LPA4/GPR23 Is a Lysophosphatidic Acid (LPA) Receptor Utilizing Gs-, Gq/Gi-mediated Calcium Signaling and G12/13-mediated Rho Activation
2006; Elsevier BV; Volume: 282; Issue: 7 Linguagem: Inglês
10.1074/jbc.m610826200
ISSN1083-351X
AutoresChang‐Wook Lee, Richard Rivera, Adrienne E. Dubin, Jerold Chun,
Tópico(s)Receptor Mechanisms and Signaling
ResumoLysophosphatidic acid (LPA) is a bioactive lysophospholipid that signals through G protein-coupled receptors (GPCRs) to produce a range of biological responses. A recently reported fourth receptor, LPA4/GPR23, was notable for its low homology to the previously identified receptors LPA1–3 and for its ability to increase intracellular concentrations of cAMP and calcium. However, the signaling pathways leading to LPA4-mediated induction of cAMP and calcium levels have not been reported. Using epitope-tagged LPA4, pharmacological intervention, and G protein mini-genes, we provide independent confirmatory evidence that supports LPA4 as a fourth LPA receptor, including LPA concentration-dependent responses and specific membrane binding. Importantly, we further demonstrate new LPA-dependent activities of LPA4 that include the following: receptor internalization; G12/13- and Rho-mediated neurite retraction and stress fiber formation; Gq protein and pertussis toxin-sensitive calcium mobilization and activation of a nonselective cation conductance; and cAMP increases mediated by Gs. The receptor is broadly expressed in embryonic tissues, including brain, as determined by Northern blot and reverse transcription-PCR analysis. Adult tissues have increased expression in skin, heart, and to a lesser extent, thymus. These data confirm the identification and extend the functionality of LPA4 as an LPA receptor, bringing the number of independently verified LPA receptors to five, with both overlapping and distinct signaling properties and tissue expression. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that signals through G protein-coupled receptors (GPCRs) to produce a range of biological responses. A recently reported fourth receptor, LPA4/GPR23, was notable for its low homology to the previously identified receptors LPA1–3 and for its ability to increase intracellular concentrations of cAMP and calcium. However, the signaling pathways leading to LPA4-mediated induction of cAMP and calcium levels have not been reported. Using epitope-tagged LPA4, pharmacological intervention, and G protein mini-genes, we provide independent confirmatory evidence that supports LPA4 as a fourth LPA receptor, including LPA concentration-dependent responses and specific membrane binding. Importantly, we further demonstrate new LPA-dependent activities of LPA4 that include the following: receptor internalization; G12/13- and Rho-mediated neurite retraction and stress fiber formation; Gq protein and pertussis toxin-sensitive calcium mobilization and activation of a nonselective cation conductance; and cAMP increases mediated by Gs. The receptor is broadly expressed in embryonic tissues, including brain, as determined by Northern blot and reverse transcription-PCR analysis. Adult tissues have increased expression in skin, heart, and to a lesser extent, thymus. These data confirm the identification and extend the functionality of LPA4 as an LPA receptor, bringing the number of independently verified LPA receptors to five, with both overlapping and distinct signaling properties and tissue expression. Lysophosphatidic acid (LPA, 2The abbreviations used are: LPA, lysophosphatidic acid; GPCR, G protein-coupled receptors; RT, reverse transcription; PTX, pertussis toxin; HA, hemagglutinin; EGFP, enhanced green fluorescent protein; BSA, bovine serum albumin; PBS, phosphate-buffered saline; pF, picofarad.2The abbreviations used are: LPA, lysophosphatidic acid; GPCR, G protein-coupled receptors; RT, reverse transcription; PTX, pertussis toxin; HA, hemagglutinin; EGFP, enhanced green fluorescent protein; BSA, bovine serum albumin; PBS, phosphate-buffered saline; pF, picofarad. 