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Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLC 2 in B lymphocytes

2003; Springer Nature; Volume: 22; Issue: 18 Linguagem: Inglês

10.1093/emboj/cdg457

1460-2075

Motohiro Nishida,

Protein Kinase Regulation and GTPase Signaling

Article15 September 2003free access Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCγ2 in B lymphocytes Motohiro Nishida Motohiro Nishida Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan Search for more papers by this author Kenji Sugimoto Kenji Sugimoto Institute of Advanced Energy, Kyoto University, and PRESTO, Japan Science and Technology Corporation, Uji, Kyoto, 611-0011 Japan Search for more papers by this author Yuji Hara Yuji Hara Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501 Japan Search for more papers by this author Emiko Mori Emiko Mori Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501 Japan Search for more papers by this author Takashi Morii Takashi Morii Institute of Advanced Energy, Kyoto University, and PRESTO, Japan Science and Technology Corporation, Uji, Kyoto, 611-0011 Japan Search for more papers by this author Tomohiro Kurosaki Tomohiro Kurosaki Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, and Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Moriguchi, 570-8506 Japan Search for more papers by this author Yasuo Mori Corresponding Author Yasuo Mori Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501 Japan Search for more papers by this author Motohiro Nishida Motohiro Nishida Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan Search for more papers by this author Kenji Sugimoto Kenji Sugimoto Institute of Advanced Energy, Kyoto University, and PRESTO, Japan Science and Technology Corporation, Uji, Kyoto, 611-0011 Japan Search for more papers by this author Yuji Hara Yuji Hara Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501 Japan Search for more papers by this author Emiko Mori Emiko Mori Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501 Japan Search for more papers by this author Takashi Morii Takashi Morii Institute of Advanced Energy, Kyoto University, and PRESTO, Japan Science and Technology Corporation, Uji, Kyoto, 611-0011 Japan Search for more papers by this author Tomohiro Kurosaki Tomohiro Kurosaki Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, and Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Moriguchi, 570-8506 Japan Search for more papers by this author Yasuo Mori Corresponding Author Yasuo Mori Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501 Japan Search for more papers by this author Author Information Motohiro Nishida1, Kenji Sugimoto3, Yuji Hara1,2, Emiko Mori1,2, Takashi Morii3, Tomohiro Kurosaki4 and Yasuo Mori 1,2 1Division of Molecular and Cellular Physiology, Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi, 444-8585 Japan 2Laboratory of Molecular Biology, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 606-8501 Japan 3Institute of Advanced Energy, Kyoto University, and PRESTO, Japan Science and Technology Corporation, Uji, Kyoto, 611-0011 Japan 4Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, and Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Moriguchi, 570-8506 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:4677-4688https://doi.org/10.1093/emboj/cdg457 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In non-excitable cells, receptor-activated Ca2+ signalling comprises initial transient responses followed by a Ca2+ entry-dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5-trisphosphate (IP3) sensor revealed that in B lymphocytes, receptor-activated and store-operated Ca2+ entry greatly enhanced IP3 production, which terminated in phospholipase Cγ2 (PLCγ2)-deficient cells. Association between receptor-activated TRPC3 Ca2+ channels and PLCγ2, which cooperate in potentiating Ca2+ responses, was demonstrated by co-immunoprecipitation. PLCγ2-deficient cells