The Ca2+-sensing receptor couples to G{alpha}12/13 to activate phospholipase D in Madin-Darby canine kidney cells

Chunfa Huang, Kristine M. Hujer, Zhenzhen Wu, and R. Tyler Miller

Division of Nephrology, Department of Medicine, Case Western Reserve University, and Louis Stokes Veteran Affairs Medical Center, Cleveland, Ohio 44106

Submitted 2 June 2003 ; accepted in final form 29 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Ca2+-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: G{alpha}i to inhibit the activity of adenylyl cyclase and activate ERK, G{alpha}q to stimulate phospholipase C and phospholipase A2, and G{beta}{gamma} to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to G{alpha}12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known G{alpha}12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaRWT) or the nonfunctional mutant CaRR796W as a negative control, prelabeled these cells with [3H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [3H]phosphatidylethanol (PEt). The formation of [3H]PEt increased in a time-dependent manner in the cells that overexpress the CaRWT but not the CaRR796W. Treatment of the cells with C3 exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of G{alpha}12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate G{alpha}i or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for G{alpha}i and G{alpha}q, and a p115RhoGEF construct containing the RGS domain for G{alpha}12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaRWT inhibited extracellular Ca2+-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to G{alpha}12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of G{alpha}i and G{alpha}q. This suggests that the CaR may regulate cytoskeleton via G{alpha}12/13, Rho, and PLD.

calcium-sensing receptor; G proteins; RGS proteins


THE CA2+-SENSING RECEPTOR (CaR), a member of the G protein-coupled receptor (GPCR) superfamily, was first cloned in 1993 from a bovine parathyroid gland cDNA library (4). The physiological function of the CaR is to regulate the secretion of parathyroid hormone and cell proliferation in the parathyroid glands (5) and to modulate Na+, Cl, Ca2+, and H2O transport in the kidney (6). Human genetic studies have demonstrated that mutations in the CaR gene lead to two different types of human disease: inactivating mutations cause familial hypocalciuric hypercalcemia (the heterozygous state) and neonatal severe hyperparathyroidism (the homozygous state) (39), and activating mutations cause autosomal dominant hypocalcemia (40, 49). In addition to sensing extracellular Ca2+ ([Ca2+]o) under physiological conditions, the CaR also responds to Pb2+, Cd2+, and Fe2+ (7, 21). The fact that these heavy metal compounds are environmental toxins suggests that the receptor may be involved in the regulation of cell and tissue injury.

A number of studies have addressed the intracellular signaling pathways regulated by the CaR. In Madin-Darby canine kidney (MDCK) cells that express the CaR endogenously, activation of the CaR with [Ca2+]o and Gd3+ leads to the increasing incorporation of [32P]azidoanalide-GTP into G{alpha}q and G{alpha}i (2). In HEK-293 cells that overexpress the CaR, it couples to members of the G{alpha}i family to inhibit the activity of adenylyl cyclase and activate ERK (11, 21, 29), and members of the G{alpha}q family to stimulate phospholipase C and phospholipase A2 (20). One recent report also showed that in parafollicular cells, phosphatidylinositol (PI) 3-kinase can be stimulated by a CaR-G{beta}{gamma}-dependent signaling pathway (33). We recently demonstrated that the CaR stimulates PI 4-kinase via a Rho-dependent and Gi- and Gq-independent pathway in HEK-293 cells (26). These data indicate that the CaR may act via G{alpha}12/13 or via a novel heterotrimeric G protein-independent pathway.

Recent reports show that expression of constitutively active G{alpha}12/13 strongly stimulates phospholipase D (PLD) activity and that expression of dominant negative Rho family proteins inhibits it (38, 43). PLD is a known G{alpha}12/13 target and is one of the key enzymes in the generation of lipid second messengers. Although a subset of GPCRs such as the AT1, LPA, M3 muscarinic, sphingosine 1-phosphate, and thrombin receptors couple to G{alpha}12/13 signaling, none of these has been shown to directly regulate PLD activity via G{alpha}12/13 (13, 47, 54). The signaling pathways regulated by G{alpha}12/13 are not well understood, but they play a crucial role in agonist-stimulated cellular responses such as morphology, motility, endocytosis, exocytosis, and ion channels activity (1, 35).

