G-protein-coupled Receptor Agonists Activate EndogenousPhospholipase C� and Phospholipase C�3 in a TemporallyDistinct Manner*

Received for publication, July 15, 2005, and in revised form, November 23, 2005 Published, JBC Papers in Press, November 28, 2005, DOI 10.1074/jbc.M507681200

Grant G. Kelley‡§1, Katherine A. Kaproth-Joslin‡§, Sarah E. Reks‡, Alan V. Smrcka¶, and Richard J. H. Wojcikiewicz§

From the Departments of ‡Medicine and §Pharmacology, State University of New York Upstate Medical University, Syracuse,New York 13210 and the ¶Department of Pharmacology, University of Rochester School of Medicine and Dentistry,Rochester, New York 14642

Phospholipase C� (PLC�) is one of the newest members of thephosphatidylinositol-specific phospholipase C (PLC) family. Previ-ous studies have suggested that G-protein-coupled receptors(GPCRs) stimulate phosphoinositide (PI) hydrolysis by activatingPLC� isoforms through Gq family G proteins and G�� subunits.Using RNA interference to knock down PLC isoforms, we demon-strate that the GPCR agonists endothelin (ET-1), lysophosphatidicacid (LPA), and thrombin, acting through endogenous receptors,couple to both endogenous PLC� and the PLC� isoform, PLC�3, inRat-1 fibroblasts. Examination of the temporal activation of thesePLC isoforms, however, reveals agonist- and isoform-specific pro-files. PLC�3 is activated acutely within the first minute of ET-1,LPA, or thrombin stimulation but does not contribute to sustainedPI hydrolysis induced by LPA or thrombin and accounts for onlypart of ET-1 sustained stimulation. PLC�, on the other hand, pre-dominantly accounts for sustained PI hydrolysis. Consistent withthis observation, reconstitution of PLC� in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulatedPI hydrolysis. Furthermore, combined knockdown of both PLC�

and PLC�3 additively inhibits PI hydrolysis, suggesting independ-ent regulation of each isoform. Importantly, ubiquitination of ino-sitol 1,4,5-trisphosphate receptors correlates with sustained, butnot acute, activation of PLC� or PLC�3. In conclusion, GPCR ago-nists ET-1, LPA, and thrombin activate endogenous PLC� andPLC�3 in Rat-1 fibroblasts. Activation of these PLC isoforms dis-plays agonist-specific temporal profiles; however, PLC�3 is pre-dominantly involved in acute and PLC� in sustained PI hydrolysis.

The phosphatidylinositol-specific phospholipase C (PLC)2 family is agroup of critical cellular signaling enzymes that hydrolyze phosphatidy-linositol 4,5-bisphosphate (PI) to generate inositol 1,4,5-trisphosphate(IP3) and diacylglycerol, which increase the intracellular free Ca2� con-centration ([Ca2�]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-cou-pled receptor (GPCR) activation of heterotrimeric Gq family G-proteinsand G�� subunits. PLC� is regulated by tyrosine phosphorylation byreceptor tyrosine kinases (e.g. epidermal growth factor and platelet-derived growth factor) and nonreceptor tyrosine kinases (e.g. Src) acti-vated by immunoglobulin and cytokines. Regulation of PLC� is less wellunderstood but is probably regulated by changes in [Ca2�]i, possiblydownstream from activation of other PLC isoforms, and by highmolec-ular weight G-protein, Gh. PLC� is also regulated by [Ca2�]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) andRho (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 regu-lated by receptor tyrosine kinases and GPCRs. Receptor tyrosine kinaseagonists, epidermal growth factor (9), and platelet-derived growth fac-tor (8) have been shown to stimulate PLC� throughRas andRap. GPCR-mediated activation of PLC� can be subdivided into two groups. Onegroup, �2 adrenergic, prostanoid, and muscarinic M3 receptor agonists,hasbeenproposedtostimulatePLC� throughRap2BmediatedbycAMP-dependent activation of the Rap GTP exchange factor EPAC (12, 13).The second group, lysophosphatidic acid (LPA), sphingosine 1-phos-phate (S1P), and thrombin receptor agonists, activate PLC� throughG�12/13 and Rap (9).

Accumulating evidence suggests that activation of PLC by hormonesis more complex than a simple linear activation from receptor to onePLC isoform (1, 2). For example, GPCR receptor activation of PLC�

isoforms may lead to activation of PLC� isoforms through increasedlevels of [Ca2�]i (14, 15). Alternatively, �1B-adrenergic receptor activa-tion has been shown to stimulate PLC� through G�q/11 and to a lesserextent PLC�1 through Gh (16). GPCRs have been shown to transacti-vate receptor tyrosine kinases (17, 18) with potential activation of PLC�

and PLC�, respectively. In addition, angiotensin II activation of theGPCRAT1 has been proposed to activate PLC�1 (19) and possibly tem-porally activate PLC�1 (�30 s) followed by PLC�1 (�30 s) (20). Fur-thermore, recent studies suggest that PLC�2 and PLC�1 exist as a het-erodimer that sequesters and inhibits PLC�1 activity (21). Upon G��

activation of PLC�2, PLC�1 is released, allowing the enzyme to hydro-lyze its substrate. Thus, receptor activation of cellular PLC activity isprobably the composite of a complex signaling network that involvesthe activation of multiple PLC isoforms. Since similar receptor agonistshave been postulated to stimulate PLC� and the classic isoforms, PLC�

and PLC�, it is possible that novel signaling pathways, that redefinecurrent PLC dogma, participate in this network.In the present studies, we used RNA interference to elucidate the

* This work was supported by National Institutes of Health Grants DK56294 (to G. G. K.)and DK49194 (to R. J. H. W.) and American Diabetes Association Physician ScientistTraining Award 7-03-PS-01 (K. A. K.-J.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 To whom correspondence should be addressed: Dept. of Medicine, SUNY Upstate Med-ical University, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-5725; Fax: 315-464-5797; E-mail: kelleyg@upstate.edu.

2 The abbreviations used are: PLC, phosphoinositide-specific phospholipase C; LPA, lyso-phosphatidic acid; ET-1, endothelin; siRNA, short interfering RNA; FBS, fetal bovineserum; PI, phosphatidylinositol 4,5-bisphosphate; IP, inositol phosphate; IP3, inositol1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; S1P, sphingosine1-phosphate; GPCR, G-protein-coupled receptor.

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physiologic regulation of PLC� and the PLC network regulated byGPCR agonists in Rat-1 fibroblasts. Rat-1 fibroblasts are a classic modelfor examining hormonal regulation of downstream effectors, and weshow that these cells are easily manipulated by RNA interference usingretrovirally transduced short interfering hairpin RNAs to generate sta-ble and almost complete knockdown of signaling proteins. The GPCRagonists, endothelin, LPA, and thrombin, have been shown to stimulatePLC in Rat-1 fibroblasts through endogenous receptors (22–24), andprevious studies suggest that these agonists couple to PLC� isoforms(25–27). Here, we demonstrate for the first time that these GPCR ago-nists activate both endogenous PLC� and PLC�3 through endogenousreceptors in Rat-1 fibroblasts. Activation of these isoforms, however,exhibits distinct agonist- and isoform-specific temporal profileswhereby PLC�3 predominantly participates in acute and PLC� sus-tained PLC activity. In addition, sustained activation of PLC� is func-tionally correlatedwith IP3 receptor ubiquitination, a protective processassociated with sustained activation of PLC.