1-acyl-2-sn-glycerol-3-phosphate) is a water-soluble bioactive phospholipid that can be generated by many cell types and has been shown to influence multiple intracellular signaling pathways, including stimulation of phospholipase C and D, activation of small GTPases, MAPK (mitogen-activated protein kinase), and phosphoinositide 3-kinase (1.Moolenaar W.H. J. Biol. 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Here we confirm the finding that GPR23 is indeed a biologically relevant receptor for LPA and report several novel aspects of LPA4 signaling that extend its functional roles.MATERIALS AND METHODSCell Culture and Stable Transfection−B103 neuroblastoma cells and RH7777 hepatoma cells (23.Fukushima N. Kimura Y. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6151-6156Crossref PubMed Scopus (251) Google Scholar) were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT) and antibiotics (Invitrogen). LPA4-expressing stable cell lines were generated by transfecting B103 cells with linearized HA-tagged mouse LPA4-pcDNA3.1 (Invitrogen) using Effectene transfection reagent (Qiagen, Valencia, CA). Stable transfectants were selected for by adding 1 mg/ml geneticin (Invitrogen) to the culture media for 2 weeks.Production of LPA4 Retrovirus and G Protein Minigenes−HA-tagged mouse and human LPA4 cDNAs were amplified using the Expand High Fidelity PCR system (Roche Applied Science) using the following primers: 5′-ATGTACCCATACGATGTTCCAGATTACGCTATGGGTGACAGAAGATTTATTG-3′ (forward) and 5′-CTAGAAGGTGGATTCCAGCATT-3′ (reverse). PCR products were subcloned into the pGEM-T Easy T vector (Promega, Madison, WI), and the cDNA insert was sequenced at the The Scripps Research Institute (TSRI) sequencing core facility. The HA-tagged LPA4 cDNA was subsequently cloned into the NotI site of the LZRS-EGFP Moloney murine leukemia retroviral vector. The Phoenix ecotropic packaging cell line (24.Pear W. Scott M. Nolan G. Methods in Molecular Medicine: Gene Therapy Protocols. Humana Press Inc., Totowa, NJ1997: 41-57Google Scholar) was transfected with the retroviral construct using FuGENE 6 transfection reagent (Roche Applied Science). Retrovirus expression vector (LZRS-EGFP) and Phoenix retrovirus packaging cell lines were provided by Dr. Garry P. Nolan (Stanford University, Stanford, CA). At 48 h post-transfection, retroviral supernatant was filtered through a 0.45-μm filter and frozen in aliquots. Construction of Gq/11, Gs, G12, and G13 minigene retroviruses was described previously (22.Lee C.W. Rivera R. Gardell S. Dubin A.E. Chun J. J. Biol. Chem. 2006; 281: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 25.Gilchrist A. Li A. Hamm H.E. Sci. STKE 2002. 2002; : PL1Google Scholar).Western Blotting of Membrane Protein Fraction−HA-LPA4-expressing cells were homogenized in 20 mm Tris buffer, pH 7.5, containing 1 mm EGTA, 1 mm EDTA, and protease inhibitor mixture (Roche Applied Science) using a Dounce homogenizer. The sample was pre-cleared by centrifugation at 2,000 rpm for 5 min at 4 °C. The supernatant was then spun at 15,000 rpm for 90 min. Pellets were resuspended in ice-cold homogenization buffer containing 1% Triton X-100 and then centrifuged at 15,000 rpm for 20 min. The supernatant containing the membrane fraction was separated on a 4–12% SDS-polyacrylamide gel (Invitrogen) under reducing, denaturing conditions and transferred to polyvinylidene difluoride membrane (Millipore, Woburn, MA). HA-tagged LPA4 receptor expression was detected using an anti-HA antibody (Covance, Berkeley, CA) and horseradish peroxidase-conjugated anti-mouse secondary antibody and visualized with ECL Plus (Amersham Biosciences).F-actin Detection and Receptor Internalization Assay−Cells were grown overnight on poly-l-lysine-coated 12-mm glass coverslips. The following night, cells were switched to serum-free medium for 16–24 h. The next day, cells were incubated with LPA (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate; Avanti Polar-Lipids, Alabaster, AL) for 30 min and then fixed with 4% paraformaldehyde/PBS. Fixed cells were permeabilized in 0.1% Triton X-100/PBS for 15 min and then blocked in 3% BSA/PBS for 