G-protein-coupled Receptor Agonists Activate Endogenous Phospholipase Cϵ and Phospholipase Cβ3 in a Temporally Distinct Manner*

Phospholipase Cϵ (PLCϵ) is one of the newest members of the phosphatidylinositol-specific phospholipase C (PLC) family. Previous studies have suggested that G-protein-coupled receptors (GPCRs) stimulate phosphoinositide (PI) hydrolysis by activating PLCβ isoforms through Gq family G proteins and Gβγ subunits. Using RNA interference to knock down PLC isoforms, we demonstrate that the GPCR agonists endothelin (ET-1), lysophosphatidic acid (LPA), and thrombin, acting through endogenous receptors, couple to both endogenous PLCϵ and the PLCβ isoform, PLCβ3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist- and isoform-specific profiles. PLCβ3 is activated acutely within the first minute of ET-1, LPA, or thrombin stimulation but does not contribute to sustained PI hydrolysis induced by LPA or thrombin and accounts for only part of ET-1 sustained stimulation. PLCϵ, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLCϵ in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLCϵ and PLCβ3 additively inhibits PI hydrolysis, suggesting independent regulation of each isoform. Importantly, ubiquitination of inositol 1,4,5-trisphosphate receptors correlates with sustained, but not acute, activation of PLCϵ or PLCβ3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLCϵ and PLCβ3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLCβ3 is predominantly involved in acute and PLCϵ in sustained PI hydrolysis.

The phosphatidylinositol-specific phospholipase C (PLC) 2 family is a group of critical cellular signaling enzymes that hydrolyze phosphatidylinositol 4,5-bisphosphate (PI) to generate inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol, which increase the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) and activate protein kinase C, respectively (1,2). Eleven isoforms of PLC, representing five distinct, differentially regu-lated classes, have been identified: PLC␤1 to ␤4; PLC␥1 and ␥2; PLC␦1, ␦3, and ␦4; and PLC⑀ and PLC. PLC␤ is regulated by G-protein-coupled receptor (GPCR) activation of heterotrimeric G q family G-proteins and G␤␥ subunits. PLC␥ is regulated by tyrosine phosphorylation by receptor tyrosine kinases (e.g. epidermal growth factor and plateletderived growth factor) and nonreceptor tyrosine kinases (e.g. Src) activated by immunoglobulin and cytokines. Regulation of PLC␦ is less well understood but is probably regulated by changes in [Ca 2ϩ ] i , possibly downstream from activation of other PLC isoforms, and by high molecular weight G-protein, G h . PLC is also regulated by [Ca 2ϩ ] i (3).
PLC⑀ was discovered only recently and is the largest member of the PLC family (4 -7). PLC⑀ is regulated by the monomeric Ras (4,8,9) and Rho (9, 10) families, the heterotrimeric G12 family (5,9), and G␤␥ subunits (11). Consistent with its diverse regulation by G-proteins, studies utilizing overexpressed PLC⑀ suggest that this isoform is regulated by receptor tyrosine kinases and GPCRs. Receptor tyrosine kinase agonists, epidermal growth factor (9), and platelet-derived growth factor (8) have been shown to stimulate PLC⑀ through Ras and Rap. GPCRmediated activation of PLC⑀ can be subdivided into two groups. One group, ␤ 2 adrenergic, prostanoid, and muscarinic M 3 receptor agonists, has been proposed to stimulate PLC⑀ through Rap2B mediated by cAMPdependent activation of the Rap GTP exchange factor EPAC (12,13). The second group, lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and thrombin receptor agonists, activate PLC⑀ through G␣ 12/13 and Rap (9).
Accumulating evidence suggests that activation of PLC by hormones is more complex than a simple linear activation from receptor to one PLC isoform (1,2). For example, GPCR receptor activation of PLC␤ isoforms may lead to activation of PLC␦ isoforms through increased levels of [Ca 2ϩ ] i (14,15). Alternatively, ␣ 1B -adrenergic receptor activation has been shown to stimulate PLC␤ through G␣ q/11 and to a lesser extent PLC␦1 through G h (16). GPCRs have been shown to transactivate receptor tyrosine kinases (17,18) with potential activation of PLC␤ and PLC␥, respectively. In addition, angiotensin II activation of the GPCR AT 1 has been proposed to activate PLC␥1 (19) and possibly temporally activate PLC␤1 (Ͻ30 s) followed by PLC␥1 (Ͼ30 s) (20). Furthermore, recent studies suggest that PLC␤2 and PLC␦1 exist as a heterodimer that sequesters and inhibits PLC␦1 activity (21). Upon G␤␥ activation of PLC␤2, PLC␦1 is released, allowing the enzyme to hydrolyze its substrate. Thus, receptor activation of cellular PLC activity is probably the composite of a complex signaling network that involves the activation of multiple PLC isoforms. Since similar receptor agonists have been postulated to stimulate PLC⑀ and the classic isoforms, PLC␤ and PLC␥, it is possible that novel signaling pathways, that redefine current PLC dogma, participate in this network.