On the basis of the formation of phosphatidylbutanol, the CaR seems to activate PLD in HEK-293 cells that overexpress the CaR and parathyroid cells (28), but the molecular mechanisms of its regulation remain largely undefined. We (26) and others (37) have demonstrated that the CaR controls a Rho-dependent signaling pathway in HEK-293 cells. To test the hypothesis that the CaR couples to G{alpha}12/13 to activate Rho and PLD, we investigated CaR-stimulated PLD activity in MDCK cells that stably overexpress the wild-type CaR (CaRWT) and the nonfunctional mutant CaR (CaRR796W). Using appropriate radiolabeling and treatment with different inhibitors or cooverexpression of regulators of G protein signaling (RGS4 for G{alpha}i and G{alpha}q and p115RhoGEF-RGS for G{alpha}12/13) to attenuate their respective signaling pathways, we found that the CaR couples to G{alpha}12/13 to regulate PLD.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. All chemicals were purchased from Sigma Chemicals or Fisher Scientific unless specified otherwise. Brefeldin A, wortmannin, phorbol 12-myristate 13-acetate (PMA), and C3 exoenzyme were purchased from BIOMOL Research (Plymouth Meeting, PA). U-73122 was obtained from Calbiochem-Novachem (La Jolla, CA). G-418 sulfate, hygromycin, and cell culture reagents were purchased from Life Technologies. [3H]palmitic acid (43 Ci/mmol) and myo-[2-3H(N)]inositol (22 Ci/mmol) were purchased from PerkinElmer Life Sciences. FuGene 6 reagent was supplied by Roche Diagnostics. SuperSignal West Pico chemiluminescent substrate was obtained from Pierce. The monoclonal anti-CaR and anti-HA antibodies were described previously (20). The rabbit anti-c-Myc antibody (A-14) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal antibody against Rho (-A, -B, -C) was supplied by Upstate Biotechnology (Lake Placid, NY). The polyclonal antibody (B-087) against G{alpha}i was described previously (32). The polyclonal antibodies against total ERK and phospho-ERK were purchased from New England Biolabs (Beverly, MA).

Construction of plasmids. The cDNAs encoding human CaRWT and CaRR796W were subcloned into the XbaI and KpnI sites of pMaRXTMIVfNeo from CaRWTpcDNA3 and CaRR796WpcDNA3. The RGS4-MycpCB6+ was digested with BamHI, blunt-ended, and digested with EcoRI and then ligated into pMaRXTMIVfHygro with EcoRI and HpaI. The cDNA encoding the human p115RhoGEF RGS domain (amino acids 1–252) in pCMV5 were generously provided by Dr. Paul Sternweis (University of Texas Southwestern Medical Center, Dallas, TX; Ref. 53). PCR was used to generate the HA-tagged p115RhoGEF RGS domain constructs. The sense PCR primer began with a HindIII site, followed by codons encoding the nine amino acids of the HA epitope tag (underlined) and the seven amino acids of the NH2 terminus of the p115RhoGEF: 5'-TTTAAGCTT ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT GAA GAC TTC GCC CGA GGG GCG-3'. The anti-sense PCR primer was 3'-GTG-GAATTC TCA GTT CCC CAT CAC-3' coding for the amino acids 252–249 of p115 RhoGEF, a stop codon, an EcoRI site and three-bp tail. The PCR product was purified and ligated into the HindIII and EcoRI sites of pMaRXIVfHygro.

Cell culture and transfection. The MDCK cells that stably co-overexpress the CaRWT or the CaRR796W with vector, the HA-tagged p115RhoGEF-RGS, or the Myc-tagged RGS4 were established by retroviral infection (19). Briefly, DNA constructs were transiently transfected into phoenix 293 packaging cells using the FuGene 6 reagent and incubated at 37°C for 12–24 h. The cultures were removed to a 32°C incubator overnight, and the medium was collected into 15-ml tubes, centrifuged at 1,500 rpm for 5 min to remove cell debris, and filtered through a 0.45-µm syringe filter. Polybrene was added to a final concentration of 5 µg/ml. To infect the MDCK cells, equal amounts of fresh medium and filtered packaging cell medium were added to 40–60% confluent cultures and incubated in a 32°C incubator for 36–48 h. Infected cells were selected with 0.2 mg/ml G-418 and hygromycin in DMEM supplemented with 10% fetal calf serum, 5 U/ml of penicillin, and 5 µg/ml of streptomycin. Overexpression of the desired proteins was documented by immunoblotting. The experiments were performed with several different clones, and similar results were obtained with all clones.

Cell prelabeling, treatment, and PLD activity measurement. MDCK cells that stably overexpress the CaR (CaRWT or CaRR796W) and RGS proteins (Myc-tagged RGS4 or HA-tagged p115 RhoGEF-RGS) were cultured in 12-well plates prelabeled with ~5 µCi/ml myo-[3H]inositol in 0.5 ml of 10% fetal bovine serum DMEM for 48 h or six-well plates prelabeled with ~1 µCi/ml [3H]palmitic acid in 1 ml of serum-free DMEM for 24 h. Measurement of [3H]IP3 release was described previously (26). To measure [3H]phosphatidylethanol ([3H]PEt) production, the cultures were equilibrated in serum-free DMEM containing 20 mM HEPES (pH 7.4) in the presence or absence of inhibitors for 1 h and then incubated in serum-free DMEM containing 2% ethanol at 37°C for the time periods indicated in the presence or absence of 5 mM CaCl2. For ADP-ribosylation experiments and PMA downregulation, MDCK cells that stably overexpress the CaRWT or the CaRR796W were prelabeled with [3H]palmitic acid for 1 day and incubated in the presence or absence of 100 ng/ml pertussis toxin during the last 12–15 h or 100 nM PMA for 20 h. The cells were then incubated in serum-free DMEM containing 2% ethanol in the presence or absence of 5 mM CaCl2 for 2 h. In the experiments with C3 exoenzyme treatment, MDCK cells that stably overexpress the CaRWT or the CaRR796W were prelabeled with [3H]palmitic acid for 1 day and incubated in the presence or absence of 20 µM digitonin and 100 ng/ml C3 exoenzyme during the last 12–15 h. The cells were then incubated in medium containing 2% ethanol in the presence or absence of 5 mM CaCl2 for 2 h. The reaction was terminated by adding 0.6 ml of cold 1% HCl in methanol, and total cellular lipids were extracted with chloroform, 1% HCl in methanol and water (6:6:5.5, vol/vol). [3H]PEt was resolved from the total cellular lipids by thin-layer chromatography (TLC) and identified by comigration with a commercial standard in a solvent system containing chloroform/methanol/ammonium hydroxide (65: 25:3, vol/vol) (25). The standard was visualized with iodine vapor, and the area corresponding to PEt was scraped into scintillation vials and quantitated with liquid scintillation spectrometry.