30 min. F-actin was visualized by staining with 25 μg/ml rhodamine-phalloidin (Sigma) in PBS/1% BSA for 40 min. Images were acquired using a fluorescence microscope fitted with an AxioCam camera (Carl Zeiss, Thornwood, NY). Receptor internalization was detected by treating serum-starved cells with BSA, LPA, or sphingosine 1-phosphate (Avanti Polar Lipids) for 15 min. Treated cells were then fixed with 4% paraformaldehyde in PBS for 1 h and permeabilized with 0.1% (w/v) Triton X-100 plus 3% BSA in PBS. HA-tagged LPA4 localization was detected by staining with an anti-HA antibody and Cy3-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA) using confocal microscopy (Carl Zeiss, Thornwood, NY).[3H]LPA Binding to Isolated Membranes−The LPA-binding assay has been described previously by Fukushima et al. (23.Fukushima N. Kimura Y. Chun J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6151-6156Crossref PubMed Scopus (251) Google Scholar). Briefly, membranes were isolated from transfected RH7777 cells harvested in ice-cold homogenization buffer (20 mm Tris-HCl, pH 7.5) containing 1 mm EDTA and protease inhibitors (Roche Applied Science) and centrifuged at 2,000 rpm for 10 min at 4 °C. The supernatant was then centrifuged at 15,000 rpm for 30 min at 4 °C. 40 μg of membrane fraction was incubated with [3H]LPA (1-oleoyl-[9,10-3H]LPA, 47 Ci/mmol; PerkinElmer Life Sciences) in LPA-binding buffer containing 0.1% fatty acid-free BSA (Sigma) and 0.5 mm CuSO4 for 30 min at room temperature. The [3H]LPA membrane fraction mixture was collected onto a Unifilter 96-GF/B (PerkinElmer Life Sciences). The filter was washed 10 times with binding buffer containing 1% BSA and dried for 30 min at 50 °C. Thirty microliters of MicroScint-O was added to each well of the filter, and radioactivity was measured using a microplate liquid scintillation counter (PerkinElmer Life Sciences). Total and nonspecific binding were evaluated in the absence and presence of 10 μm unlabeled LPA, respectively.G Proteins and Rho Inhibition in Cultured Cells−To investigate G protein coupling with LPA4, stable LPA4-expressing B103 cells were infected with several G protein minigenes and tested 2 days later or treated with PTX (200 ng/ml; List Biological Laboratories, Campbell, CA) for 12 h. To inhibit the Rho pathway, LPA4-expressing cells were treated with either the Rho inhibitor C3 transferase (10 μg/ml; Cytoskeleton Denver, CO) for 24 h or the ROCK inhibitor Y27632 (10 μm; Calbiochem) for 45 min. Rounded cells were counted following treatment with 1 μm LPA for 30 min in serum-free conditions.cAMP Measurements−Both acutely infected and stable transfectants expressing LPA4 were used in these experiments. LPA4-expressing and control B103 cells were serum-starved overnight in 24-well plates with or without PTX (200 ng/ml; List Biological Laboratories). Following treatment with 0.5 mm 3-isobutyl-1-methylxanthine for 20 min, cells were exposed to LPA (0, 1, 10, and 100 nm and 1 μm) for 30 min with or without forskolin (5 μm). Cells were then lysed in 0.1 n HCl, and cellular cAMP levels were quantified using an enzyme-linked immunosorbent assay-based detection kit (Cayman Chemicals, Ann Arbor, MI) according to the manufacturer's directions.Determination of Intracellular Calcium Mobilization−B103 cells stably expressing HA-tagged LPA4 were infected with G protein minigenes 2 days prior to testing and/or exposed to PTX overnight, and control cells were plated on glass coverslips and loaded with Fura-2 acetoxymethyl ester (Fura2-AM) (2.5 μm) for ratiometric calcium imaging studies. Cells lacking LPA4 expression served as controls. Cells were incubated for 30–60 min at 37 °C in Opti-MEM (Invitrogen) containing Fura2-AM (2.5 μm) and 1.5 μm of pluronic acid (Molecular Probes, Eugene, OR) and then briefly washed with Opti-MEM. Coverslips were perfused with Opti-MEM in a laminar flow perfusion chamber (Warner Instrument Corp., Hamden, CT). LPA (1 μm) was bath-applied by gravity perfusion when indicated. Images of Fura-2-loaded cells with the excitation wavelength alternating between 