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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
calcium-sensing receptor; G proteins; RGS proteins
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 Gq and G
i (2). In HEK-293 cells that overexpress the CaR, it couples to members of the G
i family to inhibit the activity of adenylyl cyclase and activate ERK (11, 21, 29), and members of the G
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
-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
12/13 or via a novel heterotrimeric G protein-independent pathway.
Recent reports show that expression of constitutively active G12/13 strongly stimulates phospholipase D (PLD) activity and that expression of dominant negative Rho family proteins inhibits it (38, 43). PLD is a known G
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
12/13 signaling, none of these has been shown to directly regulate PLD activity via G
12/13 (13, 47, 54). The signaling pathways regulated by G
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 G12/13 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
i and G
q and p115RhoGEF-RGS for G
12/13) to attenuate their respective signaling pathways, we found that the CaR couples to G
12/13 to regulate PLD.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1252) 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 252249 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 1224 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 4060% confluent cultures and incubated in a 32°C incubator for 3648 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 1215 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 1215 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 4050 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 Gi subunits in the cells that were pretreated with pertussis toxin, cellular proteins from these samples were resolved with 11% SDS-PAGE containing a linear 48 M urea gradient and processed for immunoblotting with a polyclonal anti-G
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.
|
|
|
|
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of pertussis toxin on CaR-stimulated PLD activity. Heterotrimeric G proteins, including Gi, G
q, and G
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
i subunits and G
q subunits (2, 11, 20, 29). To determine whether members of the G
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
i family proteins by pertussis toxin was documented using 48 M urea gradient SDS-PAGE. Figure 3A shows the slower migrating ADP-ribosylated band of G
i in MDCK cells that were pretreated with pertussis toxin. Earlier reports showed that the CaR stimulated ERK via a G
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
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 Gi and G
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
i or G
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 G12/13 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
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 1252) in MDCK cells. The p115RhoGEF-RGS construct should attenuate G
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
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
In MDCK cells that express an endogenous CaR, it has been shown to activate Gi and G
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 Gi and G
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
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
12/13. The G
12/13 proteins appear to be involved in processes that affect cytoskeletal structure, cell motility, and growth (1, 30, 35). G
13 has clearly been shown to activate PLD via Rho using expression of a constitutively active G
13 mutant and a dominant negative Rho (38). Inhibition of G
12 action with either expression of Lsc-RGS (an RGS protein for G
12) (43) or incubation with anti-G
12 antibodies inhibited carbachol and angiotensin-stimulated PLD activity (48), respectively.
One of the few downstream proteins that has been identified for the G12/13 family is p115RhoGEF, a protein that contains both an RGS domain serving as a GAP for the G
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
12/13, but Rho can also be activated via G
i and G
q family members (1618, 50, 51). Consequently, it was important to demonstrate not only that the CaR activates PLD via Rho but that G
i, G
q, and other signaling systems are not involved and that G
12/13 is required for the process.
To demonstrate that G12/13 is involved in the stimulation of PLD by the CaR, we had to exclude contributions from G
i and G
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
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
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
i subunits were not involved in PLD activation by the CaR. The G
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
q activation by expression of RGS4, a GAP for both the G
i and G
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
i or G
q and indicate that the CaR may act via G
12/13 or possibly another heterotrimeric G protein-independent mechanism.
Like the Gq family, chemical inhibitors do not exist for the G
12/13 family of proteins. In an approach similar to that used for our studies of the CaR and G
q, we expressed a truncated form of p115RhoGEF (amino acids 1252) that contains the RGS box (amino acids 45161) that acts as a GAP function for G
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
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 1252) has the same GAP activity for G
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
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
12/13 to stimulate PLD activity in MDCK cells, demonstrating that the CaR can couple to G
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 G12/13 and Rho and demonstrate clearly that the CaR is capable of coupling to G
12/13. This pathway does not require input from G
i or G
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
i to regulate inhibition of adenylyl cyclase and ERK activation, G
q to stimulate PLC and PLA2, an undefined G protein (not G
i or G
q) to stimulate PI 4-kinase, and G
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
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
12/13, Rho, and PLD.
![]() |
ACKNOWLEDGMENTS |
---|
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 |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Arthur JM, Collinsworth GP, Gettys TW, Quarles LD, and Raymond JR. Specific coupling of a cation-sensing receptor to G protein -subunits in MDCK cells. Am J Physiol Renal Physiol 273: F129F135, 1997.
3. Blankenship KA, Williams JJ, Lawrence MS, McLeish KR, Dean WL, and Arthur JM. The calcium-sensing receptor regulates calcium absorption in MDCK cells by inhibition of PMCA. Am J Physiol Renal Physiol 280: F815F822, 2001.
4. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, and Hebert SC. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366: 575580, 1993.[CrossRef][ISI][Medline]
5. Brown EM and MacLeod RJ. Extracellular calcium sensing and extracellular calcium signaling. Physiol Rev 81: 239297, 2001.