EXPERIMENTAL PROCEDURES

Materials—PLC�1 and PLC�1 antibodies were from Upstate Bio-technologies, Inc. (Lake Placid, NY). PLC�2, PLC�3, PLC�4, PLC�2,PLC�1, PLC�2, and PLC�3 antibodies were from Santa Cruz Biotech-nology, Inc. (Santa Cruz, CA). LPA was from Sigma, �-thrombin wasfrom Hematologic Technologies, Inc. (Essex Junction, VT), and endo-thelin-1 was from Calbiochem. Anti-ubiquitin, FK2, was from BiomolInternational L.P. (Plymouth Meeting, PA). The PLC� antibody used inthese studies was a rabbit polyclonal antibody generated from the RA1domain. The type 1 IP3 receptor antibody was a rabbit polyclonal anti-body generated to a C-terminal peptide (28).

Tissue Culture—Rat-1 fibroblasts and all HEK293 cell lines weremaintained in Dulbecco’s modified Eagle’s medium supplemented with10% fetal bovine serum. For reconstitution studies, Rat-1 clonal cell lineswere seeded into 24-well dishes at 8 � 104 6 h prior to adenoviraltransduction. Medium was then removed, and adenoviruses at theappropriate concentrations were applied in 250 �l of complete Dulbec-co’smodified Eagle’smedium for 24 h.Cellswere then labeled for 18–24h in inositol-free, serum-free Dulbecco’s modified Eagle’s medium. Forall other PLC assays, Rat-1 cells were seeded into 24-well dishes at 4 �104, grown overnight, and labeled the following day for 18–24 h ininositol-free, serum-free Dulbecco’s modified Eagle’s medium.

Plasmid Constructs—Short interfering hairpin RNAs (siRNAs)were designed for PLC� and PLC�3 targeting rat (r), mouse (m), orhuman (h) sequences: siRNAPLC�#1, GCCGGCATTCCTAAGA-CAC (r); siRNAPLC�#3, GGCACCAAGGCAAAGCAGC (rmh);siRNAPLC�#5, GCCAAATATTCCTACAGCA (rmh); siRNAPLC�3#2,CATGGAGGTGGACACACTG (rmh); siRNAPLC�3#3, GGTCTGGT-CAGAGGAGTTG (rm); siRNAPLC�3#5, GCTTCTGACTACATC-CCAG (rm); siRNARan#5, GTAACATCGCCACGCGTCA (scrambledsiRNAPLC�#1); siRNARan#6, CAGACACCGCAGCAGGAGA (scram-bled siRNAPLC�#3); siRNARan#3, ACTGTCACAAGTACCTACA(scrambled siRNAPLC�#5).

Sense (5�-gatcccc(siRNA)ttcaagaga(antisense siRNA)tttttggaaa andantisense (5�-agcttttccaaaaa(siRNA)tctcttgaa(antisense siRNA)ggg) oli-gonucleotides incorporating these siRNAs were synthesized (Sigma)and cloned into pSUPER and/or pSUPER-Retro (29), generous gifts ofDr. R. Agami.Silent mutations were introduced into rat PLC�, pcDNA-

PLC�-FLAG, to generate pcDNA-PLC�SI#5. Mutations were intro-duced by site-directed mutagenesis using the sense primer,5�-CAAAGCCAAATAcagtTACAGCATCC, to mutate TTCC to cagt

(shown in lowercase type) (QuickMutagenesis Kit, Stratagene, La Jolla,CA). The mutated region was sequenced and subcloned into the origi-nal plasmid.The cDNA for PLC�SI#5 was subcloned into the pShuttle-CMV vec-

tor, and recombinant adenovirus was generated following themanufac-turer’s protocol for the Stratagene AdEasy Adenoviral Vector System(Stratagene).Similarly, the lipase-inactive mutant (5), H1433L PLC�, was created

by site-directed mutagenesis. The region encompassing this mutationwas subcloned into PLC�SI#5, which was subsequently subcloned intopShuttle-CMV, and recombinant adenovirus, Adsi#5/H1433L PLC�,was produced. All constructs were sequenced (DNA Sequencing andSynthesis Facility, Iowa State University, Ames, IA).

Stable Retroviral Knockdown—To generate retroviruses, HEK293Tcells were seeded into T75 flasks at 8 � 106 the previous day. Cells weretransfected with pSUPER-Retro siRNA constructs, pVPackECO, andpVPackGP plasmids by Ca2PO4 precipitation. Viruses were harvestedafter 48 h by filteringmedium and quick freezing on dry ice. Stocks werestored at �80 °C. Viruses were titered by transducing Rat-1 cells withserial dilutions, killing untransduced cells with 3 �g/ml puromycin, andcounting remaining colonies.Rat-1 fibroblasts were seeded in 12-well plates at 4� 104. The follow-

ing day, the conditioned medium was aspirated off, and 1 ml of viralsupernatant and 8�g/ml Polybrenewere applied to eachwell for 6–8 h.The virus was replaced with complete medium after incubation. Cellswere expanded to T75 flasks the next day, and 3 �g/ml puromycin wasadded to medium 48 h after transduction.

Adenovirus Production—Generation of primary adenovirus was per-formed according to theAdEasyAdenoviral Vector System (Stratagene)protocol, except that the transfected cultureswere incubated for 18 daysor until a visible cytopathic effect was observed. Viruses were amplifiedby several rounds of infection inAD293 cells. Viral titerswere calculatedby an immunoreactivity spot assay (30).

Phospholipase C Assay—The PLC assay was performed under twodifferent conditions, 20 and 100mMLiCl. Experiments using 20mMLi�

were performed as previously described (4, 9). Basically, agonists wereadded to the conditioned labeling medium brought to a final concen-tration of 20 mM LiCl, and the cells were incubated for 60 min unlessindicated 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; andendothelin-1 (ET-1), double-distilled H2O.

To increase trapping of inositol phosphates (IPs), 100 mM LiCl hasbeen used in Rat-1 cells (22, 31). For experiments performed in 100 mM

LiCl (Figs. 3–7), conditioned labeling medium was replaced with KrebsBuffer 1 (4.7 mM KCl, 1.2 mM KH2PO4, 25 mM NaHCO3, 1.2 mM

MgSO4, 10 mM HEPES, 1.3 mM CaCl2, 118 mM NaCl, pH 7.4, aeratedwith 5% CO2/95% O2 for 10 min), and the Rat-1 cells were allowed toequilibrate for 60 min in a CO2 incubator at 37 °C. Prior to the additionof agonists, cells were preincubated in Krebs Buffer 2 (4.7 mM KCl, 1.2mM KH2PO4, 25 mM NaHCO3, 1.2 mM MgSO4, 10 mM HEPES, 1.3 mM

CaCl2, 18 mMNaCl, 100 mM LiCl, pH 7.4, aerated with 5% CO2/95%O2

for 10 min) for 10 min. The reaction was stopped by aspirating off theincubation medium and adding 375 �l of ice-cold 50 mM formic acid.After 30 min of incubation on ice, samples were removed, neutralizedwith 125 �l of 150 mM ammonium hydroxide, and diluted with 5 ml ofice-cold water. Total inositol phosphates were separated by columnchromatography using AG 1-X8 200–400 mesh, formate form, andquantitated by liquid scintillation counting. Protein determination (Bio-Rad DC Protein Assay Kit) and expression by Western blot analysis

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were determined in parallel wells carried through the PLC assay butwithout radioactivity. Steady state labeling of phosphatidylinositol wasnot significantly different between conditions.