In the present studies, we used RNA interference to elucidate the physiologic regulation of PLC⑀ and the PLC network regulated by GPCR agonists in Rat-1 fibroblasts. Rat-1 fibroblasts are a classic model for examining hormonal regulation of downstream effectors, and we show that these cells are easily manipulated by RNA interference using retrovirally transduced short interfering hairpin RNAs to generate stable and almost complete knockdown of signaling proteins. The GPCR agonists, endothelin, LPA, and thrombin, have been shown to stimulate PLC in Rat-1 fibroblasts through endogenous receptors (22)(23)(24), and previous studies suggest that these agonists couple to PLC␤ isoforms (25)(26)(27). Here, we demonstrate for the first time that these GPCR agonists activate both endogenous PLC⑀ and PLC␤3 through endogenous receptors in Rat-1 fibroblasts. Activation of these isoforms, however, exhibits distinct agonist-and isoform-specific temporal profiles whereby PLC␤3 predominantly participates in acute and PLC⑀ sustained PLC activity. In addition, sustained activation of PLC⑀ is functionally correlated with IP 3 receptor ubiquitination, a protective process associated with sustained activation of PLC.
Tissue Culture-Rat-1 fibroblasts and all HEK293 cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For reconstitution studies, Rat-1 clonal cell lines were seeded into 24-well dishes at 8 ϫ 10 4 6 h prior to adenoviral transduction. Medium was then removed, and adenoviruses at the appropriate concentrations were applied in 250 l of complete Dulbecco's modified Eagle's medium for 24 h. Cells were then labeled for 18 -24 h in inositol-free, serum-free Dulbecco's modified Eagle's medium. For all other PLC assays, Rat-1 cells were seeded into 24-well dishes at 4 ϫ 10 4 , grown overnight, and labeled the following day for 18 -24 h in inositol-free, serum-free Dulbecco's modified Eagle's medium.
Silent mutations were introduced into rat PLC⑀, pcDNA-PLC⑀-FLAG, to generate pcDNA-PLC⑀SI#5. Mutations were introduced by site-directed mutagenesis using the sense primer, 5Ј-CAAAGCCAAATAcagtTACAGCATCC, to mutate TTCC to cagt (shown in lowercase type) (Quick Mutagenesis Kit, Stratagene, La Jolla, CA). The mutated region was sequenced and subcloned into the original plasmid.
The cDNA for PLC⑀SI#5 was subcloned into the pShuttle-CMV vector, and recombinant adenovirus was generated following the manufacturer's protocol for the Stratagene AdEasy Adenoviral Vector System (Stratagene).
Stable Retroviral Knockdown-To generate retroviruses, HEK293T cells were seeded into T75 flasks at 8 ϫ 10 6 the previous day. Cells were transfected with pSUPER-Retro siRNA constructs, pVPackECO, and pVPackGP plasmids by Ca 2 PO 4 precipitation. Viruses were harvested after 48 h by filtering medium and quick freezing on dry ice. Stocks were stored at Ϫ80°C. Viruses were titered by transducing Rat-1 cells with serial dilutions, killing untransduced cells with 3 g/ml puromycin, and counting remaining colonies.
Rat-1 fibroblasts were seeded in 12-well plates at 4 ϫ 10 4 . The following day, the conditioned medium was aspirated off, and 1 ml of viral supernatant and 8 g/ml Polybrene were applied to each well for 6 -8 h.
The virus was replaced with complete medium after incubation. Cells were expanded to T75 flasks the next day, and 3 g/ml puromycin was added to medium 48 h after transduction.
Adenovirus Production-Generation of primary adenovirus was performed according to the AdEasy Adenoviral Vector System (Stratagene) protocol, except that the transfected cultures were incubated for 18 days or until a visible cytopathic effect was observed. Viruses were amplified by several rounds of infection in AD293 cells. Viral titers were calculated by an immunoreactivity spot assay (30).
Phospholipase C Assay-The PLC assay was performed under two different conditions, 20 and 100 mM LiCl. Experiments using 20 mM Li ϩ were performed as previously described (4,9). Basically, agonists were added to the conditioned labeling medium brought to a final concentration of 20 mM LiCl, and the cells were incubated for 60 min unless indicated otherwise. Agonists were dissolved as follows: LPA, 0.1% bovine serum albumin in phosphate-buffered saline; thrombin, 0.1% PEG, 1 mg/ml bovine serum albumin in phosphate-buffered saline; and endothelin-1 (ET-1), double-distilled H 2 O.
To increase trapping of inositol phosphates (IPs), 100 mM LiCl has been used in Rat-1 cells (22,31). For experiments performed in 100 mM LiCl (Figs. 3-7), conditioned labeling medium was replaced with Krebs Buffer 1 (4.7 mM KCl, 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , 1.2 mM MgSO 4 , 10 mM HEPES, 1.3 mM CaCl 2 , 118 mM NaCl, pH 7.4, aerated with 5% CO 2 /95% O 2 for 10 min), and the Rat-1 cells were allowed to equilibrate for 60 min in a CO 2 incubator at 37°C. Prior to the addition of agonists, cells were preincubated in Krebs Buffer 2 (4.7 mM KCl, 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , 1.2 mM MgSO 4 , 10 mM HEPES, 1.3 mM CaCl 2 , 18 mM NaCl, 100 mM LiCl, pH 7.4, aerated with 5% CO 2 /95% O 2 for 10 min) for 10 min. The reaction was stopped by aspirating off the incubation medium and adding 375 l of ice-cold 50 mM formic acid. After 30 min of incubation on ice, samples were removed, neutralized with 125 l of 150 mM ammonium hydroxide, and diluted with 5 ml of ice-cold water. Total inositol phosphates were separated by column chromatography using AG 1-X8 200 -400 mesh, formate form, and quantitated by liquid scintillation counting. Protein determination (Bio-Rad DC Protein Assay Kit) and expression by Western blot analysis were determined in parallel wells carried through the PLC assay but without radioactivity. Steady state labeling of phosphatidylinositol was not significantly different between conditions. IP 3 Mass Assay-Change in IP 3 mass was determined as previously described (32). Rat-1 cells were seeded at a density of 1.9 ϫ 10 5 /6-well plate and then incubated in a 5% CO 2 incubator at 37°C for 24 h and then serum-starved for an additional 24 h. Incubations were initiated by the addition of agonist and were terminated by removing medium and adding 250 l of ice-cold 0.5 M trichloroacetic acid. After 2 h at 4°C, the 250 l was removed and mixed with 50 l of 10 mM EDTA and 250 l of Freon/N-trioctylamine (1:1), vortexed, incubated at 4°C for 15 min, and centrifuged (16,000 ϫ g for 5 min at 4°C). A portion of the aqueous phase (150 l) was then removed and neutralized with 75 l of 25 mM NaHCO 3 , and IP 3 concentration was determined with a radioreceptor assay as described (33).