displayed diminished Ca2+ entry-induced Ca2+ responses. However, this defect was canceled by suppressing IP3-induced Ca2+ release, implying that IP3 and IP3 receptors mediate the second Ca2+ phase. Furthermore, confocal visualization of PLCγ2 mutants demonstrated that Ca2+ entry evoked a C2 domain-mediated PLCγ2 translocation towards the plasma membrane in a lipase-independent manner to activate PLCγ2. Strikingly, Ca2+ entry-activated PLCγ2 maintained Ca2+ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca2+ entry with PLCγ2 translocation and activation controls the amplification and co-ordination of receptor signalling. Introduction In non-excitable cells, stimulation of surface membrane receptors induces Ca2+ signals via activation of phospholipase C (PLC), which hydrolyses phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) (Berridge, 1993; Clapham, 1995). IP3 triggers rapid Ca2+ release from internal Ca2+ stores of the endoplasmic reticulum (ER) by activating IP3 receptors (IP3R) and a consequent transient increase in cytosolic Ca2+ concentration ([Ca2+]i). The initial phase of receptor-activated Ca2+ signalling is followed by sustained and/or oscillatory [Ca2+]i changes. The molecular mechanism underlying the second phase is still elusive (Feske et al., 2001; Mori et al., 2002), despite the vital role played by this phase in various biological responses (Miyazaki et al., 1992; Thorn et al., 1993; Dolmetsch et al., 1998; Li et al., 1998). The second phase of Ca2+ signalling is critically controlled by receptor-activated Ca2+ entry processes such as capacitative Ca2+ entry (CCE) mediated by store-operated Ca2+ channels (SOCs) coupled to IP3-induced Ca2+ release or consequent depletion of Ca2+ stores (Putney and Bird, 1993). In electrophysiological measurement, SOCs and other Ca2+ channels activated by DG in a membrane-delimited and protein kinase C (PKC)-independent manner have been identified (Hofmann et al., 1999; Okada et al., 1999; Inoue et al., 2001; Gamberucci et al., 2002). In [Ca2+]i measurement, it has been postulated that CCE is distinguished as an 'off' response elicited by readministration of extracellular Ca2+ to cells after pretreatment with store-depleting agents such as thapsigargin (TG) in the absence of Ca2+ (Parekh et al., 1997). Importantly, there is a large body of evidence that the properties of IP3R Ca2+ release channels affect the temporal patterns of Ca2+ signals/oscillations (Tsien and Tsien, 1990; Miyazaki et al., 1992; Miyakawa et al., 1999). Thus, together with direct elevation of [Ca2+]i by the Ca2+ entered through SOCs, the 'capacitative' function of CCE, which refills Ca2+ stores to maintain Ca2+ release, is a major role played by Ca2+ entry in eliciting the second phase. In receptor-induced Ca2+ signalling, activation of PLC isoforms is controlled through distinct coupling mechanisms (Rhee, 2001). It has been commonly understood that receptor and non-receptor tyrosine kinases are responsible for activation of PLCγs. Recent evidence has added the novel view that PLCγs have multifunctional properties, and directly interact with numerous target proteins (Smith et al., 1994; Kim et al., 2000; Rebecchi and Pentyala, 2000; Rhee, 2001; Patterson et al., 2002; Putney, 2002; Runnels et al., 2002; Ye et al., 2002; Trebak et al., 2003). Here, we reveal that PLCγ2, beyond its role in initiating the Ca2+ response upon stimulation of tyrosine kinase-coupled receptors, mediates the second phase of Ca2+ signals in B lymphocytes. B cell receptor (BCR)-induced and store-operated Ca2+ entry elicited the translocation of PLCγ2 towards the plasma membrane in a lipase-independent manner, and enhanced PLCγ2 activation to maintain IP3 production and IP3-induced Ca2+ release. We suggest that Ca2+ entry is coupled with PLCγ2 translocation and activation to control physiological co-ordination and amplification of receptor-mediated signals. Results Activation of PLCγ2 by BCR-induced Ca2+ entry In chicken DT40 B lymphocytes, ligation of BCR with anti-IgM antibody (anti-IgM) induced rapid rises in [Ca2+]i followed by sustained elevation of [Ca2+]i in the presence of extracellular Ca2+ (Figure 1A). Since the sustained phase was eliminated by omitting extracellular Ca2+, the sustained phase must derive from Ca2+ entry. Real time changes in [IP3]i were assessed in individual cells, using a new IP3-selective sensor R9-PHIP3-D106 constructed based on the pleckstrin homology (PH) domain of PLCδ1 (Morii et al., 2002). Stimulation of BCR triggered a rapid decrease in R9-PHIP3-D106 fluorescence, indicative of an initial [IP3]i increase in the wild-type (WT) cells (Figure 1B). The increase in [IP3]i was sustained or slowly enhanced for >5 min in the presence of extracellular Ca2+, but was transient when Ca2+ was removed. Population IP3 measurement confirmed the observation (Supplementary figure 1C, available at The EMBO Journal Online). Since PLCγ2 mediates BCR-induced Ca2+ signalling (Takata et al., 1995), the PLCγ2-deficient (PLCγ2−) DT40 cell line was employed to examine whether the observed IP3 production is in fact due to PLCγ2 activation. The BCR-induced [IP3]i and [Ca2+]i rises terminated in the PLCγ2− cells (Figure 1C and D). The defects were resolved by heterologous expression of rat PLCγ2 in the PLCγ2− cells, but not by a lipase-dead PLCγ2 mutant (LD; see also Figure 4A). Figure 1.Extracellular Ca2+ elicits sustained PLCγ2 activation and receptor-evoked [Ca2+]i mobilization. (A) Left, representative time courses of Ca2+ responses in DT40 cells upon BCR stimulation with anti-IgM (1 μg/ml) in 2 mM Ca2+-containing external (n = 32 cells) or EGTA-containing, Ca2+-free (n = 32) solution. Right, peak BCR-induced [Ca2+]i rises and [Ca2+]i increases sustained after 5 min stimulation. (B) Left, representative time courses of BCR-induced changes in florescence intensities of IP3 indicator R9-PHIP3-D106 (n = 12–15). F is the fluorescence intensity and F0 is the initial F. Right, BCR-induced [IP3]i changes at peak and sustained after 5 min. (C and D) BCR-induced [Ca2+]i rises (C) and [IP3]i rises (D) at peak and sustained after 6 min stimulation in WT cells expressing GFP (GFP/WT), and in PLCγ2− cells expressing GFP (GFP/PLCγ2−), PLCγ2 (PLCγ2/PLCγ2−) or LD mutant (LD/PLCγ2−) (n = 22–60). Ca2+ is present in external solution. (E) PLCγ2 enhances Ca2+ responses induced by Ca2+ entry upon M1R stimulation. Ca2+ release was first evoked in Ca2+-free solution, and Ca2+ entry-induced Ca2+ responses were induced by readministration of 2 mM Ca2+ in WT cells expressing GFP and in PLCγ2− cells expressing GFP, PLCγ2, LD or ΔSH3 (n = 5–13). Left, average time courses. Right, peak [Ca2+]i rises in Ca2+-free solution and after Ca2+ readministration. Significance difference from control: *P < 0.05. Download figure Download PowerPoint Ca2+ entry induced by G protein-coupled receptor stimulation activates PLCγ2 To characterize the extracellular Ca2+-dependent sustained phase separately from the initial BCR-evoked phase in PLCγ2 activation, heterologously expressed M1 muscarinic acetylcholine receptor (M1R) (Sugawara et al., 1997) was stimulated with carbachol (CCh). This is because the different isoform PLCβ mediates PIP2 hydrolysis to trigger Ca2+ release upon M1R and Gq protein activation (Parekh et al., 1997). In the WT and PLCγ2− cells, stimulation of M5R triggers Ca2+ mobilization via Gq and PLCβ (Patterson et al., 2002). Stimulation of M1R induced smaller [Ca2+]i increases in the PLCγ2− cells compared with those in the WT cells (Supplementary figure 2). However, omission of extracellular Ca2+ abolished the difference between the WT and PLCγ2− cells in M1R-activated Ca2+ responses (Figure 1E). The data support the view that PLCγ2 is responsible for external Ca2+-dependent enhancement of Ca2+ responses, and that Ca2+ entry induced by PLCβ-coupled receptor leads to late sustained PLCγ2 activation. Furthermore, the