CaR-stimulated [3H]AA
release by ~80% when the CaR was activated with neomycin, indicating
that Ca2+o and a rise in Ca2+i
are required for activation of cPLA2 by the CaR (28). In
studies similar to those shown in Fig. 5, A and
B, preincubation of cells with 2 mM EGTA for 20 min in nominally Ca2+-free medium prevented bradykinin and
ATP-stimulated rises in Ca2+i (data not shown).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
CaR-regulated Ca2+i and
inhibition of cPLA2 activity by EGTA. A,
cells expressing either the wild-type or mutant (R796W) CaR were
suspended and loaded with Fura-2-AM. The cells were washed and
resuspended in medium containing 1.0 mM
Ca2+o and then stimulated with Ca2+
(4.0 mM). When the tracings returned to base line, ATP (100 µM) was added. B, effect of pertussis toxin on
the CaR-stimulated Ca2+i signal. Cells expressing
the CaR were treated with pertussis toxin (PTx, 100 ng/ml)
for 12-15 h before the experiments. The Ca2+i
response to activation of the CaR was studied as described above.
C, effect of removal of Ca2+o and
Ca2+i depletion on CaR-stimulated cPLA2
activity. Cells grown in 24-well trays were washed in buffer and
treated with EGTA (0-10 mM) for 30 min before addition of
neomycin (500 µM). [3H]AA release was
measured over 15 min as described above. The study shown is
representative of three similar experiments.
|
|
Role of ERK--
Many receptors act through the ERK pathway to
activate cPLA2 (14). We assessed the role of ERK in the
activation of cPLA2 using chemical inhibitors of MEK, the
activator of the ERKs, and a dominant negative form of MEK
(MEKK97R). Preincubation of the cells with either PD98059
or U0126 had no effect on the ability of the CaR to activate
cPLA2 (Fig. 6A) but inhibited activation of the ERKs by the CaR (Fig. 6B).
Treatment of the cells with the inactive U0126 analogue, U0124, did not affect cPLA2 or ERK activation. Similarly, coexpression of
the CaR with the dominant negative MEK, MEKK97R, and
HA-tagged ERK-1 did not inhibit activation of cPLA2 (Fig. 6C) but resulted in inhibition of ERK activation by the CaR
(Fig. 6D). These results indicate that activation of the ERK
pathway and presumably phosphorylation of cPLA2 by p42/44
ERK is not required for activation of cPLA2 by the CaR.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of inhibitors of ERK activation on
PLA2 activity. A, cells that stably express
the CaR were treated with PD98059 (30 µM), U0126 (10 µM), or U0124 (10 µM) for 30 min and
stimulated with 5.0 mM Ca2+, and
[3H]AA release was measured as described above.
B, ERK activation was measured by immunoblotting with an
anti-phospho-ERK antibody in extracts of cells pretreated with PD98059
(30 µM), U0126 (10 µM), or U0124 (10 µM) for 30 min and stimulated for 5 min with 5 mM Ca2+. A representative blot is shown
above B. C, HEK-293 cells were
cotransfected with cDNAs coding for the CaR, HA-tagged ERK-1, and
pcDNA3 or MEKK97R. After 48 h, cells were
stimulated with 5.0 mM Ca2+ for 15 min, and
[3H]AA release was measured as described above. Results
represent the means of triplicate measurements ± S.D.
D, in parallel plates, cells were stimulated with
Ca2+ (5.0 mM) for 5 min, and ERK activity was
measured in cell extracts by a gel shift assay with the monoclonal
antibody 12CA5 that recognizes the HA epitope tag on the expressed
ERK-1. Activation was quantitated by densitometry, and calculation of
the ratio of the shifted band to the total ERK was expressed. A
representative blot is shown below D.
|
|
Role of PKC--
The PLC product, DAG, activates PKC in a
Ca2+-dependent manner. To test for a role of
the conventional forms of PKC (those that are both DAG- and
Ca2+-dependent), we treated cells with
calphostin, a PKC inhibitor, and down-regulated PKC by overnight
treatment of the cells with 100 nM PMA and then measured
CaR-stimulated cPLA2 activity. As shown in Fig.