IP 3 Receptor Ubiquitination-IP 3 receptor ubiquitination was measured essentially as previously described (34). Rat-1 cells were grown to near confluence in 15-cm diameter dishes and were serum-starved for 15 h. For reconstitution studies, cells were transduced 6 h after seeding for 24 h with the indicated adenoviruses prior to serum starvation. Cells were then preincubated with the proteasome inhibitor bortezomib and exposed to stimuli. After removing culture medium, cells were solubilized by adding lysis buffer (50 mM Tris-base, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, 10 M pepstatin, 0.2 M soybean trypsin inhibitor, 1 mM dithiothreitol, pH 8) directly to the monolayers, followed by vigorous scraping. After 30 min at 4°C, lysates were clarified by centrifugation (16,000 ϫ g for 10 min at 4°C), and IP 3 R1 was immunoprecipitated by incubating overnight with anti-IP 3 R1 and Protein A-Sepharose CL-4B. Immunocomplexes were washed thoroughly with lysis buffer, resuspended in gel loading buffer, electrophoresed, and probed in immunoblots with anti-ubiquitin and anti-IP 3 R1.
Calculations and Statistical Analysis-Percentage change induced by knockdown of PLC⑀ or PLC␤3 compared with random control cell lines (percentage change of siRNARan) was determined to summarize effects of PLC isoform knockdown. To calculate this value, basal levels (activity without added GPCR agonist) were subtracted from stimulated levels. Activity in PLC⑀ or PLC␤3 cell lines was then divided by the activity in random siRNA-treated control cells, and from this value percentage change was calculated.
Paired or unpaired Student's t test, Tukey-Kramer multiple comparisons test, and one sample t test were performed where appropriate. A value of p Ͻ 0.05 was considered significant.

Serum and G-protein-coupled Receptor Agonists Are Coupled to
Endogenous PLC⑀ in Rat-1 Fibroblasts-To find a cell line in which GPCR agonists couple to endogenous PLC⑀, various cell lines were screened by Western blotting, and Rat-1 fibroblasts were determined to express high levels of PLC⑀. Rat-1 fibroblasts are a classic cell line to study hormonal regulation of PLC, and the GPCR agonists endothelin (ET-1) and thrombin have been shown to stimulate PLC in this cell line (22)(23)(24). To determine whether these agonists activate PLC⑀, RNA interference was used to knock down endogenous PLC⑀. Eight siRNAs targeting PLC⑀ were generated, which knocked down PLC⑀ with different efficiencies. Of these, three (siRNAPLC⑀#1, siRNAPLC⑀#3, and siRNAPLC⑀#5) were used to generate stable cell populations that knock down PLC⑀ in Rat-1 fibroblasts by 53-97% (Fig. 1B). Random sequences of these siRNAs were used as controls. Knockdown of PLC⑀ did not affect the expression of the other PLC isoforms present in this cell line, PLC␤3, PLC␥1, or PLC␦1 (Fig. 1B); expression of G-proteins, G 12 , G 13 , Rho, Ras, or Rap (data not shown); or cell growth (data not shown). Fig. 1A shows the effect of knocking down PLC⑀ on fetal bovine serum (FBS), ET-1, and thrombin-stimulated PLC activity. With increasing knockdown, agonist-stimulated PLC activity was increasingly inhibited compared with three different random siRNA control cell populations. The two most effective siRNAs, siRNAPLC⑀#1 and siRNAPLC⑀#5, significantly inhibited FBS-stimulated PI hydrolysis by 39 and 74%, respectively, ET-1 by 30 and 69%, and thrombin by 69 and 94%. Similar results were obtained in an independently generated set of stable cell lines (data not shown). Significant inhibition by at least two distinct siRNAs suggests that the observed inhibition was not due to off-target effects. Moreover, uncoupling was not due to siRNA-induced nonspecific activation of interferon, since an increase in STAT-1 phosphorylation was not observed in these stable cell populations (data not shown). In addition, the comparable agonist response in each of the three random cell lines (Fig. 1A) suggests that the inhibition was unlikely to be due to cell population variability induced by retroviral transduction and stable cell population selection. Taken together, these studies demonstrate that FBS, ET-1, and thrombin are coupled to PLC⑀ in Rat-1 fibroblasts.