Cellular fractionation and immunoblotting. To detect the expression of the CaR, the stable MDCK cloned cells were harvested in SDS-PAGE loading buffer and boiled for 5 min. The samples were subjected to 6% SDS-PAGE and processed for immunoblotting using a monoclonal anti-CaR antibody (26) and visualized with enhanced chemiluminescence. In experiments that determined the effect of RGS proteins on CaR-activated PLD, we used the same radiolabeled cells from which total lipids were extracted for immunoblotting. After the organic phases were transferred to glass tubes for the [3H]PEt assay, the aqueous phases were carefully removed from the tubes. The protein pellets were washed with acetone once, and the pellets were dried with air. The pellet was dissolved in 40–50 µl of SDS-PAGE loading buffer. The pH of the samples was adjusted to neutral. They were boiled for 5 min, subjected to 11% SDS-PAGE, and processed for immunoblotting with the appropriate antibodies. To detect ADP ribosylation of G{alpha}i subunits in the cells that were pretreated with pertussis toxin, cellular proteins from these samples were resolved with 11% SDS-PAGE containing a linear 4–8 M urea gradient and processed for immunoblotting with a polyclonal anti-G{alpha}i antibody (B-087) (32). This system optimizes separation of ADP ribosylated from unmodified protein. To determine active ERK, MDCK cells that stably express the CaRWT or the CaRR796W were starved in 0.5% fetal bovine serum DMEM overnight, stimulated with 5 mM CaCl2 for 5 min, and harvested in a buffer containing 20 mM Tris·HCl, pH 6.8, 1% SDS, and 40% glycerol, and protein concentrations were determined using BCA with bovine serum albumin as the standard. Equal amounts of protein from lysates were analyzed by immunoblotting using polyclonal antibodies against total ERK and phospho-ERK. To demonstrate Rho translocation, the cells were starved, stimulated, and harvested in a buffer containing 20 mM HEPES, pH 7.5, 2 mM MgCl2, 1 mM EDTA, and protease inhibitors, and homogenized with 50 strokes of a Dounce homogenizer. Homogenates were centrifuged at 1,500 rpm for 10 min to yield a postnuclear pellet. The resultant supernatants were centrifuged at 15,000 rpm for 1 h at 4°C to yield particulate and soluble fractions. Protein concentrations were determined and equal amounts of protein from the particulate fractions were analyzed by immunoblotting using a polyclonal antibody against Rho (-A, -B, -C).

Data analysis. The data in Figs. 1 (points) and 2 (light or dark hatched bars) represent the means of three experiments performed with duplicate or triplicate samples and the error bars represent SE. The data were analyzed for significance using one-way repeated measures of ANOVA followed by Tukey's test for comparisons between the experimental groups shown in the figures. In Figs. 3, 4, 5, the bars (light or dark hatched) represent the means of two experiments performed with duplicate or triplicate samples, and the error bars represent the experimental range.



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Fig. 1. Extracellular Ca2+ ([Ca2+]o)-stimulated phospholipase D (PLD) activity in Madin-Darby canine kidney (MDCK) cells. A: expression of the Ca2+-sensing receptor (CaR) in MDCK cells. The parental MDCK cells (P) or MDCK cells stably overexpressing either the CaRR796W (Mut) or the CaRWT (WT) were lysed with loading buffer and processed for immunoblotting using the anti-CaR antibody. B: time course of [Ca2+]o-induced PLD activity measured as [3H]phosphatidylethanol ([3H]PEt) formation in MDCK cells. MDCK cells that stably overexpress the CaRWT or the CaRR796W were prelabeled with [3H]palmitic acid for 24 h and were treated with 5 mM CaCl2 for different periods of time. The total cellular lipids were extracted, and [3H]PEt was separated by TLC and quantitated using liquid scintillation counting. The data represent the average of 3 experiments performed with duplicate or triplicate samples. The values for [3H]PEt formation in the CaRWT cells were statistically different from those in the CaRR796W cells. *P < 0.05; **P < 0.01; NS, not significant by ANOVA.