340 and 380 nm were captured with a cooled CCD camera (Carl Zeiss). The ratio of fluorescence intensity at the two wavelengths was calculated after subtraction of background fluorescence. Ratio levels were determined from groups of 20–40 individual cells and analyzed using MetaFluor (Universal Imaging Corp., West Chester, PA).Electrophysiology−The whole cell patch clamp technique was used to record and measure LPA-induced effects on whole cell currents of B103 cells stably expressing LPA4 receptor. The involvement of Gq in the modulations of cellular conductance was determined with stable LPA4-B103 cells after infection with virus expressing the Gq minigene or empty vector that served as the control for minigene infection. Some LPA4-B103 cells were treated overnight with PTX as described above. The extracellular solution (pH 7.4 with NaOH) contained the following: NaCl 145 mm, KCl 2.5 mm, CaCl2 1.5 mm, MgSO4 1.5 mm, HEPES 10 mm, dextrose 10 mm. LPA and vehicle were added to the bath by gravity perfusion at room temperature. Recording electrodes were fabricated and coated with dental periphery wax as described previously (22.Lee C.W. Rivera R. Gardell S. Dubin A.E. Chun J. J. Biol. Chem. 2006; 281: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Intracellular solution (pH 7.4) contained potassium gluconate 100 mm, KCl 25 mm, MgCl2 3 mm, CaCl2 0.483 mm, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-K4 1.0 mm, hemi-Na-HEPES 10 mm. The resistive whole cell configuration and data acquisition were achieved as described in Ref. 22.Lee C.W. Rivera R. Gardell S. Dubin A.E. Chun J. J. Biol. Chem. 2006; 281: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar. Cells were chosen for study if depolarization-activated peak inward current density was less than –30 pA/pF. During application of LPA (1 μm) or washout, cells were held at –50 mV, and the Vm value was stepped to –120 mV for 60 ms and ramped to +120 mV (at a rate of 1 mV/ms) every 2 s. All data are expressed in terms of the Cm value during stimulus application (current density).Reverse Transcription-PCR−TRIzol reagent (Invitrogen) was used to extract RNA from cultured cells or tissues as described by the manufacturer. Five micrograms of total RNA was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen). An equal quantity of cDNA was used to amplify LPA4 and β-actin transcripts using the following conditions: 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 45 s for a total of 30–35 cycles. The primers were used as follows: LPA4, 5′-AGGCATGAGCACATTCTCTC-3′ (forward) and 5′-CAACCTGGGTCTGAGACTTG-3′ (reverse); β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ (forward) and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (reverse). The PCR products were analyzed by electrophoresis on a 1.2% agarose gel.Northern Blot Analysis−A Northern blot (OriGene Technologies, Rockville, MD) containing 2 μg/lane of adult mouse poly(A)+ RNA from several mouse tissues was probed with a random primed full-length 32P-labeled mouse LPA4 and human β-actin cDNA. DNA labeling was performed with the high prime DNA labeling kit (Roche Applied Science), and unincorporated nucleotides were separated out by passing the reaction mixture over a Sephadex G-50 quick spin column (Roche Applied Science). The Northern blot was then probed overnight in ULTRAhyb hybridization solution (Origen), washed several times, and analyzed using a PhosphorImager detection system.Statistical Analysis of Data−Each data point was calculated from triplicate samples unless otherwise indicated. The data are presented ±S.D. Statistical analysis was performed by one-way analysis of variance and Dunnett's method, or Student's t test.RESULTSMouse LPA4 cDNA was epitope-tagged with hemagglutinin (HA) sequence at the 5′-end of the extracellular domain, and the construct was introduced into a murine leukemia, replication-deficient, bicistronic retroviral vector (Fig. 1A). This construct co-expresses tagged LPA4 and EGFP, thus allowing for the identification of receptor expression in living and fixed cells by fluorescence