6. Brown EM, Vassilev PM, and Hebert SC. Calcium ions as extracellular messengers. Cell 83: 679682, 1995.[ISI][Medline]
7. Bruce JI, Yang X, Ferguson CJ, Elliott AC, Steward MC, Case RM, and Riccardi D. Molecular and functional identification of a Ca2+ (polyvalent cation)-sensing receptor in rat pancreas. J Biol Chem 274: 2056120568, 1999.
8. Chung Sekiya F JK, Kang HS, Lee C, Han JS, Kim SR, Bae YS, Morris AJ, and Rhee SG. Synaptojanin inhibition of phospholipase D activity by hydrolysis of phosphatidylinositol 4,5-bisphosphate. J Biol Chem 272: 1598015985, 1997.
9. Cockcroft S. Signalling roles of mammalian phospholipase D1 and D2. Cell Mol Life Sci 58: 16741687, 2001.[ISI][Medline]
10. Conricode KM, Brewer KA, and Exton JH. Activation of phospholipase D by protein kinase C. Evidence for a phosphorylation-independent mechanism. J Biol Chem 267: 71997202, 1992.
11. De Jesus Ferreira MC, Helies-Toussaint C, Imbert-Teboul M, Bailly C, Verbavatz JM, Bellanger AC, and Chabardes D. Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca2+. J Biol Chem 273: 1519215202, 1998.
12. Donaldson JG, Finazzi D, and Klausner RD. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature 360: 350352, 1992.[CrossRef][ISI][Medline]
13. Dorsam RT, Kim S, Jin J, and Kunapuli SP. Coordinated signaling through both G12/13 and Gi pathways is sufficient to activate GPIIb/IIIa in human platelets. J Biol Chem 277: 4758847595, 2002.
14. Exton JH. Phospholipase D-structure, regulation and function. Rev Physiol Biochem Pharmacol 144: 194, 2002.[ISI][Medline]
15. Fensome A, Whatmore J, Morgan C, Jones D, and Cockcroft S. ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils. J Biol Chem 273: 1315713164, 1998.
16. Fleming IN, Elliott CM, and Exton JH. Differential translocation of Rho family GTPases by lysophosphatidic acid, endothelin-1, and platelet-derived growth factor. J Biol Chem 271: 3306733073, 1996.
17. Gohla A, Harhammer R, and Schultz G. The G-protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho. J Biol Chem 273: 46534659, 1998.
18. Gohla A, Offermanns S, Wilkie TM, and Schultz G. Differential involvement of G12 and G
13 in receptor-mediated stress fiber formation. J Biol Chem 274: 1790117907, 1999.
19. Grignani F, Kinsella T, Mencarelli A, Valtieri M, Riganelli D, Grignani F, Lanfrancone L, Peschle C, Nolan GP, and Pelicci PG. High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein. Cancer Res 58: 1419, 1998.[Abstract]
20. Handlogten ME, Huang C, Shiraishi N, Awata H, and Miller RT. The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gq-dependent ERK-independent pathway. J Biol Chem 276: 1394113948, 2001.
21. Handlogten ME, Shiraishi N, Awata H, Huang C, and Miller RT. Extracellular Ca2+-sensing receptor is a promiscuous divalent cation sensor that responds to lead. Am J Physiol Renal Physiol 279: F1083F1091, 2000.
22. Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, and Bollag G. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by G13. Science 280: 21122114, 1998.
23. Helms JB and Rothman JE. Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature 360: 352354, 1992.[CrossRef][ISI][Medline]
24. Honda A, Nogami M, Yokozeki T, Yamazaki M, Nakamura H, Watanabe H, Kawamoto K, Nakayama K, Morris AJ, Frohman MA, and Kanaho Y. Phosphatidylinositol 4-phosphate 5-kinase is a downstream effector of the small G protein ARF6 in membrane ruffle formation. Cell 99: 521532, 1999.[ISI][Medline]
25. Huang C and Cabot MC. Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacylglycerol by a phospholipase C pathway. J Biol Chem 265: 1485814863, 1990.
26. Huang C, Handlogten ME, and Miller RT. Parallel activation of phosphatidylinositol 4-kinase and phospholipase C by the extracellular calcium-sensing receptor. J Biol Chem 277: 2029320300, 2002.
27. Huang C, Hepler JR, Gilman AG, and Mumby SM. Attenuation of Gi- and Gq-mediated signaling by expression of RGS4 or GAIP in mammalian cells. Proc Natl Acad Sci USA 94: 61596163, 1997.
28. Kifor O, Diaz R, Butters R, and Brown EM. The Ca2+-sensing receptor (CaR) activates phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryonic kidney (HEK293) cells. J Bone Miner Res 12: 715725, 1997.[ISI][Medline]
29. Kifor O, MacLeod RJ, Diaz R, Bai M, Yamaguchi T, Yao T, Kifor I, and Brown EM. Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells. Am J Physiol Renal Physiol 280: F291F302, 2001.
