Amplification of Ca2+ Signaling by Diacylglycerol-mediated Inositol 1,4,5-Trisphosphate Production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP3 produced is particularly critical to determining the spatio-temporally coordinated Ca2+-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca2+-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity. Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP3 produced is particularly critical to determining the spatio-temporally coordinated Ca2+-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca2+-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity. Stimulation of a wide variety of cells by hormones, neurotransmitters, or growth factors leads to the activation of phospholipase C (PLC) 1The abbreviations used are: PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; OAG, 1-oleoyl-2-acyl-sn-glycerol; DAG, diacylglycerol; 2-APB, 2-aminoethyl diphenylborinate; CPA, cyclopiazonic acid; DMEM, Dulbecco's modified essential medium; SAG, 1-stearoyl-2-arachidonoyl-sn-glycerol; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PTK, protein tyrosine kinase; TRPC, canonical transient receptor potential; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo3,4-dpyrimidine; PP3, 4-amino-7-phenylpyrazol-3,4-dpyrimidine. and triggers inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ signaling via activation of IP3 receptors (IP3Rs) on the endoplasmic reticulum (1Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3311) Google Scholar, 2Berridge M.J. Lipp P. Bootman M.D. Science. 2000; 287: 1604-1605Crossref PubMed Scopus (164) Google Scholar). Proper regulation of receptor-IP3/Ca2+ signaling is very important, because the IP3-induced Ca2+ release underlying a variety of spatio-temporal Ca2+ dynamics has been shown to considerably affect various cellular functions such as smooth muscle contraction, fertilization, immune response, gene expression, synaptic plasticity, development, and so on (3Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1769) Google Scholar, 4Bootman M.D. Lipp P. Berridge M.J. J. Cell Sci. 2001; 114: 2213-2222Crossref PubMed Google Scholar). The molecular mechanisms responsible for the versatility of IP3-mediated Ca2+ signaling include (i) a plasma membrane receptor-PLC system that generates the second messenger IP3 (5Hollinger S. Hepler J.R. Pharmacol. Rev. 2002; 54: 527-559Crossref PubMed Scopus (601) Google Scholar, 6Chen C.A. Manning D.R. Oncogene. 2001; 20: 1643-1652Crossref PubMed Scopus (169) Google Scholar, 7Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1219) Google Scholar), (ii) IP3Rs (8Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (371) Google Scholar, 9Taylor C.W. Genazzani A.A. Morris S.A. Cell Calcium. 1999; 26: 237-251Crossref PubMed Scopus (242) Google Scholar), (iii) Ca2+ sequestration mechanisms (10Arai M. Jpn. Heart J. 2000; 41: 1-13Crossref PubMed Scopus (12) Google Scholar, 11Misquitta C.M. Mack D.P. Grover A.K. Cell Calcium. 1999; 25: 277-290Crossref PubMed Scopus (111) Google Scholar), (iv) IP3 phosphatases and kinases (12Joseph S.K. Williamson J.R. Arch. Biochem. Biophys. 1989; 273: 1-15Crossref PubMed Scopus (52) Google Scholar, 13Nalaskowski M.M. Mayr G.W. Curr. Mol. Med. 2004; 4: 277-290Crossref PubMed Scopus (25) Google Scholar), and (v) Ca2+ influx machinery (14Venkatachalam K. Van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (337) Google Scholar). These molecular mechanisms interact with each other, and thus, they all affect each other. For example, intracellular Ca2+ released from Ca2+ stores has a positive feedback effect on PLC activity (15Meyer T. Stryer L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5051-5055Crossref PubMed Scopus (323) Google Scholar, 16Berridge M. Lipp P. Bootman M. Curr. Biol. 1999; 9: R157-R159Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). IP3R activity is positively or negatively regulated by Ca2+ depending on the intracellular Ca2+ level (17Michikawa T. Hirota J. Kawano S. Hiraoka M. Yamada M. Furuichi T. Mikoshiba K. Neuron. 