G-protein-coupled Receptor Agonists Are Coupled to PLC␤3 in Rat-1 Fibroblasts-Classically, endothelin and thrombin are thought to activate PLC␤ isoforms through G q family G-proteins and G␤␥ subunits (25,27). We found that Rat-1 fibroblasts express high levels of PLC␤3 (Fig. 2B), but the other three PLC␤ isoforms, PLC␤1, -2, and -4, were not detected (data not shown). Because knockdown of PLC⑀ did not completely inhibit stimulation of PLC by these agonists, PLC␤3 was knocked down to determine whether these agonists couple to this PLC isoform. Seven siRNAs were screened, and the three most effective, siRNAPLC␤3#2, siRNAPLC␤3#3, and siRNAPLC␤3#5, were used to generate stable cell populations. These siRNAs knocked down PLC␤3 by 88 -96% (Fig. 2B). Fig. 2A shows that knocking down PLC␤3 with each of these siRNAs significantly inhibited ET-1-and thrombin-stimulated PLC activity. The two most effective siRNAs, siRNAPLC␤3#3 and siRNAPLC␤3#5, significantly inhibited ET-1-stimulated PI hydrolysis by 44 and 39%, respectively, and thrombin by 66 and 69%. FBS stimulation, on the other hand, was less affected by knockdown of PLC␤3 and was only significantly inhibited by 32% with siRNAPLC␤3#3 but not by siRNAPLC␤3#5. Thus, in addition to being coupled to PLC⑀, ET-1 and thrombin are also coupled to PLC␤3 in Rat-1 fibroblasts, consistent with previous studies that have suggested that these G q and G i familycoupled GPCR agonists activate PLC␤ isoforms in these cells. FBS, however, appears to predominantly regulate PLC⑀.
Distinct Temporal Activation of PLC␤3 and PLC⑀-Because knockdown experiments demonstrated that ET-1 and thrombin are coupled to both PLC␤3 and PLC⑀, the kinetics of the hormonal regulation of each isoform was examined to determine its unique contribution to total cellular PLC activity. In addition, LPA has been shown to stimulate PLC activity in Rat-1 fibroblasts (35), and its temporal regulation of PLC⑀ and PLC␤3 was also determined. For these experiments, a set of stable cell populations was generated with the most effective PLC-specific siRNAs, siRNAPLC⑀#5 and siRNAPLC␤3#3, and a random siRNA control. To increase knockdown, cells were transduced twice with retrovirus, which increased knockdown of each PLC isoform to greater than 99% compared with a random siRNA control (Fig. 3D). In addition, initial studies demonstrated that 20 mM Li ϩ was not sufficient to block degradation of sustained IP generation (data not shown). Lithium is a noncompetitive inhibitor of inositol monophosphatase, and therefore high concentrations are required to inhibit degradation of low concentrations of inositol phosphates (IPs). Thus, the concentration of Li ϩ was increased to 100 mM as previously described in these cells (22,31). Fig. 3A shows the temporal effect of knocking down PLC⑀ or PLC␤3 on FBS-, ET-1-, LPA-, and thrombin-stimulated total IP accumulation compared with a random control. FBS and each agonist effectively increased total inositol phosphate accumulation. The stimulation by ET-1, LPA, and thrombin was biphasic with an initial acute response (1 min) followed by a sustained increase (3-60 min). FBS stimulation was monophasic with no significant acute response. As shown above (Figs. 1 and 2), knockdown of PLC⑀ or PLC␤3 inhibited serum-, ET-1-, and thrombin-stimulated PLC activity. In addition, LPA stimulation was also inhibited. Fig. 3, however, shows that knockdown of PLC⑀ or PLC␤3 had differential effects on the temporal stimulation induced by these agonists.
This was most apparent for LPA and thrombin, which showed a crossover at 3 min in the temporal activation profiles of the PLC⑀ or PLC␤3 knockdown cell lines (Fig. 3A). PLC⑀ knockdown had no significant effect on the acute stimulation by LPA or thrombin (Fig. 3B); however, it markedly inhibited the sustained response to these agonists by ϳ80% at 60 min (Fig. 3C). When trapped IPs at 1 min are subtracted from later time points, knockdown of PLC⑀ effectively abolished the sustained stimulatory response. Conversely, PLC␤3 knockdown inhibited LPA and thrombin acutely by ϳ50% but had no effect on sustained stimulation. Thus, LPA and thrombin predominantly activate PLC␤3 during acute stimulation and PLC⑀ during sustained stimulation.
Similarly, acute stimulation of PI hydrolysis by ET-1 was significantly inhibited by knockdown of PLC␤3 and the sustained stimulation by knockdown of PLC⑀. However, differences were noted. Whereas knockdown of PLC␤3 inhibited LPA or thrombin stimulation by ϳ50%, ET-1 was only inhibited by 20% (Fig. 3B). Furthermore, in contrast to LPA and thrombin, knockdown of PLC⑀ only inhibited the sustained response by 44% and PLC␤3 inhibited the sustained response by 27% (Fig. 3C). Thus, whereas the activation profiles of these PLC isoforms by ET-1 are similar to LPA and thrombin, agonist-dependent differences exist.
Effect of PLC⑀ and PLC␤3 Knockdown on Acute PI Hydrolysis-Since there appeared to be a differential effect of PLC⑀ or PLC␤3 knockdown on acute stimulation of PI hydrolysis, a shorter time course was performed to better define the boundaries of the acute stimulatory component. In addition, changes in total IP accumulation were correlated with IP 3 generation. Fig. 4 shows the effect of knocking down PLC⑀ or PLC␤3 on GPCR agonist-stimulated total IP accumulation and IP 3 mass. ET-1, LPA, and thrombin effectively stimulated total IP accumulation acutely, and the responses were near maximal by 30 s (Fig. 4A). Knockdown of PLC␤3, but not PLC⑀, inhibited this acute stimulation (Fig. 4A) by 30 -60% (Fig. 4C).