defect in Ca2+ entry-induced Ca2+ response in PLCγ2− cells was partially restored by the LD mutant in the PLCγ2− cells (Figure 1E), suggesting the lipase-independent (Patterson et al., 2002) and lipase-dependent roles of PLCγ2 in receptor-activated Ca2+ response. Activation of PLCγ2 by store-operated Ca2+ influx To analyse further the Ca2+ entry-induced mechanism in BCR-induced PLCγ2 activation, we used SERCA Ca2+-ATPase inhibitor TG, which activates SOCs via passive depletion of Ca2+ stores. In the presence of extracellular Ca2+, TG produced dramatic [Ca2+]i increases in the WT cells, but only moderate [Ca2+]i increases in the PLCγ2− cells (Figure 2A and B). In the absence of Ca2+, TG caused indistinguishable transient Ca2+ rises in the WT and PLCγ2− cells, indicating that TG-sensitive Ca2+ stores are not affected by the loss of PLCγ2. As shown in Figure 2C, treating the WT cells with TG induced marked [IP3]i rises in the presence of extracellular Ca2+, but elicited small [IP3]i rises in the absence of Ca2+. The [IP3]i increase induced by TG (1.43 ± 0.1 μM) was about half that induced by BCR (2.70 ± 0.3 μM) (Figure 2D). However, in the PLCγ2− cells, TG produced small [IP3]i increases, regardless of extracellular Ca2+ (Figure 2C and D). These results suggest that Ca2+ entry activated by TG-induced store depletion leads to IP3 production by PLCγ2. Figure 2.Store-operated Ca2+ entry induced by passive depletion of Ca2+ stores activates PLCγ2. (A) Average time courses of Ca2+-responses induced by TG (2 μM) in WT (left) and PLCγ2− (right) cells in Ca2+-free or Ca2+-containing external solution (n = 18–33). (B) Peak [Ca2+]i rises induced by TG in WT and PLCγ2− cells. (C) Typical time courses of TG-induced changes in F/F0 of R9-PHIP3-D106 in WT (left) and PLCγ2− cells (right) in the presence (top) or absence (bottom) of extracellular Ca2+ (n = 7–15). (D) Peak TG-induced [IP3]i rises. (E) Dependence of TG-induced 'off' Ca2+ responses on extracellular Ca2+ concentrations in WT and PLCγ2− cells. The 'off' responses were induced after 12.5 min exposure to TG in Ca2+-free solution (n = 34–53). Right, peak [Ca2+]i rises plotted against extracellular Ca2+ concentration. Download figure Download PowerPoint In Figure 2E, the Ca2+ entry-evoked 'off' Ca2+ response was induced separately from the preceding passive Ca2+ release/depletion by TG (Parekh et al., 1997). In the physiological range of extracellular Ca2+ concentrations (1–10 mM), the Ca2+ entry-induced 'off' responses were significantly higher in the WT cells than those in the PLCγ2− cells. Replacing Ca2+ with Ba2+ failed to discriminate the 'off' responses between the WT and the PLCγ2− cells (Supplementary figure 3A), suggesting a specific positive role of Ca2+ in PLCγ2 activation or a lack of Ba2+ permeability of the Ca2+ entry channel. We also tested the possibility that the suppressed 'off' response in the PLCγ2− cells derives from a decrease in the electrical driving force for Ca2+ by membrane depolarization through cation channels such as TRPM7, which associates with PLCβ via the C2 domain (Runnels et al., 2002). In the solution containing K+ ionophore valinomycin, which maintains cell membrane potential at about −75 mV (Mori et al., 2002), and in the membrane-depolarizing high K+ (30 mM) solution, the 'off' [Ca2+]i increase remained significantly smaller in the PLCγ2− cells than in the WT cells (Supplementary figure 3B). Thus, the reduced 'off' response in the PLCγ2− cells was not due to membrane depolarization. IP3-mediated Ca2+ release is involved inTG-induced 'off' Ca2+ responses We next examined whether IP3R-mediated Ca2+ release is involved in TG-induced 'off' Ca2+ responses in DT40 B cells. Importantly, larger 'off' Ca2+ responses in the WT cells compared with those in the PLCγ2− cells were not observed after pretreatment with the IP3R antagonists, Xestospongin C (Xest C) (Figure 3A and B) and 2-aminoethoxydiphenyl borate (2-APB) (Supplementary