 


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Fig. 2. Rho translocation and the effect of C3 exoenzyme on PLD activity in MDCK cells. A: translocation of Rho in [Ca2+]o-stimulated MDCK cells. MDCK cells that overexpress the CaRWT (WT) or the CaRR796W (Mut) were starved, stimulated, and then fractionated to obtain crude membranes. Equal amounts of membrane protein were subjected to 11% SDS-PAGE and processed for immunoblotting using an anti-Rho antibody. The experiment shown is representative of 3. B: effect of C3 exoenzyme on PLD activity. MDCK cells that stably overexpress the CaRWT or the CaRR796W were prelabeled with [3H]palmitic acid for 24 h and incubated in the presence of 20 µM digitonin with or without 100 ng/ml C3 exoenzyme during the last 12–15 h. Cells were then incubated in medium containing 2% ethanol in the presence or absence of 5 mM CaCl2 for 2 h. Total lipids were extracted, resolved by TLC, and quantitated with liquid scintillation spectrometry. The results represent the average of 3 experiments performed with duplicate samples. The values for [3H]PEt formation in the cells that express the CaRWT and that were treated with CaCl2 were statistically different from those in the cells that express the CaRR796W and that were treated with C3 exozyme and CaCl2. **P < 0.01; NS, not significant by ANOVA.

 


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Fig. 3. Effect of pertussis toxin on ERK activation and [3H]PEt formation in [Ca2+]o-stimulated MDCK cells. MDCK cells that stably overexpress the CaRWT (WT) or the CaRR796W (Mut) were prelabeled with [3H]palmitic acid for 24 h, the last 12–15 h with or without 100 ng/ml pertussis toxin (PTx), and then incubated in the presence or absence of 5 mM CaCl2 for 2 h. A: using 4–8 M urea gradient SDS-PAGE, the slower migrating ADP-ribosylated band of G{alpha}i in pertussis toxin-pretreated MDCK cells was identified by immunoblotting. The experiment shown is representative of 3. B: cells were treated, and cellular proteins were determined. Equal amounts of cellular proteins were subjected to 11% SDS-PAGE and processed for immunoblotting using an anti-phospho-ERK or anti-total ERK antibody. C: cells were extracted, the lipids were resolved by TLC, and [3H]PEt formation was quantitated by liquid scintillation counting. Data represent the average of 2 experiments performed with duplicate samples.

 


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Fig. 4. Effect of RGS4 on the formation of [3H]PEt in [Ca2+]o-stimulated MDCK cells. A: determination of RGS4 expression. MDCK cells that stably co-overexpress the CaRWT (WT) or the CaRR796W (Mut) with vector alone or RGS4 were lysed with loading buffer and processed for immunoblotting using the anti-Myc antibody. Cells were prelabeled with myo-[3H]inositol for 48 h or [3H]palmitic acid for 24 h and then incubated in the presence or absence of 5 mM CaCl2 for 5 min to measure [3H]IP3 release or for 2 h to measure [3H]PEt formation. Either the release of [3H]IP3 (B) or the formation of [3H]PEt (C) was quantitated by liquid scintillation counting. Data represent the average of 2 experiments performed with triplicate samples.

 


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Fig. 5. Effect of p115RhoGEF-RGS on the formation of [3H]PEt in [Ca2+]o-stimulated MDCK cells. A: MDCK cells that stably co-overexpress the CaRWT (W) or the CaRR796W (M) with either vector alone (V) or the p115 RhoGEF-RGS domain (R) were fractionated to obtain crude membranes and cytosol. Equal amounts of cellular proteins from the cytosolic or crude membrane fractions were subjected to 11% SDS-PAGE and processed for immunoblotting using an anti-HA antibody. B: these cells were prelabeled with [3H]palmitic acid for 24 h and then incubated in the presence or absence of 5 mM CaCl2 for 2 h. The formation of [3H]PEt (B) was quantitated by liquid scintillation counting. The expression of the p115RhoGEF-RGS domain was measured by immunoblotting using an antibody against the HA tag (A). Data represent the average of 2 experiments performed with triplicate samples.

 