1999; 23: 799-808Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), and Ca2+ entry machinery is regulated by store depletion, conformational changes of IP3Rs, or direct binding of phospholipids and DAG (14Venkatachalam K. Van Rossum D.B. Patterson R.L. Ma H.T. Gill D.L. Nat. Cell Biol. 2002; 4: E263-E272Crossref PubMed Scopus (337) Google Scholar). Thus, numerous regulatory mechanisms of these signaling molecules and their interactions allow cells to establish precise and complex Ca2+ patterns that contribute to various physiological phenomena. In this paper, we report a novel regulatory mechanism involved in receptor-IP3-Ca2+ signaling, i.e. DAG-mediated positive feedback stimulation of PLC. We unexpectedly found that 1-oleoyl-2-acyl-sn-glycerol (OAG), a membrane-permeable derivative of DAG, induces Ca2+ oscillation in COS-7 cells independently of PKC activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was also observed in HeLa cells, CHO-K1 cells, and astrocytes, suggesting that the OAG-induced Ca2+ oscillation is a general phenomenon in various types of cells. The OAG-induced Ca2+ oscillation was dependent on Ca2+ release through IP3Rs, because 2-aminoethyl diphenylborinate (2-APB) (18Maruyama T. Kanaji T. Nakade S. Kanno T. Mikoshiba K. J. Biochem. (Tokyo). 1997; 122: 498-505Crossref PubMed Scopus (774) Google Scholar), an IP3R inhibitor, or depletion of Ca2+ stores with cyclopiazonic acid (CPA) abolished the Ca2+ mobilization. In addition, we found that treatment of COS-7 cells with a PLC inhibitor or expression of the IP3-absorbent protein “IP3-sponge” completely abolished the OAG-induced Ca2+ oscillation, indicating that OAG stimulates IP3 production via PLC activation. We also discovered that accumulation of endogenous DAG as a result of exposure to a DAG-lipase inhibitor increases the sensitivity of the Ca2+ response in COS-7 cells to low-dose ATP stimulation. These findings suggested that DAG produced via the receptor-PLC signaling cascade leads to further PLC activation, resulting in the increased IP3 production and amplification of receptor-Ca2+ signaling. We propose that, in addition to the well known positive feedback effect of Ca2+ on PLC activity, this novel signal cross-talk between DAG and PLC activity may be a crucial mechanism regulating the amount of IP3, which is an important factor in determining the threshold of Ca2+ signaling generation and Ca2+ dynamics in response to agonist stimulation, and that it may play an important role in physiological phenomena that are dependent on IP3-induced Ca2+ release. Culture—COS-7 cells, CHO-K1, and HeLa cells were cultured in Dulbecco's modified essential medium (DMEM) (Nakarai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin (Nakarai). Astrocytes were prepared from the cerebral cortex of postnatal 1 day mice or postnatal 1 day Wistar rats using the standard method (19Miller S. Romano C. Cotman C.W. J. Neurosci. 1995; 15: 6103-6109Crossref PubMed Google Scholar). Following trypsin treatment, cortices were dissociated by trituration and cultured in 75-mm2 flask with DMEM containing 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. The confluent cells then were treated with trypsin after overnight shaking (280 rpm) and replated on 3.5 cm of poly-l-lysine-coated glass-bottom dishes (Matsunami, Osaka, Japan) at a density of 1 × 104/dish. After 4–5 days from the replating, Ca2+ imaging was performed. We confirmed that the cultured cells were astrocytes by immunostaining with mouse anti-glial fibrillary acidic protein antibody (Sigma) (data not shown). Pharmacological Reagents—The following reagents were used in study: 1-oleoyl-2-acyl-sn-glycerol (OAG) (Calbiochem, Nakarai Tesque, and Avanti Polar Lipids, Alabaster, AL). To neglect the possible effect caused by contaminated substances, we used OAG purchased from the three companies and confirmed their same effect on Ca2+ mobilization: PP3 and PP2 (Calbiochem); 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) (Sigma); U73122 and U73343 (Calbiochem); RHC80267 (Calbiochem); 2-APB; staurosporine (Calbiochem); phorbol 12-myristate 13-acetate (PMA) (Calbiochem); CPA (Calbiochem); inositol 1,4,5-trisphosphate (Dojindo, Kumamoto, Japan); and ATP. GFP and IP3-Sponge Transfection—COS-7 cells plated on the 3.5-cm poly-l-lysine-coated glass bottom dish (Matsunami) were transfected with totally 1.0 μg