7, both calphostin and down-regulation of
PKC with PMA pretreatment reduced CaR-stimulated cPLA2
activity by ~50% but did not eliminate it. In control experiments,
15 min of exposure of the HEK-293 cells that express the wild-type and
mutant forms of the CaR to 100 nM PMA stimulated
[3H]AA release to a similar degree as treatment of the
cells that express the wild-type CaR with 5.0 mM
Ca2+. However, pretreatment of the cells overnight with 100 nM PMA prevented significant stimulation of
[3H]AA release by 15 min of exposure to 100 nM PMA, demonstrating that our protocol for down-regulation
of PMA inhibits activation of the conventional forms of PKC. These
results indicate that the conventional PKC isoforms (DAG- and
Ca2+-dependent) contribute to activation of
cPLA2 but that PKC-dependent mechanisms cannot
account for the majority of its activation by the CaR.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of PKC on CaR-stimulated
cPLA2 activity. Cells that stably express the CaR were
pretreated with calphostin C (250 nM for 30 min) or PMA
(100 nM) for 20-24 h. They were then stimulated with
Ca2+ (5.0 mM) for 15 min, and
[3H]AA release was measured as described above. Values
shown are means of triplicate points and are presented as percentage of
activity stimulated by 5.0 mM Ca2+.
|
|
Role of CaM and CaMK--
Another mechanism by which a rise in
Ca2+i could activate cPLA2 is via
calmodulin (13). To test for a role for calmodulin in
CaR-dependent activation of cPLA2, we
pretreated cells with W7, a calmodulin antagonist that competitively
inhibits interaction of Ca2+-calmodulin with its target
proteins. Pretreatment of the cells with W-7 for 45 min before
activation of the CaR eliminated ~90% of the CaR-stimulated
cPLA2 activity (IC50 7 µM),
indicating that activation of calmodulin is an essential step in
activation of cPLA2 by the CaR (Fig.
8A). The effects of calmodulin
could be mediated by Ca2+, calmodulin-dependent
protein kinase (CaMK), or other proteins. To test for a role for CaMK,
we pretreated cells with KN-93, a specific inhibitor of the CaMK
enzymes at the concentrations used (29, 30). We found that over a
concentration range up to 25 µM, KN-93 inhibited ~90%
of the CaR-stimulated cPLA2 activity with an
IC50 of ~6.3 µM (Fig. 8B). These
results demonstrate that in contrast to other G protein-coupled
receptors, the CaR stimulates cPLA2 via a
CaM/CaMK-dependent pathway that is independent of the
ERK pathway.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 8.
Role of calmodulin and
Ca2+-calmodulin-dependent protein kinase in
CaR-stimulated cPLA2 activity. A, cells
that stably express the CaR were treated with W-7 (0-50
µM) for 45 min, exposed to 0.5 mM neomycin
(closed circles) or vehicle (closed squares) for
15 min, and [3H]AA release measured as described above.
B, cells that stably express the CaR were treated with KN-93
(0-25 µM) for 3 h, exposed to 0.5 mM
neomycin (closed circles) or vehicle (closed
squares) for 15 min, and [3H]AA release was measured
as described above. In both A and B, the values
represent means of triplicate points ± S.D. and are
representative of three separate experiments.
|
|
 |
DISCUSSION |
Although different G protein-coupled receptors may act by similar
mechanisms such as activation of PLC, raising
Ca2+i, and activation of PKC, each receptor has
unique properties that include its location in the cell, its cycling
and processing by the cell, and the set of proteins with which it
interacts. These unique characteristics allow it to have discrete
functions such as activation of specific effector enzymes. For example, in MDCK cells,
1-adrenergic and P2U
receptors activate protein kinase C and MAP kinases but activate
cPLA2 by different mechanisms (31, 32). Similarly, although
the CaR activates PLC, increases Ca2+i, and
activates PKC and MAP kinases, it activates cPLA2 by
mechanisms that are particular to the CaR. To characterize the
mechanism by which the CaR activates PLA2 and determine
which type of PLA2 was activated by it, we studied
activation of PLA2 in HEK-293 cells that stably express the
CaR or a nonfunctional mutant form of the receptor, CaR R796W, to
control for nonspecific effects of the receptor ligands. We determined
that the CaR activated cPLA2 via a novel pathway that
involves G
q, PLC, Ca2+, calmodulin, and
CaMK. In contrast to many other receptors, activation of the ERKs was
not required for CaR-dependent activation of
cPLA2.