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The CaR stimulates Rho to activate PLD. To evaluate CaR-stimulated PLD activity, we first investigated the effect of [Ca2+]o on the generation of [3H]PEt in MDCK cells that stably overexpress the CaRWT or the CaRR796W (shown in Fig. 1A). The cells were prelabeled with [3H]palmitic acid for 24 h and then incubated in serum-free DMEM containing 5 mM CaCl2 and 2% ethanol for various time periods. PLD activity was measured as the formation of [3H]PEt. Figure 1B illustrates that [Ca2+]o induced a time-dependent accumulation of [3H]PEt in MDCK cells that stably overexpress the CaRWT but not the CaRR796W. Similar results were also observed in the MDCK cells when CaR-stimulated [3H]choline release, also an index of PLD activity, was measured. Recently, we demonstrated that the CaR stimulates PI 4-kinase in HEK-293 cells via a Rho-dependent and Gi- and Gq-independent signaling pathway. In that study, C3 toxin blocked CaR-stimulated PI 4-kinase activity, and Rho was coimmunoprecipitated with the CaR using an anti-CaR antibody (26). All these data indicate that the Rho family of small G proteins can be activated by the CaR. Rho has been implicated in the activation of PLD in vitro by addition of recombinant RhoA and in vivo by hormone stimulation (9, 14, 42, 45). The activation of Rho requires its translocation from the cytosol to the plasma membrane (1618, 44). One recent report (37) showed that the CaR induces a marked increase in Rho membrane association. To assess Rho activation by the CaR, MDCK cells that stably overexpress the CaRWT and the CaRR796W were stimulated with 5 mM CaCl2 for 5 min and fractionated to yield crude membranes and cytosol. Equal amounts of membrane protein were processed for immunoblotting to assess the translocation of Rho. The results from Fig. 2A showed an increase in Rho membrane association by [Ca2+]o stimulation in MDCK cells that overexpress the CaRWT, but not the CaRR796W. C3 exoenzyme selectively ADP ribosylates Rho at asparagine-41, thereby blocking its function. We prelabeled MDCK cells that stably overexpress the CaRWT or the CaRR796W with [3H]palmitic acid, permeabilized them with 20 µM digitonin, incubated them in the presence or absence of 100 ng/ml C3 exoenzyme for 12–15 h, and then stimulated them with or without 5 mM CaCl2 for 2 h. The formation of [3H]PEt was measured to determine the effect of C3 exoenzyme on CaR-stimulated PLD activity. Figure 2B shows that C3 exoenzyme leads to reduced [3H]PEt formation. The blockade of CaR-stimulated PLD activity by C3 exoenzyme indicates that the CaR stimulates PLD activation via the Rho family of small G proteins.

Effect of pertussis toxin on CaR-stimulated PLD activity. Heterotrimeric G proteins, including G{alpha}i, G{alpha}q, and G{alpha}12/13, are the major upstream entities involved in agonist-induced Rho activation (1618, 37, 38, 4244, 50). The CaR couples to at least two G protein families: G{alpha}i subunits and G{alpha}q subunits (2, 11, 20, 29). To determine whether members of the G{alpha}i family are stimulated by the CaR to activate PLD, we prelabeled the CaR-expressing MDCK cells with [3H]palmitic acid for 24 h and pretreated the cells with 100 ng/ml of pertussis toxin or vehicle for the last 12 h of the prelabeling period and then incubated the cultures in the presence or absence of 5 mM CaCl2 for 2 h to stimulate PLD activity. ADP ribosylation of G{alpha}i family proteins by pertussis toxin was documented using 4–8 M urea gradient SDS-PAGE. Figure 3A shows the slower migrating ADP-ribosylated band of G{alpha}i in MDCK cells that were pretreated with pertussis toxin. Earlier reports showed that the CaR stimulated ERK via a G{alpha}i-mediated signaling pathway in HEK-293 cells (29). The active and total ERK1/2 were determined by immunoblotting using appropriate antibodies in Ca2+-stimulated MDCK cells that stably overexpress the CaRWT or the CaRR796W. The decrease in ERK1/2 phosphorylation by pertussis toxin treatment was only observed in CaRWT-expressing MDCK cells (Fig. 3B). When these cells were prelabeled with [3H]palmitic acid, the formation of [3H]PEt (PLD activity) was not significantly changed in the pertussis toxin-pretreated cells (Fig. 3C). These results demonstrate that the CaR does not signal through G{alpha}i to stimulate PLD activity.

Effect of RGS4 on CaR-stimulated PLD activity. Further assessment of heterotrimeric G protein-activated PLD activity by the CaR was carried out using stable co-overexpression of the CaR and RGS4, a regulator of G protein signaling that activates the GTPase activity of G{alpha}i and G{alpha}q (27). Either RGS4-Myc or empty vector was stably co-overexpressed in CaR-expressing MDCK cells. RGS4 protein expression was documented by immunoblotting with an anti-Myc antibody (Fig. 4A). In a similar study, we have shown that RGS4 attenuates CaR-Gq-phospholipase C signaling to reduce IP3 release in HEK-293 cells (20, 26). Here, we also observed the same effect of RGS4 on CaR-stimulated [3H]IP3 release in MDCK cells (Fig. 4B). To assess the effect of RGS4 on CaR-stimulated PLD activation, we prelabeled these cells with [3H]palmitic acid and then incubated them with 5 mM CaCl2 for 2 h to stimulate PLD activity, measured as the formation of [3H]PEt (Fig. 4C). Although RGS4 was easily detectable and [3H]IP3 was significantly reduced, it did not significantly alter CaR-stimulated PLD activity. Taken together, these results demonstrate that the CaR does not act through members of the G{alpha}i or G{alpha}q families to stimulate PLD.