Changes in total IP accumulation correlated with changes in IP 3 mass. ET-1, LPA, and thrombin markedly stimulated IP 3 generation, which peaked at 15 s and decreased to levels modestly above basal levels by 90 s (Fig. 4B). Knockdown of PLC␤3, but not PLC⑀, inhibited this IP 3 generation by 30 -50% (Fig. 4D), comparable with inhibition of total inositol phosphate accumulation (Fig. 4C). These studies confirm the selective role of PLC␤3 during the acute GPCR stimulation of PI hydrolysis.
Because sustained IP 3 levels are low, quantitation of the effects of PLC isoform knockdown by measuring IP 3 is not as sensitive as measuring total IP accumulation in the presence of Li ϩ . Therefore, changes in the Percentage knockdown of PLC⑀ relative to random controls, determined by densitometric measurements of at least three experiments, is shown. mass of IP 3 at sustained time points were not determined except for the effect of PLC⑀ knockdown on ET-1-stimulated PI hydrolysis, which appeared elevated at 90 s (Fig. 4B). In this set of experiments, PLC⑀ knockdown significantly inhibited the sustained ET-1 stimulation of IP 3 measured at 20 min by 90 Ϯ 8% from 75 pmol/mg protein to 9 Ϯ 6 (p Ͻ 0.02; n ϭ 3), consistent with total IP measurements and a role for PLC⑀ in sustained IP 3 formation.
Dose-and Agonist-dependent, Differential Effects of PLC⑀ and PLC␤3 Knockdown on Acute and Sustained PLC Activity-To further assess the effects of PLC⑀ and PLC␤3 knockdown on agonist-stimulated acute and sustained PLC activity and to determine whether lower agonist concentrations would differentially affect coupling to either isoform, a set of experiments was performed with increasing concentrations of agonist, and total IP accumulation was determined. Acute stimulation was determined by measuring IP accumulation at 1 min after agonist addition. Cells were preincubated with Li ϩ for 10 min. For sustained stimulation, agonist was added similarly, but Li ϩ was not added until 3 min after agonist addition, and the experiment was terminated at 60 min. This effectively measures sustained PLC activity from greater than 3 min (time for Li ϩ to block IP degradation) to 60 min; IPs formed at times less than 3 min are not trapped, because Li ϩ is not present. Fig. 5 shows dose responses to ET-1, LPA, and thrombin and the effect of knocking down PLC⑀ or PLC␤3 on acute (1-min) and sustained (3-60-min) IP accumulation. ET-1 stimulated acute PLC activity with an EC 50 of ϳ20 nM (Fig. 5A) and sustained activity with an EC 50 of 0.4 nM (Fig. 5B), ϳ50-fold more potent than acute. At 1 nM, sustained stimulation was near maximal, whereas there was almost no acute stimulation. At all stimulatory doses, there was no shift in contribution of PLC⑀ or PLC␤3 to total PLC activity; PLC␤3 knockdown inhibited acute and PLC⑀ and PLC␤3 knockdown both inhibited sustained PI hydrolysis.
Similarly, thrombin stimulated acute PI hydrolysis with an EC 50 of 200 pM (Fig. 5A) and sustained Ͼ50-fold more potently with an EC 50 of 3 pM (Fig. 5B). At 10 pM, sustained PLC activity was maximal with very little acute stimulation. At all stimulatory concentrations, acute stimulation was inhibited by PLC␤3 knockdown but not PLC⑀ knockdown (Fig. 5A), in contrast to sustained stimulation, which was almost completely abrogated with PLC⑀ knockdown and not affected by PLC␤3 knockdown (Fig. 5B).
The dose response to LPA was somewhat more complicated. Both acute and sustained stimulation appeared biphasic with an initial EC 50 of 30 -100 nM for acute (Fig. 5A) and sustained stimulation (Fig. 5B) followed by a second phase with an EC 50 of 0.6 -3 M. The EC 50 of the first phase is comparable with known LPA receptors, whereas the second is possibly mediated by a low affinity LPA receptor that appears important for mitogenesis but has yet to be identified (26). Acute stimulation was inhibited by ϳ50% at all stimulatory concentrations by knockdown of PLC␤3 but not affected by knockdown of PLC⑀ (Fig. 5A). In contrast, knockdown of PLC␤3 had no effect on sustained stimulation. The first phase of the sustained stimulation, however, was entirely dependent on PLC⑀, since knockdown of PLC⑀ completely inhibited PI hydrolysis (Fig. 5B). The second sustained phase, on the other hand, was accompanied by a component that was not inhibited by PLC⑀ or PLC␤3 knockdown, suggesting that another PLC isoform is activated at high LPA concentrations. Reconstitution and Overexpression of PLC⑀-To further examine the role of PLC⑀ in sustained PI hydrolysis, a PLC⑀ construct, AdPLC⑀#5, was generated with silent mutations in the region targeted by siRNAPLC⑀#5 to prevent siRNA-mediated degradation. Transduction of Rat-1 cells reduced PLC activity for unknown reasons, but the agonist stimulatory profiles remained intact. Fig. 5, A and C, show that when PLC⑀ is reconstituted to levels present in random siRNA-treated cells (Fig. 5, B and E), the profiles of LPA (Fig. 5A) and thrombin (Fig. 5C) stimulation of sustained IP accumulation are restored. These studies confirm that the observed effects of knocking down PLC⑀ in the siRNAPLC⑀#5 cell line on the sustained agonist-stimulatory profiles are specific and not due to off-target effects of the siRNA.