Both cPLA2 and iPLA2 could be activated by G
protein-dependent signaling systems, and both enzymes could
be activated simultaneously (33). To identify the type of
PLA2 that is activated by the CaR, we tested the ability of
AACOCF3, an arachidonic acid analogue that inhibits both
cPLA2 and iPLA2, and BEL, a "suicide
substrate" that is specific for iPLA2 to inhibit
CaR-stimulated [3H]AA release from our cells (1). We
found that AACOCF3 inhibited all of the CaR-stimulated
activity with an IC50 of 25 µM, which is
comparable to its IC50 in other systems (12). BEL, used at a concentration that was considerably higher than that required to
inhibit completely iPLA2 in other cell types (10 µM), had no effect on CaR-stimulated [3H]AA
release and inhibited LPS-stimulated [3H]AA release from
RAW 264.7 cells (12, 34). Additionally, we expressed human
cPLA2 with the CaR and demonstrated that CaR-stimulated [3H]AA release increased in parallel with increasing
amounts of expressed cPLA2. These results indicate that in
our experimental system, CaR-stimulated [3H]AA release is
a result of increased cPLA2 activity.
Activation of cPLA2 by the CaR requires stimulation of PLC
activity which could occur through members of the G
i
and/or G
q families. Pretreatment of cells with pertussis
toxin had a minimal effect on the ability of the CaR to stimulate PLC
and cPLA2, indicating that the CaR does not activate
cPLA2 through members of the G
i family.
Consequently, the CaR must act through a pertussis toxin-insensitive pathway probably involving members of the G
q family, but
possibly through another mechanism. Chemical inhibitors of the
G
q family do not exist, so we used a protein that
inhibits G
q activity, RGS4, to demonstrate specifically
that the CaR activates PLC, and consequently cPLA2, through
G
q family proteins. RGS4 inhibits activation of
G
i and G
q family proteins by stimulating
their GTPase activity but acts preferentially on the G
q
family (20, 27). The fact that CaR-stimulated PLC activity is almost
completely inhibited by expression of the CaR in cells that stably
express RGS4 demonstrates that the CaR must act through
G
q family members to activate PLC and consequently
cPLA2.
A rise in Ca2+i is required for activation of
cPLA2 by most receptor-mediated mechanisms. Activation of
the CaR results in a significant rise in Ca2+i via
release from intracellular stores and influx across the cell membrane.
We attempted to buffer intracellular Ca2+ with BAPTA but
surprisingly found that BAPTA treatment resulted in a slight increase
in [3H]AA release. This result can be rationalized by the
recent finding that BAPTA may lower Ca2+i to the
point that Ca2+ influx is stimulated (35, 36). However, by
adding EGTA to the medium for 30 min, we were able to inhibit
CaR-stimulated cPLA2 activity by ~80%, demonstrating
dependence on Ca2+o. Treatment of the cells for
this period with 5-10 mM EGTA presumably also reduced, but
did not completely deplete, the Ca2+ in the intracellular
stores so that the CaR-stimulated rise in Ca2+ due to
release of the intracellular stores was reduced. Consequently, we
cannot determine whether the 20% of CaR-stimulated cPLA2
activity that remained was due to incomplete inhibition of the
Ca2+i signal or a Ca2+-independent mechanism.
In many cell types, cPLA2 is activated by a p42/44
ERK-dependent pathway. Phosphorylation of cPLA2
by ERK in vitro at Ser-505 results in a change in its
mobility in gels (retardation) and increased activity, whereas
phosphorylation by PKA or PKC does not result in a mobility shift or
increased activity. Similarly, in studies of cPLA2 from
intact cells, p42/44 ERK reduces the mobility of cPLA2 in
gels and increases its activity. Mutation of Ser-505 results in an
enzyme that is not activated by and that does not undergo a mobility
shift in response to ERK, PMA, ATP, or thrombin (14). The PKC (phorbol
ester)-induced activation of cPLA2 and CaMK-stimulated
activation of cPLA2 appear to occur via activation of the
ERKs by PKC or CaMK (13, 14, 31, 32, 37).