IP3 Mass Assay—Change in IP3 mass was determined as previouslydescribed (32). Rat-1 cells were seeded at a density of 1.9 � 105/6-wellplate and then incubated in a 5% CO2 incubator at 37 °C for 24 h andthen serum-starved for an additional 24 h. Incubations were initiated bythe addition of agonist and were terminated by removing medium andadding 250�l of ice-cold 0.5 M trichloroacetic acid. After 2 h at 4 °C, the250�l was removed andmixedwith 50�l of 10mMEDTA and 250�l ofFreon/N-trioctylamine (1:1), vortexed, incubated at 4 °C for 15min, andcentrifuged (16,000 � g for 5 min at 4 °C). A portion of the aqueousphase (150 �l) was then removed and neutralized with 75 �l of 25 mM

NaHCO3, and IP3 concentration was determined with a radioreceptorassay as described (33).

IP3 Receptor Ubiquitination—IP3 receptor ubiquitination was meas-ured essentially as previously described (34). Rat-1 cells were grown tonear confluence in 15-cm diameter dishes and were serum-starved for15 h. For reconstitution studies, cells were transduced 6 h after seedingfor 24 h with the indicated adenoviruses prior to serum starvation. Cellswere then preincubated with the proteasome inhibitor bortezomib andexposed to stimuli. After removing culture medium, cells were solubi-lized by adding lysis buffer (50 mM Tris-base, 150 mM NaCl, 1% TritonX-100, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 �M leu-peptin, 10 �M pepstatin, 0.2 �M soybean trypsin inhibitor, 1 mM dithi-othreitol, pH 8) directly to the monolayers, followed by vigorous scrap-ing. After 30 min at 4 °C, lysates were clarified by centrifugation(16,000 � g for 10 min at 4 °C), and IP3R1 was immunoprecipitated byincubating overnight with anti-IP3R1 and Protein A-Sepharose CL-4B.Immunocomplexes were washed thoroughly with lysis buffer, resus-pended in gel loading buffer, electrophoresed, and probed in immuno-blots with anti-ubiquitin and anti-IP3R1.

Calculations and Statistical Analysis—Percentage change inducedby knockdown of PLC� or PLC�3 compared with random control celllines (percentage change of siRNARan) was determined to summarizeeffects of PLC isoform knockdown. To calculate this value, basal levels(activity without addedGPCR agonist) were subtracted from stimulatedlevels. Activity in PLC� or PLC�3 cell lines was then divided by theactivity in random siRNA-treated control cells, and from this value per-centage change was calculated.Paired or unpaired Student’s t test, Tukey-Kramer multiple compar-

isons test, and one sample t test were performed where appropriate. Avalue of p � 0.05 was considered significant.

RESULTS

Serum and G-protein-coupled Receptor Agonists Are Coupled toEndogenous PLC� in Rat-1 Fibroblasts—To find a cell line in whichGPCR agonists couple to endogenous PLC�, various cell lines werescreened byWestern blotting, and Rat-1 fibroblasts were determined toexpress high levels of PLC�. Rat-1 fibroblasts are a classic cell line tostudy hormonal regulation of PLC, and the GPCR agonists endothelin(ET-1) and thrombin have been shown to stimulate PLC in this cell line(22–24). To determine whether these agonists activate PLC�, RNAinterference was used to knock down endogenous PLC�. Eight siRNAstargeting PLC� were generated, which knocked down PLC� with differ-ent efficiencies. Of these, three (siRNAPLC�#1, siRNAPLC�#3, andsiRNAPLC�#5) were used to generate stable cell populations that knockdownPLC� in Rat-1 fibroblasts by 53–97% (Fig. 1B). Random sequencesof these siRNAs were used as controls. Knockdown of PLC� did notaffect 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, G12, G13,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. Withincreasing knockdown, agonist-stimulated PLC activity was increas-ingly inhibited compared with three different random siRNA controlcell populations. The two most effective siRNAs, siRNAPLC�#1 andsiRNAPLC�#5, significantly inhibited FBS-stimulated PI hydrolysis by39 and 74%, respectively, ET-1 by 30 and 69%, and thrombin by 69 and94%. Similar results were obtained in an independently generated set ofstable cell lines (data not shown). Significant inhibition by at least twodistinct siRNAs suggests that the observed inhibition was not due tooff-target effects. Moreover, uncoupling was not due to siRNA-inducednonspecific activation of interferon, since an increase in STAT-1 phos-phorylation was not observed in these stable cell populations (data notshown). In addition, the comparable agonist response in each of thethree random cell lines (Fig. 1A) suggests that the inhibition wasunlikely to be due to cell population variability induced by retroviraltransduction and stable cell population selection. Taken together, thesestudies 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 acti-vate PLC� isoforms through Gq 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

FIGURE 1. GPCR agonists, endothelin and thrombin, couple to endogenous PLC� through endogenous receptors. A, effect of siRNA-mediated knockdown of PLC� on GPCRstimulation of PLC activity. Rat-1 fibroblasts were stably transduced with retrovirus (pSUPER-Retro) expressing siRNA targeting PLC� (siRNAPLC�#1, #3, or #5) or a random sequenceof these siRNAs (siRNARan#3, #5, or #6). Stable cell populations were then stimulated with FBS (10%), ET-1 (100 nM), thrombin (10 nM), or vehicle for 60 min in the presence of 20 mM

Li�, and PLC activity was determined. The average of the PLC response in the three random lines is shown (MeanRan). Values are mean � S.E. of three experiments performed intriplicate. *, p � 0.05 compared with random controls. B, specific knockdown of PLC�. Shown is an image of Western blots of cell lysates stained with anti-PLC� (250 kDa), anti-PLC�3(150 kDa), anti-PLC�1 (145 kDa), or anti-PLC�1 (85 kDa). Percentage knockdown of PLC� relative to random controls, determined by densitometric measurements of at least threeexperiments, is shown.

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not detected (data not shown). Because knockdown of PLC� did notcompletely inhibit stimulation of PLC by these agonists, PLC�3 wasknocked down to determine whether these agonists couple to this PLCisoform. Seven siRNAs were screened, and the three most effective,siRNAPLC�3#2, siRNAPLC�3#3, and siRNAPLC�3#5, were used togenerate stable cell populations. These siRNAs knocked down PLC�3by 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 otherhand, was less affected by knockdown of PLC�3 and was only sig-nificantly inhibited by 32% with siRNAPLC�3#3 but not bysiRNAPLC�3#5. Thus, in addition to being coupled to PLC�, ET-1 andthrombin are also coupled to PLC�3 in Rat-1 fibroblasts, consistentwith previous studies that have suggested that these Gq and Gi family-coupledGPCR agonists activate PLC� isoforms in these cells. FBS, how-ever, appears to predominantly regulate PLC�.