Effect of p115RhoGEF-RGS on CaR-stimulated PLD activity. P115RhoGEF is a multifunctional protein that is a GTPase-activating protein (GAP) for the G{alpha}12/13 family of heterotrimeric G proteins and a guanine nucleotide exchange factor for small G protein Rho (22, 31, 53). To investigate whether the CaR is coupled to endogenous G{alpha}12/13 subunits in the regulation of PLD activity, we stably co-overexpressed the CaR (CaRWT or CaRR796W) with empty vector or a construct containing the p115RhoGEF RGS domain (amino acids 1–252) in MDCK cells. The p115RhoGEF-RGS construct should attenuate G{alpha}12/13 signaling based on in vitro assays of GAP activity (31, 53). Overexpression of the p115RhoGEF-RGS was documented by immunoblotting using a monoclonal anti-HA antibody against the HA epitope tag engineered into the protein, and it was detected in both membrane and cytosol fractions (Fig. 5A). We prelabeled these cells with [3H]palmitic acid, stimulated with [Ca2+]o, and the formation of [3H]PEt (Fig. 5B) was measured as CaR-stimulated PLD activity. Overexpression of the p115RhoGEF-RGS significantly diminished CaR-stimulated activation of PLD in MDCK cells. These results demonstrate that the CaR stimulates PLD activity via a G{alpha}12/13-mediated signaling pathway.

Effect of brefeldin A, wortmannin, U-73122, and PMA-downregulation on [Ca2+]o-induced PLD activity. Many other factors, including Arf, PIP2, and PKC, may also participate in the regulation of PLD activity (9, 14, 42, 45). The next series of experiments was conducted to determine the effect of Arf, PIP2, and PKC on CaR-stimulated PLD activity. MDCK cells express Arf1 and Arf6, which may be required for PLD activation (42, 44). Our recent work demonstrated that the CaR stimulates phospholipase C and PI 4-kinase to modulate the levels of cellular PIP2 (26). To examine the effect of Arf, PIP2, and PKC on CaR-activated PLD, [3H]palmitate-prelabeled MDCK cells that stably overexpress the CaRWT or the CaRR796W were pretreated with brefeldin A to block Arf activation (44), wortmannin to deplete PIP2 by inhibiting PI 4-kinase, U-73122 to cause the accumulation of PIP2 by inhibiting phospholipase C, and 100 nM PMA for 20 h to downregulate PKC, and then incubated with 5 mM CaCl2 for 2 h to stimulate PLD activity via the CaR. As measured with [3H]PEt formation, none of these pretreatments altered CaR-stimulated PLD activity (Table 1). To confirm the effect of chemical inhibitors, we measured the effect of U-73122 and wortmannin on CaR-stimulated [3H]IP3 release. Table 1 shows that both U-73122 and wortmannin significantly inhibit CaR-stimulated [3H]IP3 release. The 10-min, 100 nM PMA treatment stimulated [3H]PEt formation in MDCK cells (CaRR796W-expressing cells, control 100 ± 7; PMA 390 ± 21; CaRWT-expressing cells, control 100 ± 8; PMA 346 ± 20); however, PMA downregulation (100 nM PMA for 20 h pretreatment) followed by the same stimulation dramatically reduced the formation of [3H]PEt (CaRR796W-expressing cells, control 100 ± 8; PMA 95 ± 7; CaRWT-expressing cells, control 100 ± 3; PMA 105 ± 8). Although the chemical inhibitors behaved as expected, Arf, PKC, and the alteration of cellular PIP2 levels did not affect CaR-stimulated PLD activity.


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Table 1. Role of different signaling molecules in CaR-stimulated [3H]PEt or [3H]IP3 formation

 


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although activation of the CaR leads to stimulation of PLD in intact cells (28), the signaling pathways by which it does so remain undefined. Activation of G{alpha}i and G{alpha}q family members by the CaR has been clearly documented (2, 11, 20, 29), but activation of additional G protein {alpha}-subunit families such as the G{alpha}12/13 family has not been addressed. Our previous studies indicated that the CaR acts via a G{alpha}i- and G{alpha}q-independent mechanism to activate PI 4-kinase, suggesting that it could couple to G{alpha}12/13 (26). To determine whether the CaR could in fact act via G{alpha}12/13 and to define additional components of the signaling pathway(s) controlled by the CaR, we tested its ability to activate PLD, one of the few effectors or second messenger-generating enzymes that have been clearly linked to G{alpha}12/13 activation in CaR-overexpressed MDCK cells.

In MDCK cells that express an endogenous CaR, it has been shown to activate G{alpha}i and G{alpha}q (2) and to inhibit plasma membrane calcium ATPase (3). Our MDCK cells do not express CaR protein endogenously at a level that permits detection by immunoblot. In our MDCK cells that contain the expression vector or the CaRR796W mutant, there is a small amount of [Ca2+]o-stimulated PLD activity (similar in both cell lines) that could be due either to a low level of endogenous CaR expression or increased intracellular Ca2+ ([Ca2+]i) due to increased Ca2+ entry as a result of the increased [Ca2+]o (5 mM). In either case, this response is too small to study signaling pathway. The magnitude of the CaR-stimulated PLD response in our cells that overexpress the CaRWT is comparable to the response in the freshly isolated parathyroid cells and HEK-293 cells that overexpress the CaRWT in earlier studies (28). The differences in PLD activity between MDCK cell lines that overexpress the CaRWT and the CaRR796W are attributable to signaling by the CaR. Consequently, although our studies make use of cells in which the CaR has been expressed at a relatively high level, we feel that our system is relevant to the in vivo situation.