of plasmids encoding GFP (pcDNA-N3 (Clontech, Palo Alto, CA)) and IP3-sponge (pcDNA-IP3-sponge). The G224 fragment encoding IP3-sponge (20Uchiyama T. Yoshikawa F. Hishida A. Furuichi T. Mikoshiba K. J. Biol. Chem. 2002; 277: 8106-8113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) was cloned into pcDNA3 (Clontech) (molar ratio 1:2) by lipid method using TransIT-LT1 (Mirus, Madison, WI) according to the manufacturer's instructions. After 36 h, the cells were used for Ca2+ imaging. Intracellular Ca2+Imaging—Cells plated on the 3.5-cm poly-l-lysine-coated glass bottom dish (Matsunami) were loaded with 5 μm Fura-2/AM (Dojindo) for 30 min at room temperature in the loading solution (in mm): 115 NaCl; 5.4 KCl; 1 MgCl2; 2 mm CaCl2; 20 Hepes; and 10 glucose, pH 7.42. Fura-2 fluorescence images were analyzed using an inverted microscope (IX70, Olympus, Tokyo, Japan) and a video image analysis system (Argus-50/CA, Hamamatsu Photonics, Hamamatsu, Japan) with excitation filters at 340 ± 10 and 380 ± 10 nm, a dichroic beam splitter at 400 nm, and a bandpass emission filter at 510–550 nm. The recording solution contains (in mm): 115 NaCl; 5.4 KCl; 1 MgCl2; 20 Hepes; and 10 glucose, pH 7.42 (unless mentioned otherwise). Solutions containing phospholipids (OAG and SAG) were used after brief sonication. Ca2+Release from Cerebellar Microsomes—Mice cerebellar microsome fractions were prepared as described previously (17Michikawa T. Hirota J. Kawano S. Hiraoka M. Yamada M. Furuichi T. Mikoshiba K. Neuron. 1999; 23: 799-808Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). IP3-induced Ca2+ release from cerebellar microsomes was measured with Fura-2 and a fluorospectrometer, CAF110 (Jasco, Tokyo, Japan). The membrane fractions were suspended at a concentration of 200 μg/ml protein in the buffer containing 1 μg/ml oligomycin (Sigma), 2 mm MgCl2, 25 μg/ml creatine kinase (Roche Diagnostics, Tokyo, Japan), 10 mm creatine phosphate (Sigma), and 2 μm Fura-2 (Dojindo) After loading Ca2+ into microsomes by activating Ca2+-ATPase using 1 mm ATP (Sigma), Fura-2 was alternately excited with 340 and 380 nm and the fluorescence changes at 510 ± 10 nm were detected in response to 100 μm OAG and 100 μm IP3 application. At the end of each set of experiments, maximum and minimum values of Fura-2 fluorescence were obtained in the presence of 2 mm CaCl2 and 10 mm EGTA, respectively (21Grynkiewicz M. J. Biol. Chem. Full Text PDF PubMed Scopus Google Scholar). DAG cells at in dishes were treated with 50 μm RHC80267 or for 10 min in the recording buffer containing 2 mm Ca2+ and then with μm ATP for after of the cells were with 1 of and to a The dish was with 1 of which then was to the previously and the of DAG level was using this DAG kinase was as previously J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Biophys. 1988; PubMed Scopus Google Scholar). After DAG using the was to and the density of to DAG was using a for image analysis DAG OAG, in of a novel function of DAG in intracellular signaling, we measured Ca2+ following the of a membrane-permeable DAG derivative, OAG, to COS-7 cells. we found that, in the of OAG to COS-7 cells induces Ca2+ oscillation with a of min also The of Ca2+ oscillation from cell to the solution contains very low concentration of Ca2+ the Ca2+ following OAG not been to Ca2+ influx through cell surface such as as previously T. M. C. T. Nature. 1999; PubMed Scopus Google Scholar, T. K. A. T. T. S. K. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Cell Calcium. PubMed Scopus Google Scholar) because we also detected the OAG-induced Ca2+ in the extracellular solution containing mm The of a DAG derivative, to Ca2+ oscillation The effect of OAG was shown in Ca2+ was 10 μm OAG was The OAG-induced Ca2+ oscillation was observed at 30 μm OAG, and the of Ca2+ oscillation increased as the OAG concentration was increased to and μm The between OAG and the of the Ca2+ response also to as the OAG concentration was The of the not Ca2+ (data not shown). These results were because Ca2+ OAG M. Cell Calcium. PubMed Scopus Google Scholar, L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. A. J. Neurosci. 