Distinct Temporal Activation of PLC�3 and PLC�—Because knock-down experiments demonstrated that ET-1 and thrombin are coupledto both PLC�3 and PLC�, the kinetics of the hormonal regulation ofeach isoform was examined to determine its unique contribution tototal cellular PLC activity. In addition, LPA has been shown to stimulatePLC activity in Rat-1 fibroblasts (35), and its temporal regulation ofPLC� and PLC�3 was also determined. For these experiments, a set ofstable cell populations was generated with the most effective PLC-spe-cific siRNAs, siRNAPLC�#5 and siRNAPLC�3#3, and a random siRNAcontrol. To increase knockdown, cells were transduced twice with ret-rovirus, which increased knockdown of each PLC isoform to greaterthan 99% comparedwith a random siRNA control (Fig. 3D). In addition,initial studies demonstrated that 20 mM Li� was not sufficient to blockdegradation of sustained IP generation (data not shown). Lithium is anoncompetitive inhibitor of inositol monophosphatase, and thereforehigh concentrations are required to inhibit degradation of low concen-trations of inositol phosphates (IPs). Thus, the concentration of Li� wasincreased 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 accumulationcompared with a random control. FBS and each agonist effectivelyincreased total inositol phosphate accumulation. The stimulation byET-1, LPA, and thrombin was biphasic with an initial acute response (1min) followed by a sustained increase (3–60 min). FBS stimulation wasmonophasic with no significant acute response. As shown above (Figs. 1and 2), knockdown of PLC� or PLC�3 inhibited serum-, ET-1-, and

thrombin-stimulated PLC activity. In addition, LPA stimulation wasalso inhibited. Fig. 3, however, shows that knockdown of PLC� orPLC�3 had differential effects on the temporal stimulation induced bythese agonists.This was most apparent for LPA and thrombin, which showed a

crossover at 3 min in the temporal activation profiles of the PLC� orPLC�3 knockdown cell lines (Fig. 3A). PLC� knockdown had no signif-icant effect on the acute stimulation by LPA or thrombin (Fig. 3B);however, it markedly inhibited the sustained response to these agonistsby �80% at 60 min (Fig. 3C). When trapped IPs at 1 min are subtractedfrom later time points, knockdown of PLC� effectively abolished thesustained stimulatory response. Conversely, PLC�3 knockdown inhib-ited LPA and thrombin acutely by �50% but had no effect on sustainedstimulation. Thus, LPA and thrombin predominantly activate PLC�3during 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 byknockdownof PLC�. However, differenceswere noted.Whereas knock-down of PLC�3 inhibited LPA or thrombin stimulation by �50%, ET-1was only inhibited by 20% (Fig. 3B). Furthermore, in contrast to LPAandthrombin, knockdown of PLC� only inhibited the sustained response by44% and PLC�3 inhibited the sustained response by 27% (Fig. 3C). Thus,whereas the activation profiles of these PLC isoforms by ET-1 are sim-ilar to LPA and thrombin, agonist-dependent differences exist.

Effect of PLC� and PLC�3 Knockdown on Acute PI Hydrolysis—Sincethere appeared to be a differential effect of PLC� or PLC�3 knockdownon acute stimulation of PI hydrolysis, a shorter time course was per-formed to better define the boundaries of the acute stimulatory compo-nent. In addition, changes in total IP accumulation were correlated withIP3 generation. Fig. 4 shows the effect of knocking downPLC� or PLC�3on GPCR agonist-stimulated total IP accumulation and IP3 mass. ET-1,LPA, and thrombin effectively stimulated total IP accumulation acutely,and the responses were near maximal by 30 s (Fig. 4A). Knockdown ofPLC�3, but not PLC�, inhibited this acute stimulation (Fig. 4A) by30–60% (Fig. 4C).Changes in total IP accumulation correlatedwith changes in IP3mass.

ET-1, LPA, and thrombin markedly stimulated IP3 generation, whichpeaked at 15 s and decreased to levelsmodestly above basal levels by 90 s(Fig. 4B). Knockdown of PLC�3, but not PLC�, inhibited this IP3 gen-eration by 30–50% (Fig. 4D), comparablewith inhibition of total inositolphosphate accumulation (Fig. 4C). These studies confirm the selectiverole of PLC�3 during the acute GPCR stimulation of PI hydrolysis.

Because sustained IP3 levels are low, quantitation of the effects of PLCisoform knockdown by measuring IP3 is not as sensitive as measuringtotal IP accumulation in the presence of Li�. Therefore, changes in the

FIGURE 2. Endothelin and thrombin couple to endogenous PLC�3. A, effect of siRNA-mediated knockdown of PLC�3 on GPCR stimulation of PLC activity. Rat-1 fibroblasts werestably transduced with retrovirus (pSUPER-Retro) expressing siRNA targeting PLC�3 (siRNAPLC�3#2, #3, or #5) or random sequences (siRNARan#3, #5, or #6). Stable cell populationswere then stimulated with FBS (10%), ET-1 (100 nM), thrombin (10 nM), or vehicle for 60 min in the presence of 20 mM Li�, and PLC activity was determined. The average of the PLCresponse in the three random lines is shown (MeanRan). Values are mean � S.E. of two experiments performed in triplicate. *, p � 0.05 compared with random controls. B, specificknockdown of PLC�3. Shown is an image of Western blots of cell lysates stained with anti-PLC�3 (150 kDa), anti-PLC� (250 kDa), anti-PLC�1 (145 kDa), or anti-PLC�1 (85 kDa).Percentage knockdown of PLC� relative to random controls, determined by densitometric measurements of at least three experiments, is shown.

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mass of IP3 at sustained time points were not determined except for theeffect of PLC� knockdown on ET-1-stimulated PI hydrolysis, whichappeared elevated at 90 s (Fig. 4B). In this set of experiments, PLC�

knockdown significantly inhibited the sustained ET-1 stimulation of IP3measured at 20 min by 90 � 8% from 75 pmol/mg protein to 9 � 6 (p �0.02; n 3), consistent with total IP measurements and a role for PLC�

in sustained IP3 formation.Dose- and Agonist-dependent, Differential Effects of PLC� and PLC�3

Knockdown onAcute and Sustained PLCActivity—To further assess theeffects of PLC� and PLC�3 knockdown on agonist-stimulated acute andsustained PLC activity and to determine whether lower agonist concen-trations would differentially affect coupling to either isoform, a set ofexperiments was performed with increasing concentrations of agonist,and total IP accumulation was determined. Acute stimulation wasdetermined by measuring IP accumulation at 1 min after agonist addi-tion. Cells were preincubated with Li� for 10 min. For sustained stim-ulation, agonist was added similarly, but Li� was not added until 3 minafter agonist addition, and the experiment was terminated at 60 min.This effectively measures sustained PLC activity from greater than 3min (time for Li� to block IP degradation) to 60min; IPs formed at timesless than 3 min are not trapped, because Li� is not present.

Fig. 5 shows dose responses to ET-1, LPA, and thrombin and theeffect of knocking down PLC� or PLC�3 on acute (1-min) and sustained(3–60-min) IP accumulation. ET-1 stimulated acute PLC activity withan EC50 of �20 nM (Fig. 5A) and sustained activity with an EC50 of 0.4nM (Fig. 5B), �50-fold more potent than acute. At 1 nM, sustainedstimulation was nearmaximal, whereas there was almost no acute stim-ulation. At all stimulatory doses, there was no shift in contribution of

PLC� or PLC�3 to total PLC activity; PLC�3 knockdown inhibitedacute and PLC� and PLC�3 knockdown both inhibited sustained PIhydrolysis.Similarly, thrombin stimulated acute PI hydrolysis with an EC50 of

200 pM (Fig. 5A) and sustained �50-fold more potently with an EC50 of3 pM (Fig. 5B). At 10 pM, sustained PLC activity was maximal with verylittle acute stimulation. At all stimulatory concentrations, acute stimu-lation was inhibited by PLC�3 knockdown but not PLC� knockdown(Fig. 5A), in contrast to sustained stimulation, which was almost com-pletely abrogated with PLC� knockdown and not affected by PLC�3knockdown (Fig. 5B).The dose response to LPA was somewhat more complicated. Both

acute and sustained stimulation appeared biphasic with an initial EC50

of 30–100 nM for acute (Fig. 5A) and sustained stimulation (Fig. 5B)followed by a second phase with an EC50 of 0.6–3 �M. The EC50 of thefirst phase is comparable with known LPA receptors, whereas the sec-ond is possibly mediated by a low affinity LPA receptor that appearsimportant for mitogenesis but has yet to be identified (26). Acute stim-ulation was inhibited by �50% at all stimulatory concentrations byknockdown of PLC�3 but not affected by knockdown of PLC� (Fig. 5A).In contrast, knockdown of PLC�3 had no effect on sustained stimula-tion. The first phase of the sustained stimulation, however, was entirelydependent on PLC�, since knockdown of PLC� completely inhibited PIhydrolysis (Fig. 5B). The second sustained phase, on the other hand, wasaccompanied by a component that was not inhibited by PLC� or PLC�3knockdown, suggesting that another PLC isoform is activated at highLPA concentrations.