In contrast to the many receptors that couple to G{alpha}i and G{alpha}q family proteins (30, 34), relatively few receptors (AT1, LPA, M3 muscarinic, sphingosine 1-phosphate, and thrombin receptors) have been shown to couple to G{alpha}12/13 family members (13, 47, 54). Many of these studies have relied on demonstration of receptor-G protein coupling and have not analyzed signaling pathways controlled by a receptor via G{alpha}12/13. The G{alpha}12/13 proteins appear to be involved in processes that affect cytoskeletal structure, cell motility, and growth (1, 30, 35). G{alpha}13 has clearly been shown to activate PLD via Rho using expression of a constitutively active G{alpha}13 mutant and a dominant negative Rho (38). Inhibition of G{alpha}12 action with either expression of Lsc-RGS (an RGS protein for G{alpha}12) (43) or incubation with anti-G{alpha}12 antibodies inhibited carbachol and angiotensin-stimulated PLD activity (48), respectively.

One of the few downstream proteins that has been identified for the G{alpha}12/13 family is p115RhoGEF, a protein that contains both an RGS domain serving as a GAP for the G{alpha}12/13 family and a dbl homology (DH) domain for Rho guanine nucleotide exchange (22, 31, 43, 50). Although several members of the Rho family (Rho A, Rac1, and Cdc42) are capable of binding to and activating PLD (52), the CaR has been shown to activate Rho. We demonstrated the involvement of Rho in CaR signaling by showing inhibition of CaR-stimulated PLD activity by C3 exozyme, which specifically ADP ribosylates Rho (not Rac1 or Cdc42), and showing an increase in the membrane association of Rho in response to CaR activation. The activation of Rho by a GPCR is consistent with the receptor acting via G{alpha}12/13, but Rho can also be activated via G{alpha}i and G{alpha}q family members (1618, 50, 51). Consequently, it was important to demonstrate not only that the CaR activates PLD via Rho but that G{alpha}i, G{alpha}q, and other signaling systems are not involved and that G{alpha}12/13 is required for the process.

To demonstrate that G{alpha}12/13 is involved in the stimulation of PLD by the CaR, we had to exclude contributions from G{alpha}i and G{alpha}q family proteins, both of which are known to couple to the CaR (2, 11, 20, 29) and both of which are also capable of activating Rho (16, 50). Pertussis toxin ADP ribosylates G{alpha}i family proteins and prevents their activation by receptors. Pretreatment of cells with pertussis toxin did not alter the ability of the CaR to activate PLD but did alter the migration of G{alpha}i on protein gels (evidence of covalent modification) (32) and inhibited ERK activation. These results demonstrate that pertussis toxin has had its desired effect under the experimental conditions used (29) but that the G{alpha}i subunits were not involved in PLD activation by the CaR. The G{alpha}q family has no known chemical inhibitors, and because of redundancy, inhibition of expression with RNAi-based approaches is difficult. For these reasons, we chose to inhibit G{alpha}q activation by expression of RGS4, a GAP for both the G{alpha}i and G{alpha}q families (27). Overexpression of RGS4 had no effect on CaR-stimulated PLD activity but eliminated CaR-stimulated PLC activity (Fig. 4B, Refs. 20 and 26). These results demonstrate that the CaR activates PLD by a mechanism that does not involve either G{alpha}i or G{alpha}q and indicate that the CaR may act via G{alpha}12/13 or possibly another heterotrimeric G protein-independent mechanism.

Like the G{alpha}q family, chemical inhibitors do not exist for the G{alpha}12/13 family of proteins. In an approach similar to that used for our studies of the CaR and G{alpha}q, we expressed a truncated form of p115RhoGEF (amino acids 1–252) that contains the RGS box (amino acids 45–161) that acts as a GAP function for G{alpha}12/13 (31). P115RhoGEF is a complicated phosphoprotein with a number of defined and undefined functional domains, including RGS, DH, and pleckstrin homogoly (PH) domains. It acts as a GAP for G{alpha}12/13 and as a guanine nucleotide exchange factor for Rho. Consequently, results obtained with expression of the full-length p115RhoGEF could be difficult to interpret. In in vitro assays, the p115RhoGEF-RGS (amino acids 1–252) has the same GAP activity for G{alpha}12/13 as full-length p115RhoGEF (31), but a related construct that lacks the DH and PH domains does not bind or activate Rho (53). This construct is found at significant levels in both the particulate (membrane) and soluble (cytosolic) fractions when expressed in mammalian cells (Fig. 5A, Ref. 53). Overexpression of the p115RhoGEF-RGS, documented by immunoblotting, resulted in inhibition of ~65% of the CaR-stimulated PLD activity (Fig. 5B). Partial inhibition could be due to the affinity with which the expressed p115RhoGEF-RGS interacts with its target. The PH domain, absent in this construct, also appears to be important for membrane localization. Alternatively the p115RhoGEF-Rho pathway may be only partially responsible for the observed activation. In studies of others, dominant negative Rho only partially blocked the PLD1 activity stimulated by constitutively active G{alpha}13, and the activity was completely blocked by expression of dominant negative RhoA and Rac1 (38). It is possible that some other unknown protein(s) may be involved in CaR-stimulated PLD activity. In any case, the CaR acts via G{alpha}12/13 to stimulate PLD activity in MDCK cells, demonstrating that the CaR can couple to G{alpha}12/13.

