The Ca2+-sensing Receptor Activates Cytosolic Phospholipase A2 via a Gqalpha -dependent ERK-independent Pathway*

Mary E. Handlogten, Chunfa Huang, Naoki Shiraishi, Hisataka Awata, and R. Tyler MillerDagger

From the Division of Nephrology, Department of Medicine, University of Florida, Gainesville, Florida 32610

Received for publication, August 11, 2000, and in revised form, November 20, 2000




    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Ca2+-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A2 (PLA2) by the Ca2+-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA2 activity was attributable to cytosolic PLA2 (cPLA2) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA2. No CaR-stimulated cPLA2 activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca2+ (Ca2+i), whereas inhibition of phospholipase C (PLC) with U73122 completely inhibited CaR-stimulated PLC and cPLA2 activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha q activity. CaR-stimulated cPLA2 activity was inhibited 80% by chelation of extracellular Ca2+ and depletion of intracellular Ca2+ with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca2+, calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEKK97R, had no effect on CaR-stimulated cPLA2 activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA2 via a Galpha q, PLC, Ca2+-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, Galpha i and Galpha 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, alpha 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 Galpha q, PLC, Ca2+i, CaM, and CaMK-dependent but ERK-independent signaling pathway.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



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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.



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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.



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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-beta in activation of cPLA2 by first demonstrating inhibition of CaR-stimulated [3H]AA release by U73122, an inhibitor of PLC-beta . Pretreatment of cells that express the CaR with U73122 eliminated the CaR-stimulated cPLA2 activity demonstrating that the CaR activates cPLA2 via PLC-beta (Fig. 4A). To confirm that U73122 inhibits PLC-beta activity, we measured CaR-stimulated IP3 production with and without U73122 (Fig. 4B), and we found that it was completely inhibited by U73122. PLC-beta can be activated by pertussis toxin-sensitive (Galpha i-dependent) or pertussis toxin-insensitive (presumably Galpha 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 alpha i family have a minimal role in stimulation of cPLA2 and PLC-beta by the CaR. To test specifically for a role for a Galpha q family member in the activation of PLC-beta 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 Galpha q family, accelerates their GTPase activity, and reduces their activation (20, 27). Fig. 4C shows that RGS4 eliminated CaR-stimulated PLC-beta activity. These results indicate that the CaR acts through a pertussis toxin-insensitive, Galpha q-dependent pathway to activate PLC, the products of which stimulate cPLA2.



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Fig. 4.   Role of PLC-beta and Galpha i and Galpha 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-beta 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).



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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.



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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.



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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.



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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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha 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 Galpha 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 Galpha i and/or Galpha 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 Galpha i family. Consequently, the CaR must act through a pertussis toxin-insensitive pathway probably involving members of the Galpha q family, but possibly through another mechanism. Chemical inhibitors of the Galpha q family do not exist, so we used a protein that inhibits Galpha q activity, RGS4, to demonstrate specifically that the CaR activates PLC, and consequently cPLA2, through Galpha q family proteins. RGS4 inhibits activation of Galpha i and Galpha q family proteins by stimulating their GTPase activity but acts preferentially on the Galpha 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 Galpha 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 PKCalpha 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.


    ACKNOWLEDGEMENT

We thank Ray Harris for helpful discussions.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK41726, the American Heart Association (to C. H.), and the National Kidney Foundation (to N. S. and H. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Division of Nephrology, Dept, of Medicine, Louis Stokes VAMC, Case-Western Reserve University, 10701 East Blvd., Cleveland, OH 44106. Tel.: 216-791-3800, ext. 4660; Fax; 216-421-3025.

Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M007306200


    ABBREVIATIONS

The abbreviations used are: CaR, extracellular Ca2+-sensing receptor; AACOCF3, arachidonyl trifluoromethyl ketone; BEL, bromoenol lactone; Ca2+i, intracellular Ca2+; Ca2+o, extracellular Ca2+; CaM, calmodulin; CaMK, calcium, calmodulin-dependent protein kinase; ERK, extracellular signal-regulated kinase; HA, influenza hemagglutinin antigen; HEK-293 cells, human embryonic kidney 293 cells; IP3, inositol-1,4,5 trisphosphate; LPS, lipopolysaccharide; MEK, mitogen-activated protein kinase kinase-ERK kinase; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; PKC, protein kinase C; iPLA2, Ca2+-insensitive PLA2; PMA, phorbol myristate acetate; RGS protein, Regulator of G protein Signaling protein; bp, base pairs; DAG, diacylglycerol; MDCK, Madin-Darby canine kidney cells; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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