FIGURE 3. Temporal coupling of GPCR agonist activation of PLC� and PLC�3. A, effect of siRNA-mediated knockdown of PLC� or PLC�3 on agonist stimulation of PLC activity. Rat-1fibroblasts were stably transduced with retrovirus (pSUPER-Retro) expressing siRNA targeting PLC� (siRNAPLC�#5), PLC�3 (siRNAPLC�3#3), or a random sequence (siRNARan#6) asin Figs. 1 and 2, except cells were exposed to the retrovirus a second time to increase knockdown. Stable cell populations were then stimulated with FBS (10%), ET-1 (100 nM), LPA (3�M), thrombin (Thr; 10 nM), or vehicle. PLC activity was determined at the indicated times as in Figs. 1 and 2, except the medium was changed to a Krebs buffer, and lithium wasincreased to 100 mM to prevent metabolism and low IP levels. Values are mean � S.E. of 3– 6 experiments performed in triplicate. *, p � 0.05 for PLC� knockdown cells; �, p � 0.05for PLC�3 knockdown cells compared with random cells. Shown are relative changes of agonist-stimulated PI hydrolysis induced by knockdown of PLC� or PLC�3 compared withrandom treated cells at 1 min (B) and 60 min (C) time points from A (mean � S.E.). FBS did not stimulate at 1 min. D, knockdown of PLC� or PLC�3. Image of Western blots of cell lysatesstained with anti-PLC� (250 kDa) or anti-PLC�3 (150 kDa). Percentage knockdown of PLC� or PLC�3 relative to random control, determined by densitometric measurements of at leastthree experiments, is shown.

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Reconstitution and Overexpression of PLC�—To further examine therole of PLC� in sustained PI hydrolysis, a PLC� construct, AdPLC�#5,was generated with silent mutations in the region targeted bysiRNAPLC�#5 to prevent siRNA-mediated degradation. Transductionof Rat-1 cells reduced PLC activity for unknown reasons, but the agoniststimulatory profiles remained intact. Fig. 5, A and C, show that whenPLC� 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 studiesconfirm that the observed effects of knocking down PLC� in thesiRNAPLC�#5 cell line on the sustained agonist-stimulatory profiles arespecific 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 bythrombin. In this set of experiments, a random cell line was transducedwith LacZ and a PLC� knockdown cell line with increasing amounts ofadenovirus expressing PLC� (Fig. 5E). Overexpressing PLC� slightlyincreased basal, unstimulated IP accumulation from 13.4� 1.0 cpm/�gprotein in AdLacZ-treated cells to 17.4 � 3.6 and 21.8 � 0.4 for cellsmoderately (multiplicity of infection 20), and highly (multiplicity ofinfection 60) overexpressing PLC�, respectively. However, the -foldstimulation acutely at 1 min was not different. On the other hand, thesustained PI hydrolysis was markedly potentiated. At 30 and 60 min, IPaccumulation in the moderately overexpressing cells was 1.6- and 2.2-fold above the control stimulation (-fold stimulation of the PLC�-over-expressing cell line/-fold stimulation of the Ran#6 LacZ-expressing cell

line), respectively, and in the highly expressing cells it was 3.6- and5.1-fold, respectively. Thus, overexpressing PLC� has no effect on acutethrombin stimulation but markedly potentiates sustained stimulation,consistent with the knockdown experiments.

Double Knockdown of PLC� and PLC�3—To determine whether theactivations of PLC� and PLC�3 are independent, both PLC� and PLC�3were knocked down, and agonist-stimulated PLC activitywas comparedwith a random siRNA-treated cell line. A set of stable cell populationswas generated in which PLC� and PLC�3 were knocked down �99 and92%, respectively, compared with a random control (Fig. 6B). Fig. 6Ashows the stimulatory response to ET-1, LPA, and thrombin in thesecell lines. In addition, the stimulatory response observed in singleknockdown cell lines is shown. For each agonist, IP accumulation wasinhibited to a similar extent as the additive knockdown of the singleknockdown cell lines. This would suggest that PLC� and PLC�3 areregulated independently by these GPCR agonists.

Sustained but Not Acute PLC Activation Correlates with IP3 ReceptorUbiquitination—Previous studies have demonstrated that GPCR acti-vation of PLC can induce down-regulation of IP3 receptors (IP3Rs)through the ubiquitin-proteasome pathway (28, 36, 37), and we haveshown that ET-1 stimulates IP3R ubiquitination and down-regulation inRat-1 fibroblasts (38). Since sustained PI hydrolysis appears to berequired for this process (28, 32, 37, 39), we reasoned that knockdown ofPLC isoforms coupled to sustained PI hydrolysis should inhibit IP3Rubiquitination. Fig. 8 shows the effect of knocking downPLC� or PLC�3on type 1 IP3R ubiquitination stimulated by ET-1 or thrombin. Knock-

FIGURE 4. Acute activation of PLC�3 but notPLC�. A, acute regulation of inositol phosphateproduction. Control and knockdown Rat-1 fibro-blasts described in the legend to Fig. 3 were stim-ulated with ET-1 (100 nM), LPA (3 �M), thrombin(Thr; 10 nM), or vehicle for the indicated times, andacute regulation of total inositol phosphate accu-mulation in the presence of 100 mM Li� was deter-mined. Values are mean � S.E. of four experimentsperformed in triplicate. B, correlation with IP3

mass. In a separate set of experiments, performedsimilarly but with no added Li�, IP3 mass wasmeasured. Values are mean � S.E. of four experi-ments. Shown are relative change of total inositolproduction measured at 30 s (C) and IP3 mass cal-culated as area under curve (AUC) from 0 to 30 s inPLC� and PLC�3 knockdown clones comparedwith random control cells from A and B (mean �S.E) (D). �, p � 0.05 for PLC�3 knockdown com-pared with random or PLC� knockdown cell lines.

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down of PLC� markedly inhibited ET-1-stimulated ubiquitination andcompletely blocked the effects of thrombin (Fig. 8, A and B). In addi-tional experiments, inhibition of ET-1-stimulated ubiquitination wasreversed by adenovirus-mediated reconstitution of PLC�, as in Fig. 6 butnot with a catalytically inactive mutant, H1433L PLC�, consistent withthe lipase function of PLC� mediating agonist-induced IP3R ubiquitina-tion (Fig. 8C). Similarly, knockdown of PLC�3 partially inhibited ET-1-stimulated ubiquitination, although to a lesser extent than PLC� knock-down and consistent with differences in inhibition of total IPaccumulation (see Fig. 3C). In contrast, knockdown of PLC�3 had noeffect on thrombin-stimulated ubiquitination (Fig. 8, A and B), whichonly inhibited acute PI hydrolysis. These studies show a direct correla-tion between inhibition of agonist-stimulated sustained PI hydrolysisand IP3R ubiquitination (see Figs. 3C and 8B) and that PLC� is predom-inant in mediating sustained PI hydrolysis.