microscopy.To demonstrate cell surface receptor gene expression, LPA4-infected B103 cells were labeled with an anti-HA primary antibody, and receptor was visualized using Cy3-conjugated secondary antibody (Fig. 1B). Western blot analysis with an anti-HA antibody showed that the tagged receptor was of the expected size (Fig. 1C). To determine whether the LPA4 receptor was indeed capable of binding to LPA, membranes were prepared from LPA4-expressing cells and incubated with [3H]LPA in the presence or absence of 10 μm cold LPA. Membranes prepared from LPA4-expressing cells revealed statistically significant specific [3H]LPA binding compared with membranes isolated from control cells (Fig. 1E).G protein-coupled receptors typically undergo internalization during prolonged agonist exposure (28.Ferguson S.S. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). We reasoned that if GPR23 is a physiologically relevant receptor for LPA but not other lysophospholipids, then LPA exposure would likely produce agonist-induced receptor internalization, whereas other ligands would not. We performed a standard internalization assay using B103 cells expressing HA-tagged LPA4 and visualized LPA4 localization with anti-HA immunolabeling and confocal microscopy. We found that receptor internalization occurred following LPA treatment but not with another lysophospholipid, sphingosine 1-phosphate (Fig. 1D). We noticed that during exposure to LPA the cells retracted their processes, a response observed for activation of heterologously expressed LPA1,2,5 in B103 cells (see below). LPA4 internalization was not a consequence of cell rounding because sphingosine 1-phosphate-induced retraction did not cause LPA4 internalization.We next sought to determine the signaling pathways mediating LPA4-induced morphological changes in LPA4-expressing B103 cells. A strong cell rounding response occurred within 30 min of LPA treatment (Fig. 2A). However, control vector-infected cells were unresponsive to LPA (data not shown). F-actin staining with rhodamine-phalloidin demonstrated stress fiber formation in LPA4-expressing RH7777 cells (Fig. 2B) but not control cells. In B103 cells expressing LPA4, LPA produced a concentration-dependent increase in the proportion of rounded cells (Fig. 2C), with ∼70% of LPA4-infected cells rounding after exposure to 1 μm LPA.FIGURE 2LPA4-mediated neurite retraction of B103 cells and stress fiber formation in RH7777 cells. A, serum-starved LPA4-expressing B103 cells were treated with fatty-acid free BSA (0.1%) or LPA (1 μm) for 30 min and then fixed and mounted on glass slides. B, empty vector- or LPA4-expressing RH7777 cells were stimulated with 1 μm LPA for 30 min after overnight serum starvation. The cells were fixed and stained with rhodamine-phalloidin to detect actin stress fibers. C, quantification of LPA-induced neurite retraction for LPA4-expressing B103 cells. The data are mean ± S.D. (n = 3). **, p < 0.01 (one-way analysis of variance) versus basal 0 μm LPA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PTX and G-protein minigenes were used to determine which members of the heterotrimeric G-protein family are responsible for mediating the LPA-induced cell rounding of LPA4-expressing cells (Fig. 3A). G12/13 interacts with p115 RhoGEF, the Rho guanine nucleotide exchange factor (29.Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (672) Google Scholar, 30.Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (736) Google Scholar), activating the Rho signaling pathway to produce actin cytoskeleton rearrangement (31.Sah V.P. Seasholtz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (297) Google Scholar, 32.Seasholtz T.M. Majumdar M. Brown J.H. Mol. Pharmacol. 