30. Knust E. G protein signaling and asymmetric cell division. Cell 107: 125128, 2001.[ISI][Medline]
31. Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bollag G, and Sternweis PC. p115 RhoGEF, a GTPase activating protein for G12 and G
13. Science 280: 21092111, 1998.
32. Li X, Mumby SM, Greenwood A, and Jope RS. Pertussis toxin-sensitive G protein -subunits: production of monoclonal antibodies and detection of differential increases on differentiation of PC12 and LA-N-5 cells. J Neurochem 64: 11071117, 1995.[ISI][Medline]
33. Liu KP, Russo AF, Hsiung SC, Adlersberg M, Franke TF, Gershon MD, and Tamir H. Calcium receptor-induced serotonin secretion by parafollicular cells: role of phosphatidylinositol 3-kinase-dependent signal transduction pathways. J Neurosci 23: 20492057, 2003.
34. Morris AJ and Malbon CC. Physiological regulation of G protein-linked signaling. Physiol Rev 79: 13731430, 2001.
35. Neves SR, Ram PT, and Iyengar R. G protein pathways. Science 296: 16361639, 2002.
36. Pertile P, Liscovitch M, Chalifa V, and Cantley LC. Phosphatidylinositol 4,5-bisphosphate synthesis is required for activation of phospholipase D in U937 cells. J Biol Chem 270: 51305135, 1995.
37. Pi M, Spurney RF, Tu Q, Hinson T, and Quarles LD. Calcium-sensing receptor activation of rho involves filamin and rho-guanine nucleotide exchange factor. Endocrinology 143: 38303838, 2002.
38. Plonk SG, Park SK, and Exton JH. The -subunit of the heterotrimeric G protein G13 activates a phospholipase D isozyme by a pathway requiring Rho family GTPases. J Biol Chem 273: 48234826, 1998.
39. Pollak MR, Brown EM, Chou YH, Hebert SC, Marx SJ, Steinmann B, Levi T, Seidman CE, and Seidman JG. Mutations in the human Ca2+-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75: 12971303, 1993.[ISI][Medline]
40. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE, and Seidman JG. Autosomal dominant hypocalcaemia caused by a Ca2+-sensing receptor gene mutation. Nat Genet 8: 303307, 1994.[ISI][Medline]
41. Powner DJ, Hodgkin MN, and Wakelam MJ. Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKC in RBL-2H3 cells. Mol Biol Cell 13: 12521262, 2002.
42. Rizzo MA and Romero G. Pharmacological importance of phospholipase D and phosphatidic acid in the regulation of the mitogen-activated protein kinase cascade. Pharmacol Ther 94: 3550, 2002.[CrossRef][ISI][Medline]
43. Rumenapp U, Asmus M, Schablowski H, Woznicki M, Han L, Jakobs KH, Fahimi-Vahid M, Michalek C, Wieland T, and Schmidt M. The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G12 but not Gq-type G proteins: regulators of G proteins as tools to dissect pertussis toxin-resistant G proteins in receptoreffector coupling. J Biol Chem 276: 24742479, 2001.
44. Santy LC and Casanova JE. Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J Cell Biol 154: 599610, 2001.
45. Singer WD, Brown HA, and Sternweis PC. Regulation of eukaryotic phosphatidylinositol-specific phospholipase C and phospholipase D. Annu Rev Biochem 66: 475509, 1997.[CrossRef][ISI][Medline]
46. Skippen A, Jones DH, Morgan CP, Li M, and Cockcroft S. Mechanism of ADP ribosylation factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in HL60 cells. J Biol Chem 277: 58235831, 2002.
47. Sugimoto N, Takuwa N, Okamoto H, Sakurada S, and Takuwa Y. Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform. Mol Cell Biol 23: 15341545, 2003.
48. Ushio-Fukai M, Alexander RW, Akers M, Lyons PR, Lassegue B, and Griendling KK. Angiotensin II receptor coupling to phospholipase D is mediated by the betagamma subunits of heterotrimeric G proteins in vascular smooth muscle cells. Mol Pharmacol 55: 142149, 1999.
49. Vargas-Poussou R, Huang C, Hulin P, Houillier P, Jeunemaitre X, Paillard M, Planelles G, Dechaux M, Miller RT, and Antignac C. Functional characterization of a calcium-sensing receptor mutation in severe autosomal dominant hypocalcemia with a Bartter-like syndrome. J Am Soc Nephrol 13: 22592266, 2002.
50. Vogt S, Grosse R, Schultz G, and Offermanns S. Receptor dependent RhoA activation in G12/G13 deficient cells: genetic evidence for an involvement of Gq/G11. J Biol Chem Paper in Press M304570200.
51. Voss M, Weernink PA, Haupenthal S, Moller U, Cool RH, Bauer B, Camonis JH, Jakobs KH, and Schmidt M. Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras/Ral signaling cascade. J Biol Chem 274: 3469134698, 1999.
52. Walker SJ and Brown HA. Specificity of Rho insert-mediated activation of phospholipase D1. J Biol Chem 277: 2626026267, 2002.
53. Wells CD, Gutowski S, Bollag G, and Sternweis PC. Identification of potential mechanisms for regulation of p115 RhoGEF through analysis of endogenous and mutant forms of the exchange factor. J. Biol. Chem. 276: 2889728905, 2001.
54. Yuan J, Slice LW, Gu J, and Rozengurt E. Cooperation of Gq, Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid. J Biol Chem 278: 48824891, 2003.