23: PubMed Google Scholar). we detected Ca2+ after exposure to OAG purchased from three companies the OAG-induced Ca2+ response is to been an To OAG Ca2+ oscillation in types of cells, we OAG to CHO-K1 cells, HeLa cells, and astrocytes and for a Ca2+ shown in in the of we observed OAG-induced Ca2+ in these cells, the same as in COS-7 cells, suggesting that the OAG-induced Ca2+ oscillation is a general phenomenon to COS-7 cells. Ca2+Release through IP3Rs OAG-induced Ca2+ the of Ca2+ release from Ca2+ stores to the OAG-induced Ca2+ oscillation in COS-7 cells. the Ca2+ store been with a Ca2+ inhibitor, CPA OAG OAG Ca2+ oscillation the of 2-APB, an inhibitor of IP3Rs, completely abolished the OAG-induced Ca2+ oscillation suggesting that the OAG-induced Ca2+ oscillation was caused by Ca2+ release through To OAG IP3Rs we an in Ca2+ release using mice cerebellar shown in Ca2+ release from cerebellar microsomes was detected in response to 100 μm OAG or the the of IP3 to cerebellar microsomes transient Ca2+ release. In addition, OAG not the amount of Ca2+ release by IP3 the OAG-induced Ca2+ oscillation is to be by IP3 production by direct activation of To the possible of IP3 production in the OAG-induced Ca2+ oscillation, we the cells to a PLC inhibitor, to OAG application. shown in 10 μm U73122 completely abolished the OAG-induced Ca2+ oscillation in COS-7 cells the U73343 effect Furthermore, we IP3 protein IP3-sponge (20Uchiyama T. Yoshikawa F. Hishida A. Furuichi T. Mikoshiba K. J. Biol. Chem. 2002; 277: 8106-8113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and GFP in COS-7 cells OAG Ca2+ oscillation was detected in COS-7 cells, cells that not IP3-sponge Ca2+ oscillation of GFP not affect the OAG-induced Ca2+ oscillation (data not shown). Thus, these results suggested that OAG and also DAG are of production of IP3 via PLC activation, which Ca2+ release through of the of the OAG-induced further into the molecular mechanisms DAG to PLC activation, we analyzed the of PKC activity in the OAG-induced Ca2+ shown in the OAG-induced Ca2+ oscillation independently of PKC activation, because of PMA, a of PKC to COS-7 cells not Ca2+ oscillation and of cells with a kinase inhibitor, staurosporine not the Ca2+ following OAG because cellular DAG is by DAG we treated COS-7 cells with the DAG-lipase inhibitor RHC80267 OAG application. shown in RHC80267 not affect the OAG-induced oscillation, indicating that it was not caused by of we found that a tyrosine kinase inhibitor, PP2, completely abolished the OAG-induced Ca2+ oscillation, the PP2 PP3 used as a to the OAG-induced Ca2+ oscillation DAG the to ATP Stimulation in COS-7 we the to the intracellular Ca2+ in COS-7 cells of DAG produced in response to ATP stimulation. We that of produced DAG was by DAG-lipase inhibitor, DAG further production of IP3, resulting in the in Ca2+ in response to agonist stimulation. The of the DAG-lipase inhibitor, has been a method to the effect of endogenous DAG on physiological phenomena T. M. C. T. Nature. 1999; PubMed Scopus Google Scholar, T. T. Yamada 2004; PubMed Scopus (12) Google Scholar), and we the increased endogenous DAG level by RHC80267 treatment ATP stimulation using DAG kinase To the possible effect of PKC activity on Ca2+ we COS-7 cells with nm for 36 to PKC activity J. Neurosci. PubMed Google Scholar) and Ca2+ by of various of ATP and presence and of DAG-lipase inhibitor RHC80267 shown in the results that, of the cells released Ca2+ in response to μm ATP stimulation a of cells release Ca2+ from Ca2+ stores in response to μm ATP stimulation ± ± In addition, the of cells that to and μm ATP stimulation were the of cells μm ± RHC80267 ± μm ± RHC80267 ± μm ± RHC80267 ± ± cells to Ca2+ the cells, was in Ca2+ between the cells and the cells. with a of ATP all of the and cells released Ca2+ and were between in the of cells that or the of the Ca2+ release (data not shown). Thus, these results suggested that a level of DAG produced in response to ATP stimulation further IP3 production via PLC activation, resulting in the Ca2+ from Ca2+ stores through IP3Rs in response to low-dose agonist stimulation. DAG is well known as a PKC physiological of DAG been activation of Science. 1998; PubMed Scopus Google Scholar) and T. M. C. T. Nature. 