figure 3C). The involvement of IP3-mediated Ca2+ release in the 'off' responses was confirmed by using ionomycin (IM), which depletes internal Ca2+ stores more indiscriminatingly and efficiently than TG, which selectively targets SERCA-containing stores (Supplementary figure 3D) (Parekh et al., 1997). The WT cells treated with IM showed greater Ca2+ release, but a smaller 'off' Ca2+ response compared with the WT cells treated with TG (Figure 3C). IM elicited nearly identical 'off' Ca2+ responses in the WT and the PLCγ2− cells, suggesting that thorough store depletion by IM nullified the contribution of IP3-induced Ca2+ release to the 'off' responses in the WT cells. Interestingly, IM elicited significant PLCγ2 translocation (Table I) and [IP3]i increase (1.55 ± 0.1 μM). In the cells deficient in three IP3R subtypes (IP3R-KO) (Sugawara et al., 1997), TG and IM induced nearly identical 'off' Ca2+ responses (Figure 3D). The data support the view that 'off' responses are promoted by IP3-induced Ca2+ release from Ca2+ stores, when Ca2+ stores are partially depleted (Supplementary figure 3D). Figure 3.Critical involvement of IP3-induced Ca2+ release in 'off' Ca2+ responses. (A and B) The IP3R blocker Xest C significantly suppresses TG-induced 'off' Ca2+ responses in WT (A) but not in PLCγ2− (B) cells (n = 23–48). Xest C (50 μM) was loaded with fura-2/AM using 0.1% Pluronic F-127 (Molecular Probes) for 30 min prior to [Ca2+]i measurements. During measurements, Xest C (50 μM) was added to perfusion solution for 20 min, and was omitted from Ca2+ readministration solution. Left, average time courses. Right, peak [Ca2+]i rises in Ca2+-free or Ca2+-containing external solution. (C) Left, average time courses of Ca2+ responses induced by TG and IM (1 μM) in WT and PLCγ2− cells (n = 16–33). Right, comparison between peak IM- and TG-induced [Ca2+]i rises in Ca2+-free or Ca2+-containing external solution. (D) Left, average time courses of Ca2+ responses induced by TG or IM in IP3R knockout cells. Right, peak IM- and TG-induced [Ca2+]i rises in Ca2+-free or Ca2+-containing solution. Download figure Download PowerPoint Figure 4.The C2 domain mediates Ca2+ entry-induced activation of PLCγ2. (A) Schematic diagram for various mutant constructs of PLCγ2. (B) Peak BCR-induced [Ca2+]i rises during 5 min incubation in Ca2+-free solution and after subsequent readmission of Ca2+ in PLCγ2− cells expressing respective PLCγ2 mutants (n = 11–36). (C) Peak TG-induced [Ca2+]i rises in Ca2+-free solution and after readmission of Ca2+ in PLCγ2− cells expressing respective PLCγ2 mutants (n = 12–37). Protocol is the same as in Figure 2E. (D) Left, average time courses of BCR-induced Ca2+ responses in PLCγ2− cells expressing PLCγ2, membrane-bound PLCγ2 chimera (mPLCγ2) or GFP (n = 8–14). mPLCγ2 is composed of the human CD16 extracellular domain, the human TCR ζ-chain transmembrane domain and the complete rat PLCγ2 as a cytoplasmic domain (Ishiai et al., 1999). Right, BCR- induced [Ca2+]i rises at peak and sustained after 5 min. (E) Left, average time courses of BCR-induced F/F0 changes of R9-PHIP3-D106. Right, maximal [IP3]i elevation and sustained [IP3]i rises after 5 min upon BCR stimulation. Download figure Download PowerPoint Table 1. Changes of PLCγ2 localization by BCR stimulation (% of total fluorescence) PLCγ2 mutant Before BCR stimulation (membrane area) 5 min after BCR stimulation (membrane area) WT WT (2 mM Ca2+) 37.0 ± 3.9 (n = 5) 57.9 ± 2.3* LD 32.7 ± 2.7 (n = 3) 53.4 ± 2.8* LD + U73122 30.1 ± 2.9 (n = 3) 31.0 ± 5.0 LD (EGTA) 35.0 ± 3.3 (n = 3) 37.6 ± 4.6 ΔC2 26.9 ± 8.4 (n = 3) 22.4 ± 5.1 minΔC2 33.3 ± 6.4 (n = 3) 42.1 ± 5.9 ΔPH 25.5 ± 4.2 (n = 3) 24.5 ± 3.5 E165A 31.8 ± 4.7 (n = 3) 56.5 ± 2.9* P820L 28.8 ± 3.1 (n = 3) 46.5 ± 2.2* PLCγ2− WT (2 mM Ca2+) 33.8 ± 4.2 (n = 4) 60.2 ± 9.3* WT (EGTA) 39.4 ± 5.7 (n = 5) 37.3 ± 7.5 WT (2 mM Ba2+) 35.7 ± 6.1 (n = 3) 39.1 ± 2.3 WT + U73122 29.3 ± 7.9 (n = 3) 35.0 ± 8.1 WT + U73343 