In addition, Fig. 5D shows that overexpressing PLC⑀ selectively enhances the sustained increase in IP accumulation stimulated by thrombin. In this set of experiments, a random cell line was transduced with LacZ and a PLC⑀ knockdown cell line with increasing amounts of adenovirus expressing PLC⑀ (Fig. 5E). Overexpressing PLC⑀ slightly increased basal, unstimulated IP accumulation from 13.4 Ϯ 1.0 cpm/g protein in AdLacZ-treated cells to 17.4 Ϯ 3.6 and 21.8 Ϯ 0.4 for cells moderately (multiplicity of infection 20), and highly (multiplicity of infection 60) overexpressing PLC⑀, respectively. However, the -fold stimulation acutely at 1 min was not different. On the other hand, the sustained PI hydrolysis was markedly potentiated. At 30 and 60 min, IP accumulation in the moderately overexpressing cells was 1.6-and 2.2fold above the control stimulation (-fold stimulation of the PLC⑀-overexpressing cell line/-fold stimulation of the Ran#6 LacZ-expressing cell line), respectively, and in the highly expressing cells it was 3.6-and 5.1-fold, respectively. Thus, overexpressing PLC⑀ has no effect on acute thrombin stimulation but markedly potentiates sustained stimulation, consistent with the knockdown experiments.
Double Knockdown of PLC⑀ and PLC␤3-To determine whether the activations of PLC⑀ and PLC␤3 are independent, both PLC⑀ and PLC␤3 were knocked down, and agonist-stimulated PLC activity was compared with a random siRNA-treated cell line. A set of stable cell populations was generated in which PLC⑀ and PLC␤3 were knocked down Ͼ99 and 92%, respectively, compared with a random control (Fig. 6B). Fig. 6A shows the stimulatory response to ET-1, LPA, and thrombin in these cell lines. In addition, the stimulatory response observed in single knockdown cell lines is shown. For each agonist, IP accumulation was inhibited to a similar extent as the additive knockdown of the single knockdown cell lines. This would suggest that PLC⑀ and PLC␤3 are regulated independently by these GPCR agonists.

Sustained but Not Acute PLC Activation Correlates with IP 3 Receptor Ubiquitination-Previous studies have demonstrated that GPCR activation of PLC can induce down-regulation of IP 3 receptors (IP 3 Rs)
through the ubiquitin-proteasome pathway (28,36,37), and we have shown that ET-1 stimulates IP 3 R ubiquitination and down-regulation in Rat-1 fibroblasts (38). Since sustained PI hydrolysis appears to be required for this process (28,32,37,39), we reasoned that knockdown of PLC isoforms coupled to sustained PI hydrolysis should inhibit IP 3 R ubiquitination. Fig. 8 shows the effect of knocking down PLC⑀ or PLC␤3 on type 1 IP 3 R ubiquitination stimulated by ET-1 or thrombin. Knock- down of PLC⑀ markedly inhibited ET-1-stimulated ubiquitination and completely blocked the effects of thrombin (Fig. 8, A and B). In additional experiments, inhibition of ET-1-stimulated ubiquitination was reversed by adenovirus-mediated reconstitution of PLC⑀, as in Fig. 6 but not with a catalytically inactive mutant, H1433L PLC⑀, consistent with the lipase function of PLC⑀ mediating agonist-induced IP 3 R ubiquitination (Fig. 8C). Similarly, knockdown of PLC␤3 partially inhibited ET-1stimulated ubiquitination, although to a lesser extent than PLC⑀ knockdown and consistent with differences in inhibition of total IP accumulation (see Fig. 3C). In contrast, knockdown of PLC␤3 had no effect on thrombin-stimulated ubiquitination (Fig. 8, A and B), which only inhibited acute PI hydrolysis. These studies show a direct correlation between inhibition of agonist-stimulated sustained PI hydrolysis and IP 3 R ubiquitination (see Figs. 3C and 8B) and that PLC⑀ is predominant in mediating sustained PI hydrolysis.

DISCUSSION
ET-1, LPA, and thrombin previously have been shown to stimulate cellular PLC activity (25)(26)(27). Classically, these and other hormones acting through GPCRs have been proposed to activate PLC␤ isoforms through G q family G-proteins and G␤␥ subunits (25)(26)(27). In addition, recent studies have raised the possibility that these agonists could activate PLC␥ isoforms through transactivation of receptor tyrosine kinase receptors (18,40). The discovery of PLC⑀, however, raises the possibility that these agonists might activate this novel PLC family member. PLC⑀ has been shown to be regulated by multiple G-proteins, including Ras (4,8,9), Rho (9, 10), and G 12 (5,9) family G proteins and by G␤␥ subunits (11). Since ET-1, LPA, and thrombin receptors couple to G q , G i , and G 12 family G proteins (25)(26)(27) and have been shown to activate Ras (18, 40 -42) and Rho (43), these agonists could potentially activate PLC␤ isoforms and/or PLC⑀, changing the view of how these GPCR agonists regulate cellular PI hydrolysis. To determine whether these agonists couple to PLC␤ isoforms and/or PLC⑀, we identified Rat-1 fibroblasts as a cell line that expresses both of these PLC family members and examined the effects of knocking down specific isoforms on agonist-stimulated PI hydrolysis.
Our studies show that ET-1, LPA, and thrombin, acting through endogenous GPCRs, couple to endogenous PLC⑀ in Rat-1 fibroblasts. This is the first demonstration that these agonists physiologically couple to PLC⑀. In addition, and consistent with previous paradigms (1,2,(25)(26)(27), we also show that these diverse agonists activate endogenous PLC␤3. Thus, under physiologic conditions, ET-1, LPA, and thrombin dually regulate both PLC⑀ and PLC␤3 in Rat-1 fibroblasts.