Several other factors such as PIP2, PKC, and Arf have been identified that can regulate PLD (9, 14, 42, 45). PIP2 has a number of functions in the cell that include its function as a substrate for PLC, PI 3-kinase, and direct regulation of the activity of a number of proteins, including Trp channels, cytoskeletal proteins, and PLD. In a number of experimental systems and assays, PIP2 stimulates PLD activity. Addition of PIP2 to PLD in exogenous substrate assays in HL-60 cells stimulates membrane-bound PLD activity (45). In vitro, addition of synaptojanan, an inositol polyphosphate 5-phospahatase, inhibits PLD activity (8). In permeabilized U-937 cells, anti-PI 4-kinase antibodies reduce PLD activity (36). To assess the role of PIP2 in CaR-stimulated PLD activity in intact MDCK cells, we increased PIP2 levels using U-73122 to block metabolism of PIP2 by PLC and reduced PIP2 levels by treating the cells with wortmannin to block PI 4-kinase. In our previous studies, we demonstrated that in the presence of U-73122, activation of the CaR leads to significant accumulation of PIP2, whereas wortmannin inhibits its accumulation (26). Neither of these maneuvers affected CaR-stimulated PLD activity, indicating that PIP2 levels, to the extent we could manipulate them experimentally, are not important for activation of PLD by the CaR.

In earlier studies, evidence for activation of PLD by PKC was provided by treatment of intact cells with phorbol esters to stimulate PKC or specific inhibitors to block PKC (25) and incubation of cell membranes with purified rat brain PKC in a cell free assay (10). We tested this possibility by downregulating PKC with long-term phorbol ester treatment. The ability of PMA to activate PLD was lost with PKC downregulation; however, the activation of PLD by the CaR was maintained under in the absence of PKC downregulation (Table 1). These data indicate that the CaR does not require PKC for activation of PLD.

On the basis of reports that the small GTP-binding protein Arf can activate PLD in a variety of cell types (15, 41), we tested for a role of Arf in stimulation of PLD by the CaR. Arf can bind to PLD and activate it directly, or it may act to stimulate PI 4-P 5-kinase and increase PIP2 levels to stimulate PLD (24, 42, 46). We tested the effect of brefeldin A, a compound that inhibits GDP-GTP exchange at a concentration that inhibits Arf-dependent events in other systems (12, 23), on CaR-stimulated PLD activity. We found that brefeldin A had no effect on PLD activity in our system.

Our results demonstrate that the CaR activates PLD via a pathway that involves G{alpha}12/13 and Rho and demonstrate clearly that the CaR is capable of coupling to G{alpha}12/13. This pathway does not require input from G{alpha}i or G{alpha}q and is not affected by the increased or decreased PIP2 levels that we were able to achieve in our studies. Additionally, we found no contribution of PKC or Arf to activation of PLD by the CaR. The CaR couples to at least three distinct heterotrimeric G proteins to regulate different signaling systems, G{alpha}i to regulate inhibition of adenylyl cyclase and ERK activation, G{alpha}q to stimulate PLC and PLA2, an undefined G protein (not G{alpha}i or G{alpha}q) to stimulate PI 4-kinase, and G{alpha}12/13 to stimulate PLD. Each of these presumably parallel pathways may be subject to modulation by separate factors, making the signaling system controlled by the CaR complex. When and which signaling pathway(s) are regulated may depend on the cell types or tissues so that the CaR may not activate G{alpha}12/13 in all cell types and at various levels of expression. Finally, these results provide a mechanism by which the CaR could effect changes in the structure of the cytoskeleton of cells that we have observed (Huang C, unpublished observations). The CaR could regulate actin assembly, vesicle transport, and secretion via G{alpha}12/13, Rho, and PLD.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by grants from the American Heart Association (to C. Huang and R. T. Miller), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41726 and DK59985 (to R. T. Miller), the Rainbow Center for Childhood PKD, and the Leonard Rosenberg Research Foundation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. Huang, Div. of Nephrology, Dept. of Medicine, Case Western Reserve Univ., Louis Stokes Veteran Affairs Medical Center, 10701 East Blvd, 151W, Cleveland, OH 44106 (E-mail: cxh87{at}po.cwru.edu).

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.


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