However, some receptors may activate cPLA2 by mechanisms
that are independent of, or only partially dependent on, the ERK pathway (32, 38, 39). P2U receptors in MDCK-D1
cells activate cPLA2 by PKC- and ERK-dependent
pathways (32). In MDCK cells, bradykinin stimulates cPLA2
activity by a mechanism that is independent of both PKC
and ERK but
that is dependent on tyrosine phosphorylation (39). In renal proximal
tubule cells and breast carcinoma cells, ERK can be activated by
PLA2-dependent AA metabolites rather than ERK-activating PLA2, reversing the conventional
relationship (40, 41).
Our results indicate that the CaR activates cPLA2 by a
pathway that is independent of ERK activation. Inhibition of CaR
stimulated ERK activation by three methods; the two chemical inhibitors
of the ERK activator MEK (PD-98059 and U0126) and expression of a dominant negative form of MEK, MEKK97R, inhibited
CaR-stimulated ERK activation but had no effect on CaR-stimulated
cPLA2 activation. Stimulation of cPLA2 by the
CaR also appears to be partially dependent on PKC (~50% inhibition with calphostin, a general PKC inhibitor or down-regulation of PKC with
PMA pretreatment).
Activation of cPLA2 by the CaR is inhibited to a similar
degree by W-7, a competitive calmodulin inhibitor, and KN-93, a CaMK inhibitor, implicating CaM and one of the CaMK isoforms in the regulatory pathway. The most likely scenario is that a
receptor-stimulated rise in Ca2+i activates CaM
which activates CaMK, and that CaMK activates cPLA2 by a
direct or indirect mechanism. CaM has many potential targets, including
kinases (CaMK, myosin light chain kinase, and phosphorylase kinase),
phosphatases (calcineurin), cytoskeletal proteins (MAP-2, Tau, fodrin,
and neuromodulin), cyclic nucleotide phosphodiesterases, adenylyl
cyclases, and Ca2+ transporters (pumps and channels), any
or all of which could contribute to cPLA2 activation (29).
However, the CaMK inhibitor KN-93 inhibited 90% of CaR-stimulated
cPLA2 activity indicating a significant role for
CaMK.
The extent to which KN-93 inhibited CaR-stimulated cPLA2
activity in our studies is comparable to that found by others (13, 30,
42) studying CaMK-dependent processes in whole cell
systems. In our studies with 3 h of exposure to KN-93, we found an
IC50 of ~6.3 µM and ~90% inhibition of
CaR-stimulated cPLA2 activity at 25 µM KN-93.
The IC50 of KN-93 for purified CaMKII is 370 nM, and it is specific up to ~30 µM (30).
In PC12 cells with a 3-day incubation, the IC50 value for
KN-93-dependent inhibition of tyrosine hydroxylase activity
was ~2 µM, and pretreatment of KCl or acetyl choline-stimulated intact PC12 cells with 10 µM KN-93 for
1 h resulted in a 60-80% inhibition of tyrosine hydroxylase
activity (30). Consequently, although we cannot exclude the possibility that some other CaM-dependent process participates in
activation of cPLA2 by the CaR, we think that it is most
likely that the CaR acts through CaM to activate one of the isoforms of
CaMK which then activates cPLA2. At this point, we cannot
determine which CaMK isoform is involved or if CaMK acts via a direct
or indirect mechanism.
Our studies demonstrate that the CaR activates cPLA2 by a
novel pathway in which Ca2+, CaM, and CaMK are of principal
importance, and ERKs are not involved. CaMK is involved in activation
of cPLA2 by other G protein-coupled receptors, but CaMK
activates the ERKs which then activate cPLA2. Clearly,
receptor-dependent mechanisms that do not involve the ERKs
can activate cPLA2 (39, 40). The different mechanisms used
by various receptors to activate cPLA2 may reflect
selective cellular localization of signaling proteins with a particular receptor.