1999; 55: 949-956Crossref PubMed Scopus (203) Google Scholar). Using G12 and G13 minigenes to inhibit G12/13 signaling, LPA-induced cell rounding was significantly reduced (Fig. 3A). Recently, Gq/11 has also been shown to activate a Rho-dependent pathway in G12/13-deficient cells (33.Vogt S. Grosse R. Schultz G. Offermanns S. J. Biol. Chem. 2003; 278: 28743-28749Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). However, blocking this pathway using a pan-Gq/11 minigene did not inhibit cell rounding in response to LPA (Fig. 3A). Blocking Gi signaling with PTX pretreatment (Fig. 3A) or Gs signaling using a Gs minigene (data not shown) also failed to inhibit LPA-induced cell rounding.FIGURE 3G12, G13, and Rho signaling is involved in LPA4-mediated neurite retraction of B103 cells. A, LPA4-expressing B103 cells were pretreated with PTX (200 ng/ml) overnight or infected with a G protein minigene 2 days prior to testing. Cells were fixed after treatment with 1 μm LPA for 30 min. B, LPA4-expressing B103 cells were pretreated with C3 (10 μg/ml) for 24 h or pretreated with Y27632 (10 μm) for 45 min and then stimulated with 1 μm LPA for 30 min. Data are the mean ± S.D. (n = 3). **, p < 0.01; ***, p < 0.001 (one-way analysis of variance) versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To test the involvement of Rho signaling in LPA receptor-mediated cell rounding, we used C3 toxin and Y27632 to inhibit Rho and Rho kinase, respectively. Similar to LPA1- and LPA2-induced Rho signaling-dependent cell rounding in infected cells (26.Ishii I. Contos J.J. Fukushima N. Chun J. Mol. Pharmacol. 2000; 58: 895-902Crossref PubMed Scopus (181) Google Scholar), the cell rounding response in LPA4-expressing B103 cells was inhibited by both C3 and Y27632 (Fig. 3B). Furthermore, LPA4-mediated stress fiber formation in RH7777 cells was completely blocked by C3 and Y27632 treatment (data not shown).Because all known lysophospholipid GPCRs can influence cAMP levels (14.Ishii I. Fukushima N. Ye X. Chun J. Annu. Rev. Biochem. 2004; 73: 321-354Crossref PubMed Scopus (636) Google Scholar), we analyzed cAMP levels in LPA4-expressing B103 cells after LPA exposure. LPA increased intracellular cAMP levels in LPA4-expressing cells either in the absence or presence of forskolin (5 μm) (Fig. 4, A and B). Because intracellular cAMP levels are regulated by Gs α subunit as well as G protein βγ subunits, (34.Tang W.J. Gilman A.G. Science. 1991; 254: 1500-1503Crossref PubMed Scopus (741) Google Scholar, 35.Federman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (480) Google Scholar), we tested whether Gs was responsible for LPA4-mediated cAMP induction using a Gs minigene (Fig. 4C). LPA4-mediated cAMP accumulation was completely blocked by a Gs minigene (Fig. 4C). Thus, the LPA4 receptor coupling to Gs activates adenylyl cyclase to increase intracellular cAMP levels.FIGURE 4LPA-induced intracellular cAMP accumulation. A, concentration-response data for basal cAMP content in acutely LPA4-infected B103 cells (▪, PTX-untreated cells; ▴, PTX-treated cells (200 ng/ml)) and empty vector infected cells (□, PTX-untreated cells; ▵, PTX-treated cells) exposed to LPA in the presence of 0.5 mm 3-isobutyl-1-methylxanthine. B, effect of forskolin on cAMP accumulation in LPA4-infected B103 cells. LPA was added to cells in the presence of 5 μm forskolin in the same manner as in A, and basal levels were about 15 times higher than forskolin-free conditions. C, cAMP accumulation in stably transfected LPA4-expressing B103 cells infected with control or Gs minigene retrovirus after treatment with 1 μm LPA under forskolin-free conditions. About 50% of the cells expressed LPA4 through acute retroviral infection in A and B, whereas stably expressing cells were used for the experiments employing minigene retroviruses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next sought to determine the signaling pathway(s) mediating LPA4-induced calcium mobilization in B103 cells (20.Noguchi K. Ishii S. Shimizu T. J. Biol. Chem. 2003; 278: 25600-25606Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). In LPA4-expressing B103 cells (Fig. 5A), but not vector control cells (Fig. 5B), LPA significantly elevated intracellular calcium levels. The response appears to desensitize in the continued presence of LPA. Interestingly, when cells were pretreated with PTX or expressed a Gq minigene for at lea
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