DISCUSSION

ET-1, LPA, and thrombin previously have been shown to stimulatecellular PLC activity (25–27). Classically, these and other hormonesacting through GPCRs have been proposed to activate PLC� isoformsthrough Gq family G-proteins and G�� subunits (25–27). In addition,recent studies have raised the possibility that these agonists could acti-vate PLC� isoforms through transactivation of receptor tyrosine kinasereceptors (18, 40). The discovery of PLC�, however, raises the possibilitythat these agonists might activate this novel PLC family member. PLC�

has been shown to be regulated bymultipleG-proteins, includingRas (4,8, 9), Rho (9, 10), and G12 (5, 9) family G proteins and by G�� subunits(11). Since ET-1, LPA, and thrombin receptors couple toGq, Gi, andG12family G proteins (25–27) 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 agonistsregulate cellular PI hydrolysis. To determine whether these agonistscouple to PLC� isoforms and/or PLC�, we identified Rat-1 fibroblasts asa cell line that expresses both of these PLC family members and exam-ined the effects of knocking down specific isoforms on agonist-stimu-lated 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 coupleto PLC�. In addition, and consistent with previous paradigms (1, 2,25–27), we also show that these diverse agonists activate endogenousPLC�3. Thus, under physiologic conditions, ET-1, LPA, and thrombindually regulate both PLC� and PLC�3 in Rat-1 fibroblasts.

GPCR agonist regulation of these PLC isoforms, however, is not asimple, simultaneous activation of PLC� and PLC�3. Importantly, wedemonstrate that the stimulation of these PLC isoforms is temporallydistinct. Our studies show that ET-1, LPA, and thrombin activatePLC�3 acutely (peak at 15 s; duration �1 min) but not PLC�. In con-trast, ET-1, LPA, and thrombin activate PLC� during sustained PIhydrolysis (�30 s to at least 60min). This distinct temporal activation ofPLC� and PLC�3 is most apparent for LPA, doses �1 �M, and throm-bin, which couple to PLC�3 acutely and almost exclusively to PLC�

during sustained stimulation. Consistent with this observation, overex-pression of PLC� had no effect on acute but markedly increased sus-tained agonist-stimulated PI hydrolysis. ET-1 was similarly coupled toPLC�3 acutely but, in addition to activating PLC� during the sustainedphase, also partly activated PLC�3, indicating an agonist dependencefor the temporal activation of these isoforms. Interestingly, a differencein the activation of PLC by these agonists is consistent with an earlierstudy that noted ET-1 and LPA regulate PLC activity by distinct mech-

FIGURE 5. Dose-dependent acute and sustained coupling of GPCR agonists to PLC� and PLC�3. Control and knockdown Rat-1 fibroblasts described in the legend to Fig. 3 werestimulated with increasing concentrations of ET-1, LPA, or thrombin. A, acute stimulation was determined by preincubating with 100 mM Li� for 10 min and then adding agonist for1 min. B, sustained stimulation was determined by stimulating with agonist for 60 min as in Fig. 3, except 100 mM Li� was added 3 min after agonist addition to trap sustained, butnot acute IPs. Basal, unstimulated activity (23–25 cpm/�g protein) was subtracted from stimulated responses. Values are mean � S.E. of 3–5 experiments performed in triplicate. *,p � 0.05 for PLC� knockdown; �, p � 0.05 for PLC�3 knockdown compared with random cells.

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anisms in Rat-1 fibroblasts (44). Overall, however, PLC�3 predomi-nantly accounts for acute and PLC� for sustained PLC responses. Inter-estingly, in vascular smoothmuscle cells, angiotensin II has been shownto stimulate PLC�1 acutely followed by activation of PLC�1 (20), sug-gesting that acute activation of PLC� isoforms may be a generalphenomenon.Whereas we have demonstrated that ET-1, LPA, and thrombin reg-

ulate PLC�3 and PLC�, our studies also suggest that these agonistsregulate other PLC isoforms. This is apparent, because acute stimula-tion was only partly inhibited by knockdown of PLC�3 (Fig. 4) or thecombined knockdown of both PLC�3 and PLC� (Fig. 7). Similarly, highdoses (3�M) of LPA activate an unknown PLC during sustained stim-ulation (Fig. 5B). It is possible that either PLC�1 or PLC�1, which weshow are present in this cell line, mediates these responses. In Rat-1fibroblasts, ET-1 and LPA have been shown to transactivate the epider-mal growth factor receptor (18, 40), and therefore PLC�1 might beactivated. In addition, whereas PLC� and PLC�3 appear to be regulatedindependently because of their distinct activation profiles and additiveeffects on cellular PLC activity in combined knockdown studies, otherPLC isoforms may be activated downstream of these enzymes. In thisregard, PLC�2 and PLC�1 have been suggested to exist as an inactiveheterodimer, and upon G�� subunit stimulation of PLC�2, PLC�1 isreleased to hydrolyze PIs (21). Whether PLC�1 is involved in PLC�3 orPLC� 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 presentstudies confirm that LPA and thrombin couple to PLC� and that theinteraction is physiological. Interestingly, in COS-7 cells overexpressing

PLC� or PLC� isoforms, these agonists stimulated PLC� to a markedlygreater extent than PLC�1 or PLC�2, suggesting preferential couplingto PLC�. However, IP accumulation was measured at 60 min, and acutestimulationwas not examined. Because the current studies demonstratethat these agonists regulate PLC�3 acutely and PLC� in a sustainedmanner, 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 mechanismmediates activation of PLC� by thrombin and LPA in Rat-1 fibroblasts,and preliminary studies examining the effects of knocking downG�12/13are consistent with this hypothesis.3 Thus, differential activation of GqandG12 or Ras family G-proteinsmaymediate the temporal stimulationof PLC�3 and PLC�, respectively. Interestingly, we observed thatthrombin stimulated PLC� at least 50-fold more potently than PLC�3(Fig. 5). Other investigators have found similar differences in thepotency for thrombin activation of Rho through G12 or G13, which is20-fold greater than activation through Gq (45). Riobo et al. (46) havesuggested that this difference may be determined by the affinity of thereceptor for a given G protein, and this phenomenon may help explainpotency differences observed in our studies. Furthermore, our studiesalso indicate that the signaling pathway(s) underlying thrombin or LPAactivation of PLC�3 desensitize acutely within the first 90 s of stimula-tion (Fig. 4B), as opposed to the pathways regulating PLC�, whichremain active for extended periods of at least 60min (Fig. 3A).Whetherthis difference reflects differential G-protein regulation (i.e. Gq versus

3 G. G. Kelley, unpublished observations.

FIGURE 6. Adenovirus-mediated reconstitution of sustained PLC� activation. Stable Rat-1 fibroblast cell lines (siRNAPLC�#5 or siRNARan#6 shown in Fig. 3) were transduced withadenovirus expressing LacZ (AdLacZ) or mutant PLC� (Adsi#5PLC�), and PLC activity was determined in response to LPA (3 �M) (A) or thrombin (10 nM) (C and D) as in Fig. 3. Themultiplicity of infection (MOI; viral plaque-forming units/cell) for ADLacZ was 6.0 and as indicated for PLC�. Values are mean � S.E. of representative experiments of three similarexperiments performed in triplicate for each agonist. Corresponding expression of PLC� in each experiment is shown (B and E). Western blots of cell lysates were stained withanti-PLC� (250 kDa).