1999; PubMed Scopus Google Scholar, T. K. A. T. T. S. K. K. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and inhibition of J. Biophys. J. 1995; Full Text PDF PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar). We propose a function of DAG, i.e. IP3 production via PLC activation. The results of the that a DAG derivative, OAG, induces Ca2+ oscillation in various types of cells, and on the following we that the OAG-induced Ca2+ oscillation was to IP3-mediated Ca2+ release through IP3Rs as the of extracellular of the oscillation by store by IP3-mediated signaling and and by PLC These findings the of a positive feedback effect of DAG on PLC activity, and with this we that the DAG-mediated positive feedback signaling to PLC to the amplification of Ca2+ signaling, suggesting a physiological of the feedback The amount of IP3 produced by OAG be from the of Ca2+ signaling, because the is dependent on the concentration of the to cells. In types of cells, of low of which result in the production of of IP3, induces a transient in Ca2+ a or Ca2+ of Ca2+ the of agonist application. at the concentration we OAG an and not a Ca2+ in COS-7 cells the amount of IP3 produced by DAG-mediated signaling been is by the that a low level of expression of IP3-sponge completely abolished the OAG-induced oscillation because of Ca2+ by IP3-sponge has been shown to be dependent on its expression level and the of the agonist (20Uchiyama T. Yoshikawa F. Hishida A. Furuichi T. Mikoshiba K. J. Biol. Chem. 2002; 277: 8106-8113Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). the amount of IP3 from the DAG-mediated signaling be in IP3-mediated Ca2+ signaling agonist stimulation is with this we that accumulation of intracellular DAG with an DAG-lipase inhibitor greatly increased the number of cells that to low and of ATP we that IP3 production is an important molecular mechanism in the regulation of Ca2+ signaling in addition to numerous molecular mechanisms of IP3R activity by and of signaling from surface receptors to M. M. A. J. Neurosci. 23: PubMed Google Scholar) the that OAG to cells Ca2+ oscillation that was dependent on the extracellular Ca2+ that the OAG-induced Ca2+ oscillation was caused by Ca2+ entry through and that Ca2+ release from stores was not involved in the Ca2+ In the presence of we observed Ca2+ in cells that were in the of Ca2+ (data not the of the cells Ca2+ oscillation in response to OAG in the presence of extracellular Ca2+ was by M. M. 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DAG and Ca2+ concentration changes may the spatio-temporally Ca2+ dynamics by The molecular mechanisms by which DAG PLC to be is the molecular of of OAG-induced Ca2+ oscillation by PP2 of protein tyrosine kinases because we were to effect of DAG on of the activity in (data not is to be a direct of DAG in the DAG-mediated PLC activation pathway. Thus, DAG may that to activation. of PLC is by At PLC been to are and M.J. Rev. 2000; PubMed Scopus Google Scholar, K. J. Biochem. (Tokyo). 2002; PubMed Scopus Google Scholar), and of has been to be by J. 2001; 20: PubMed Scopus Google Scholar). In of the that the activity of the of is increased by DAG Science. 1998; PubMed Scopus Google Scholar), may be of the for the responsible for the IP3 suggested possible of activity in the DAG-mediated PLC activation, activity is involved in the signaling is is because be by S. S.G. Science. PubMed Scopus Google Scholar, F. S.G. Biochem. Biophys. PubMed Scopus Google Scholar, F. C. Mol. Pharmacol. 2002; PubMed Scopus Google Scholar). are also involved in the activation of receptor-PLC signaling through tyrosine T. S. M. S. Mikoshiba K. T. Science. 1997; PubMed Scopus Google Scholar), the of such signaling by may the molecular mechanism of the PLC activation. In we propose a novel function of DAG as an of IP3 production through PLC activation. The novel signal cross-talk between DAG and the IP3-mediated signal Ca2+ signaling in response to extracellular Ca2+ release an important role in various physiological functions synaptic plasticity, gene expression, and development, the increased IP3 production via DAG-mediated signaling may affect numerous phenomena. We all of the of T. A. and S. for their and with