GPCR agonist regulation of these PLC isoforms, however, is not a simple, simultaneous activation of PLC⑀ and PLC␤3. Importantly, we demonstrate that the stimulation of these PLC isoforms is temporally distinct. Our studies show that ET-1, LPA, and thrombin activate PLC␤3 acutely (peak at 15 s; duration ϳ1 min) but not PLC⑀. In contrast, ET-1, LPA, and thrombin activate PLC⑀ during sustained PI hydrolysis (Ͼ30 s to at least 60 min). This distinct temporal activation of PLC⑀ and PLC␤3 is most apparent for LPA, doses Յ1 M, and thrombin, which couple to PLC␤3 acutely and almost exclusively to PLC⑀ during sustained stimulation. Consistent with this observation, overexpression of PLC⑀ had no effect on acute but markedly increased sustained agonist-stimulated PI hydrolysis. ET-1 was similarly coupled to PLC␤3 acutely but, in addition to activating PLC⑀ during the sustained phase, also partly activated PLC␤3, indicating an agonist dependence for the temporal activation of these isoforms. Interestingly, a difference in the activation of PLC by these agonists is consistent with an earlier study that noted ET-1 and LPA regulate PLC activity by distinct mech- anisms in Rat-1 fibroblasts (44). Overall, however, PLC␤3 predominantly accounts for acute and PLC⑀ for sustained PLC responses. Interestingly, in vascular smooth muscle cells, angiotensin II has been shown to stimulate PLC␤1 acutely followed by activation of PLC␥1 (20), suggesting that acute activation of PLC␤ isoforms may be a general phenomenon.
Whereas we have demonstrated that ET-1, LPA, and thrombin regulate PLC␤3 and PLC⑀, our studies also suggest that these agonists regulate other PLC isoforms. This is apparent, because acute stimulation was only partly inhibited by knockdown of PLC␤3 (Fig. 4) or the combined knockdown of both PLC␤3 and PLC⑀ (Fig. 7). Similarly, high doses (Ն3 M) of LPA activate an unknown PLC during sustained stimulation (Fig. 5B). It is possible that either PLC␦1 or PLC␥1, which we show are present in this cell line, mediates these responses. In Rat-1 fibroblasts, ET-1 and LPA have been shown to transactivate the epidermal growth factor receptor (18,40), and therefore PLC␥1 might be activated. In addition, whereas PLC⑀ and PLC␤3 appear to be regulated independently because of their distinct activation profiles and additive effects on cellular PLC activity in combined knockdown studies, other PLC isoforms may be activated downstream of these enzymes. In this regard, PLC␤2 and PLC␦1 have been suggested to exist as an inactive heterodimer, and upon G␤␥ subunit stimulation of PLC␤2, PLC␦1 is released to hydrolyze PIs (21). Whether PLC␦1 is involved in PLC␤3 or PLC⑀ signaling remains to be determined.
Previously, we demonstrated that the GPCR agonists LPA, S1P, and thrombin stimulate PLC⑀ overexpressed in COS-7 cells (9). The present studies confirm that LPA and thrombin couple to PLC⑀ and that the interaction is physiological. Interestingly, in COS-7 cells overexpressing PLC⑀ or PLC␤ isoforms, these agonists stimulated PLC⑀ to a markedly greater extent than PLC␤1 or PLC␤2, suggesting preferential coupling to PLC⑀. However, IP accumulation was measured at 60 min, and acute stimulation was not examined. Because the current studies demonstrate that these agonists regulate PLC␤3 acutely and PLC⑀ in a sustained manner, earlier time points may reveal coupling to PLC␤ isoforms.
We have also shown previously that LPA and thrombin activate PLC⑀ through G␣ 12/13 and Rap (9). It is possible that a similar mechanism mediates activation of PLC⑀ by thrombin and LPA in Rat-1 fibroblasts, and preliminary studies examining the effects of knocking down G␣ 12/13 are consistent with this hypothesis. 3 Thus, differential activation of G q and G 12 or Ras family G-proteins may mediate the temporal stimulation of PLC␤3 and PLC⑀, respectively. Interestingly, we observed that thrombin stimulated PLC⑀ at least 50-fold more potently than PLC␤3 (Fig. 5). Other investigators have found similar differences in the potency for thrombin activation of Rho through G 12 or G 13 , which is 20-fold greater than activation through G q (45). Riobo et al. (46) have suggested that this difference may be determined by the affinity of the receptor for a given G protein, and this phenomenon may help explain potency differences observed in our studies. Furthermore, our studies also indicate that the signaling pathway(s) underlying thrombin or LPA activation of PLC␤3 desensitize acutely within the first 90 s of stimulation (Fig. 4B), as opposed to the pathways regulating PLC⑀, which remain active for extended periods of at least 60 min (Fig. 3A). Whether this difference reflects differential G-protein regulation (i.e. G q versus 3 G. G. Kelley, unpublished observations. G 12/13 ), a functionally different subset of receptors or intrinsic PLC enzymatic regulation remains to be determined.