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G12/13), a functionally different subset of receptors or intrinsic PLCenzymatic regulation remains to be determined.Consistent with the temporal activation of PLC� and PLC�3, we

demonstrated a direct functional correlation with IP3R ubiquitination.Certain GPCRs that activate PLC have been shown to induce down-regulation of IP3Rs (28, 36, 37), possibly as a physiologic, protectivemechanism to prevent overstimulation through the GPCR effector sys-tem (36). This down-regulation is mediated by the ubiquitin-protea-some pathway and probably involves an IP3- and Ca2�-induced IP3Rstructural change that triggers ubiquitination and subsequent protea-some targeting (36, 47). IP3 appears to be required, because microinjec-tion of an IP3 analog causes proteasome-mediated down-regulation (48,49), and mutations in the region of the IP3R that bind IP3 inhibit ubiq-uitination (47, 50). Indeed, induction of IP3R ubiquitination and degra-dation requires sustained activation of PLC (28, 32, 39). For example,sustained activation of PLC with carbachol in SH-SY5Y neuroblastomacells (37), cholecystokinin and bombesin in AR4–2J pancreatoma cells(32), or angiotensin II in WB rat liver epithelial cells (39) induces IP3Rubiquitination and down-regulation. In contrast, agonists that do notactivate PLC in a sustainedmanner, such as carbachol or substance P inAR4–2J cells (32) or epidermal growth factor, vasopressin, or bradyki-nin in WB cells (39), fail to induce ubiquitination or down-regulation.Our data concur with these studies, since inhibition of sustained, butnot acute, PI hydrolysis stimulated by thrombin or ET-1 inhibits IP3Rubiquitination. Thus, knockdown of PLC�, which has no effect on acutePI hydrolysis but inhibits sustained PI hydrolysis, markedly inhibitedthrombin and ET-1 induced ubiquitination, an effect dependent on thelipase function of the PLC (Fig. 8). In contrast, knockdown of PLC�3had no effect on thrombin-stimulated ubiquitination and only partlyinhibited ET-1, consistent with its effects on PI hydrolysis. This is thefirst demonstration of the coupling of specific PLC isoforms to thisimportant cellular adaptive response.The physiologic role of the differential activation of PLC�3 and PLC�

is not known. However, the observed temporal and dose-dependentdifferences in their activation have multiple implications. Acutely, peakIP3 regulates Ca2� release through the IP3R (51), and PLC�3 probablycontributes to this response. The potential role for PLC� and sustainedPLC activation is less clear. During sustained stimulation, IP3 can bephosphorylated to higher IPs, including inositol 1,3,4,5-tetrakisphos-phate 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 agonist-mediated IP3 receptor ubiquitination. A, stable PLC� or PLC�3 knockdown or randomsiRNA cell lines shown in Fig. 3 were preincubated with 1 �M bortezomib for 1 h and thenwere exposed to ET-1 (100 nM) or thrombin (Thr; 10 nM) for 1 h. IP3R1 was then immuno-precipitated (IP), subjected to Western blot analysis (IB), and probed with anti-ubiquitin(upper blot) and then reprobed with anti-IP3R1 (lower blot). Results are representative offour similar experiments. B, relative changes of agonist-stimulated ubiquitination ofIP3R1 in PLC� or PLC�3 knockdown cells compared with random cells. Levels of IP3R1ubiquitination were determined by quantitation of ECL pixel density. Values are mean �S.E. of 2– 4 experiments. *, p � 0.05 for PLC� knockdown cells; �, p � 0.05 for PLC�3knockdown cells compared with random cells. C, reconstitution of ubiquitination. StablePLC� knockdown or random siRNA cells were transduced with an adenovirus expressingLacZ (AdLacZ), PLC� resistant to siRNAPLC�#5 knockdown (Adsi#5PLC�), or a lipase-in-active mutant, H1433L PLC� (Adsi#5/H1433LPLC�), and IP3R1 ubiquitination in responseto ET-1 was determined as in A. The lower blot shows PLC� expression. Results are repre-sentative of two similar experiments.

FIGURE 7. Dual knockdown of PLC� and PLC�3. Rat-1 fibroblasts were stably transduced with retrovirus (pSUPER-Retro) expressing siRNA targeting PLC� (siRNAPLC�#5) and PLC�3(siRNAPLC�3#3) or a random sequence (siRNARan#6) (A). Stable cell populations were then stimulated with ET-1 (100 nM), lysophosphatidic acid (LPA) (3 �M), thrombin (10 nM), orvehicle, and PLC activity was determined as in Fig. 3. Values are mean � S.E. of 2–5 experiments performed in triplicate. The dotted lines represent values obtained in the singleknockdown clones as indicated. B, image of Western blots of cell lysates stained with anti-PLC� (250 kDa) or anti-PLC�3 (150 kDa). Percentage knockdown of PLC� or PLC�3 relativeto random control, determined by densitometric measurements of at least three experiments, is shown.

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ulate that sustained agonist activation of PLC� may down-regulate theIP3R 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 whichpathway is activated. This is most apparent for thrombin, which acti-vates PLC� with a potency almost 2 orders of magnitude greater thanPLC�3 (Fig. 5). Thus, concentrations of 3–10 pM would activate signal-ing pathways involving PLC� but have little effect on those utilizingPLC�3. A concentration dependence of ET-1, where concentrationsless than 1 nM promote Ca2� influx but greater stimulate mobilizationof intracellular Ca2�, has been noted previously in Rat-1 cells (54).Whether differential regulation of PLC� or PLC�3 accounts for this orother dose-dependent effects remains to be determined. Elucidating thetemporally distinct products generated by isoform-specific PLCs anddefining their physiologic role is an important area of futureinvestigation.In summary, our studies demonstrate that the GPCR agonists ET-1,

LPA, and thrombin couple to both PLC� and PLC�3 in Rat-1 fibro-blasts. Whereas there is some agonist-dependent overlap, activation ofthese isoforms occurs in a temporally distinct manner whereby PLC�3is activated acutely and PLC� in a sustained manner. This temporalregulation of sustained, but not acute, activation of PLC� and PLC�3functionally correlates with IP3R ubiquitination. In addition, other PLCisoforms, possibly PLC�1 and/or PLC�1, are also simultaneously acti-vated. Clearly, these studies demonstrate thatGPCR activation of PLC iscomplex and involves an agonist- and dose-dependent, temporal acti-vation of multiple PLC isoforms, which translates into discrete regula-tory functions.

Acknowledgments—We are especially grateful to Reuven Agami for pSUPERplasmids and Anja G. Teschemacher for providing the method for titeringadenovirus. We also thank Chris Potvin for technical assistance.

REFERENCES1. Rhee, S. G. (2001) Annu. Rev. Biochem. 70, 281–3122. Rebecchi, M. J., and Pentyala, S. N. (2000) Physiol. Rev. 80, 1291–13353. Kouchi, Z., Fukami, K., Shikano, T., Oda, S., Nakamura, Y., Takenawa, T., and

Miyazaki, S. (2003) J. Biol. Chem.4. Kelley, G. G., Reks, S. E., Ondrako, J. M., and Smrcka, A. V. (2001) EMBO J. 20,

743–7545. Lopez, I., Mak, E. C., Ding, J., Hamm, H. E., and Lomasney, J. W. (2001) J. Biol. Chem.

276, 2758–27656. Song, C., Hu, C. D., Masago, M., Kariyai, K., Yamawaki-Kataoka, Y., Shibatohge, M.,

Wu, D., Satoh, T., and Kataoka, T. (2001) J. Biol. Chem. 276, 2752–27577. Kelley, G. G., and Smrcka, A. V. (2005) AfCS-Nature Molecule Pages

doi:10.1038/mp.a000046.0000018. Song, C., Satoh, T., Edamatsu,H.,Wu,D., Tadano,M.,Gao,X., andKataoka, T. (2002)

Oncogene 21, 8105–81139. Kelley, G. G., Reks, S. E., and Smrcka, A. V. (2004) Biochem. J. 378, 129–13910. Seifert, J. P., Wing, M. R., Snyder, J. T., Gershburg, S., Sondek, J., and Harden, T. K.

(2004) J. Biol. Chem.11. Wing,M. R., Houston, D., Kelley, G. G., Der, C. J., Siderovski, D. P., and Harden, T. K.

(2001) J. Biol. Chem. 276, 48257–4826112. Evellin, S., Nolte, J., Tysack, K., vomDorp, F., Thiel,M.,Weernink, P. A., Jakobs, K.H.,

Webb, E. J., Lomasney, J.W., and Schmidt,M. (2002) J. Biol. Chem. 277, 16805–1681313. Schmidt,M., Evellin, S.,Weernink, P. A., vonDorp, F., Rehmann,H., Lomasney, J.W.,

and Jakobs, K. H. (2001) Nat. Cell Biol. 3, 1020–1024

14. Banno, Y., Okano, Y., and Nozawa, Y. (1994) J. Biol. Chem. 269, 15846–1585215. Kim, Y.H., Park, T. J., Lee, Y.H., Baek, K. J., Suh, P.G., Ryu, S.H., andKim,K. T. (1999)

J. Biol. Chem. 274, 26127–2613416. Zhang, J., Tucholski, J., Lesort, M., Jope, R. S., and Johnson, G. V. (1999) Biochem. J.

343 Pt 3, 541–54917. Daub, H.,Wallasch, C., Lankenau, A., Herrlich, A., andUllrich, A. (1997) EMBO J. 16,

7032–704418. Kranenburg, O., and Moolenaar, W. H. (2001) Oncogene 20, 1540–154619. Berk, B. C., and Corson, M. A. (1997) Circ. Res. 80, 607–61620. Ushio-Fukai, M., Griendling, K. K., Akers, M., Lyons, P. R., and Alexander, R. W.

(1998) J. Biol. Chem. 273, 19772–1977721. Guo, Y., Rebecchi, M., and Scarlata, S. (2004) J. Biol. Chem.22. Muldoon, L. L., Rodland, K. D., Forsythe,M. L., andMagun, B. E. (1989) J. Biol. Chem.

264, 8529–853623. MacNulty, E. E., Plevin, R., and Wakelam, M. J. (1990) Biochem. J. 272, 761–76624. Plevin, R., MacNulty, E. E., Palmer, S., andWakelam, M. J. (1991) Biochem. J. 280 (Pt

3), 609–61525. Sugden, P. H. (2003) J. Mol. Cell Cardiol. 35, 871–88626. Chun, J., Goetzl, E. J., Hla, T., Igarashi, Y., Lynch, K. R., Moolenaar, W., Pyne, S., and

Tigyi, G. (2002) Pharmacol. Rev. 54, 265–26927. Macfarlane, S. R., Seatter, M. J., Kanke, T., Hunter, G. D., and Plevin, R. (2001)

Pharmacol. Rev. 53, 245–28228. Wojcikiewicz, R. J., Furuichi, T., Nakade, S., Mikoshiba, K., andNahorski, S. R. (1994)

J. Biol. Chem. 269, 7963–796929. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) Science 296, 550–55330. Duale, H., Kasparov, S., Paton, J. F., and Teschemacher, A. G. (2005) Exp. Physiol. 90,

71–7831. Muldoon, L. L., Rodland, K. D., and Magun, B. E. (1988) J. Biol. Chem. 263,

18834–1884132. Oberdorf, J., Webster, J. M., Zhu, C. C., Luo, S. G., and Wojcikiewicz, R. J. (1999)

Biochem. J. 339 (Pt 2), 453–46133. Challiss, R. A., and Nahorski, S. R. (1991) J. Neurochem. 57, 1042–105134. Wojcikiewicz, R. J., Xu, Q., Webster, J. M., Alzayady, K., and Gao, C. (2003) J. Biol.

Chem. 278, 940–94735. Cadwallader, K., Beltman, J., McCormick, F., and Cook, S. (1997) Biochem. J. 321 (Pt

3), 795–80436. Wojcikiewicz, R. J. (2004) Trends Pharmacol. Sci. 25, 35–4137. Wojcikiewicz, R. J., and Nahorski, S. R. (1991) J. Biol. Chem. 266, 22234–2224138. Alzayady, K. J., Panning, M. M., Kelley, G. G., and Wojcikiewicz, R. J. (2005) J. Biol.

Chem. 280, 34530–3453739. Bokkala, S., and Joseph, S. K. (1997) J. Biol. Chem. 272, 12454–1246140. Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C., and Ullrich,

A. (1999) Nature 402, 884–88841. van Corven, E. J., Hordijk, P. L., Medema, R. H., Bos, J. L., and Moolenaar, W. H.

(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1257–126142. Daub, H., Weiss, F. U., Wallasch, C., and Ullrich, A. (1996) Nature 379, 557–56043. Sah, V. P., Seasholtz, T.M., Sagi, S. A., and Brown, J. H. (2000)Annu. Rev. Pharmacol.

Toxicol. 40, 459–48944. Plevin, R.,MacNulty, E. E., Palmer, S., andWakelam,M. J. (1991)Biochem. Soc. Trans.

19, 100S45. Vogt, S., Grosse, R., Schultz, G., and Offermanns, S. (2003) J. Biol. Chem. 278,

28743–2874946. Riobo, N. A., and Manning, D. R. (2005) Trends Pharmacol. Sci. 26, 146–15447. Alzayady, K. J., and Wojcikiewicz, R. J. (2005) Biochem. J.48. Brind, S., Swann, K., and Carroll, J. (2000) Dev. Biol. 223, 251–26549. Jellerette, T., He, C. L., Wu, H., Parys, J. B., and Fissore, R. A. (2000) Dev. Biol. 223,

238–25050. Zhu, C. C., and Wojcikiewicz, R. J. (2000) Biochem. J. 348 Pt 3, 551–55651. Berridge, M. J., Bootman, M. D., and Roderick, H. L. (2003) Nat. Rev. Mol. Cell. Biol.

4, 517–52952. Shears, S. B. (2004) Biochem. J. 377, 265–28053. Hodgkin, M. N., Pettitt, T. R., Martin, A., Michell, R. H., Pemberton, A. J., and

Wakelam, M. J. (1998) Trends Biochem. Sci. 23, 200–20454. Muldoon, L. L., Enslen, H., Rodland, K. D., and Magun, B. E. (1991) Am. J. Physiol.

260, C1273–1281

GPCR Regulation of PLC� and PLC�3

2648 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 5 • FEBRUARY 3, 2006

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Richard J. H. WojcikiewiczGrant G. Kelley, Katherine A. Kaproth-Joslin, Sarah E. Reks, Alan V. Smrcka and

3 in a Temporally Distinct MannerβPhospholipase CG-protein-coupled Receptor Agonists Activate Endogenous Phospholipase C? and

doi: 10.1074/jbc.M507681200 originally published online November 28, 20052006, 281:2639-2648.J. Biol. Chem. 

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