Consistent with the temporal activation of PLC⑀ and PLC␤3, we demonstrated a direct functional correlation with IP 3 R ubiquitination. Certain GPCRs that activate PLC have been shown to induce downregulation of IP 3 Rs (28,36,37), possibly as a physiologic, protective mechanism to prevent overstimulation through the GPCR effector system (36). This down-regulation is mediated by the ubiquitin-proteasome pathway and probably involves an IP 3 -and Ca 2ϩ -induced IP 3 R structural change that triggers ubiquitination and subsequent proteasome targeting (36,47). IP 3 appears to be required, because microinjection of an IP 3 analog causes proteasome-mediated down-regulation (48,49), and mutations in the region of the IP 3 R that bind IP 3 inhibit ubiquitination (47,50). Indeed, induction of IP 3 R ubiquitination and degradation requires sustained activation of PLC (28,32,39). For example, sustained activation of PLC with carbachol in SH-SY5Y neuroblastoma cells (37), cholecystokinin and bombesin in AR4 -2J pancreatoma cells (32), or angiotensin II in WB rat liver epithelial cells (39) induces IP 3 R ubiquitination and down-regulation. In contrast, agonists that do not activate PLC in a sustained manner, such as carbachol or substance P in AR4 -2J cells (32) or epidermal growth factor, vasopressin, or bradykinin in WB cells (39), fail to induce ubiquitination or down-regulation. Our data concur with these studies, since inhibition of sustained, but not acute, PI hydrolysis stimulated by thrombin or ET-1 inhibits IP 3 R ubiquitination. Thus, knockdown of PLC⑀, which has no effect on acute PI hydrolysis but inhibits sustained PI hydrolysis, markedly inhibited thrombin and ET-1 induced ubiquitination, an effect dependent on the lipase function of the PLC (Fig. 8). In contrast, knockdown of PLC␤3 had no effect on thrombin-stimulated ubiquitination and only partly inhibited ET-1, consistent with its effects on PI hydrolysis. This is the first demonstration of the coupling of specific PLC isoforms to this important cellular adaptive response.
The physiologic role of the differential activation of PLC␤3 and PLC⑀ is not known. However, the observed temporal and dose-dependent differences in their activation have multiple implications. Acutely, peak IP 3 regulates Ca 2ϩ release through the IP 3 R (51), and PLC␤3 probably contributes to this response. The potential role for PLC⑀ and sustained PLC activation is less clear. During sustained stimulation, IP 3 can be phosphorylated to higher IPs, including inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate, which have been shown to regu-late vesicle transport and nuclear transcription factors (52). Similarly, time-dependent production of different DAG species has been noted, which likely have distinct physiologic roles (53). It is intriguing to spec-FIGURE 8. Knockdown of sustained but not acute PI hydrolysis inhibits agonistmediated IP 3 receptor ubiquitination. A, stable PLC⑀ or PLC␤3 knockdown or random siRNA cell lines shown in Fig. 3 were preincubated with 1 M bortezomib for 1 h and then were exposed to ET-1 (100 nM) or thrombin (Thr; 10 nM) for 1 h. IP 3 R1 was then immunoprecipitated (IP), subjected to Western blot analysis (IB), and probed with anti-ubiquitin (upper blot) and then reprobed with anti-IP 3 R1 (lower blot). Results are representative of four similar experiments. B, relative changes of agonist-stimulated ubiquitination of IP 3 R1 in PLC⑀ or PLC␤3 knockdown cells compared with random cells. Levels of IP 3 R1 ubiquitination were determined by quantitation of ECL pixel density. Values are mean Ϯ S.E. of 2-4 experiments. *, p Ͻ 0.05 for PLC⑀ knockdown cells; ϩ, p Ͻ 0.05 for PLC␤3 knockdown cells compared with random cells. C, reconstitution of ubiquitination. Stable PLC⑀ knockdown or random siRNA cells were transduced with an adenovirus expressing LacZ (AdLacZ), PLC⑀ resistant to siRNAPLC⑀#5 knockdown (Adsi#5PLC⑀), or a lipase-inactive mutant, H1433L PLC⑀ (Adsi#5/H1433LPLC⑀), and IP 3 R1 ubiquitination in response to ET-1 was determined as in A. The lower blot shows PLC⑀ expression. Results are representative of two similar experiments. ulate that sustained agonist activation of PLC⑀ may down-regulate the IP 3 R to redirect signaling to these alternative pathways. Furthermore, our studies show a dose-dependent stimulation of PLC⑀ and PLC␤3, which suggests that local agonist concentrations may determine which pathway is activated. This is most apparent for thrombin, which activates PLC⑀ with a potency almost 2 orders of magnitude greater than PLC␤3 (Fig. 5). Thus, concentrations of 3-10 pM would activate signaling pathways involving PLC⑀ but have little effect on those utilizing PLC␤3. A concentration dependence of ET-1, where concentrations less than 1 nM promote Ca 2ϩ influx but greater stimulate mobilization of intracellular Ca 2ϩ , has been noted previously in Rat-1 cells (54). Whether differential regulation of PLC⑀ or PLC␤3 accounts for this or other dose-dependent effects remains to be determined. Elucidating the temporally distinct products generated by isoform-specific PLCs and defining their physiologic role is an important area of future investigation.
In summary, our studies demonstrate that the GPCR agonists ET-1, LPA, and thrombin couple to both PLC⑀ and PLC␤3 in Rat-1 fibroblasts. Whereas there is some agonist-dependent overlap, activation of these isoforms occurs in a temporally distinct manner whereby PLC␤3 is activated acutely and PLC⑀ in a sustained manner. This temporal regulation of sustained, but not acute, activation of PLC⑀ and PLC␤3 functionally correlates with IP 3 R ubiquitination. In addition, other PLC isoforms, possibly PLC␦1 and/or PLC␥1, are also simultaneously activated. Clearly, these studies demonstrate that GPCR activation of PLC is complex and involves an agonist-and dose-dependent, temporal activation of multiple PLC isoforms, which translates into discrete regulatory functions.