31.2 ± 4.0 (n = 5) 56.8 ± 3.5* LD 31.7 ± 2.1 (n = 4) 34.2 ± 1.9 ΔC2 34.7 ± 8.3 (n = 3) 44.2 ± 3.8 minΔC2 36.7 ± 5.3 (n = 3) 47.6 ± 7.7 ΔPH 27.5 ± 4.8 (n = 3) 30.2 ± 8.8 E165A 29.4 ± 3.6 (n = 3) 50.0 ± 5.3* P820L 31.3 ± 5.4 (n = 3) 60.4 ± 3.7* WT (ionomycin) 38.4 ± 2.0 (n = 4) 44.9 ± 2.1* Membrane regions of fluorescent areas in DT40 cells were as described in Figure 5A. * n, number of experiment. P < 0.05, significantly different from cells before BCR stimulation. Contrary to the above notion, the IP3R-KO cells showed TG-induced 'off' Ca2+ responses indistinguishable from those in the WT cells (Supplementary figure 4A). This discrepancy seems partly due to a compensatory enhancement in DG-sensitive Ca2+ channel expression; notable La3+-sensitive 'off' [Ca2+]i increases due to the DG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 30 μM) were observed in about half of the IP3R-KO cells (18/41) (Supplementary figure 4B), while fewer WT cells (15/50) showed small, transient OAG-induced 'off' responses. As reported previously (Venkatachalam et al., 2001), extracellular application of Ba2+ for Ca2+ elicited a tiny increase in the fluorescence ratio (data not shown), suggesting the importance of extracellular Ca2+ in differentiating WT cells from IP3R-KO cells in the OAG-induced 'off' response. Thus, TG-induced 'off' Ca2+ responses in IP3R-KO cells may result from store-operated Ca2+ entry and Ca2+ entry activated by DG via PLCγ2 activation, while the contribution of the latter pathway may be minor in WT cells. C2 domain is responsible for Ca2+ influx-dependent activation of PLCγ2 The domains critical for Ca2+ entry-dependent activation were determined in PLCγ2. Three potential Ca2+-binding domains (EF-hand, catalytic domain and C2 domain), and the PH and SH3 domains are distinguished in PLCγ2 (Bairoch et al., 1990; Essen et al., 1996; Rhee, 2001; Ananthanarayanan et al., 2002). In PLCγ1, PH mediates membrane-targeting through phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding, while SH3 acts as a physiological guanine nucleotide exchange factor (GEF) for the nuclear GTPase PIKE, an activator of nuclear phosphatidylinositol 3-kinase (PI3K) (Ye et al., 2002) that produces the PIP3 required for BCR-induced [Ca2+]i elevation (Falasca et al., 1998). Mutations were introduced in the PLCγ2 domains tagged with yellow fluorescent protein (YFP) (Figure 4A). As expected, expression of the LD mutant in PLCγ2− cells failed to restore either the BCR-induced [Ca2+]i increase or to potentiate a TG-induced 'off' Ca2+ response (Figure 4B and C). Strikingly, deletion of C2 domain (ΔC2), which is involved in the formation of the Ca2+-phospholipid ternary complex in PLCδs (Ananthanarayanan et al., 2002), ablated the PLCγ2 function to restore the BCR- and TG-induced Ca2+ responses. Deleting one of the expected Ca2+ binding regions (Δ1138E-1140D) (Essen et al., 1996) in C2 (minΔC2) similarly blocked the restoration of the 'off' responses but not that of the BCR-induced Ca2+ release, strikingly differentiating Ca2+ entry-mediated responses from Ca2+ release. The PH deletion mutant (ΔPH) also failed to restore the PLCγ2− deficiency, as reported in PLCγ1 activation (Falasca et al., 1998). In contrast, the SH3 deletion (ΔSH3) mutant elicited partially impaired the Ca2+ responses, and the point SH3 mutant with leucine at proline 820 (P820L) that corresponds to P842 essential for GEF activity in PLCγ1 (Ye et al., 2002), or the EF-hand mutant (E165A), behaved like WT. Thus, the C2 domain is critical for Ca2+ entry-dependent activation of PLCγ2. C2 domain mediates Ca2+ influx-dependent translocation of PLCγ2 The above data and the requirement for C2 when various proteins target the plasma membrane (Sutton et al., 1995; Ananthanarayanan et al., 2002; Delmas et al., 2002) motivated us to visualize the localization of YFP-tagged PLCγ2 (Figure 5). Transfected PLCγ2 was diffusively distributed in the cytosol from the perinuclear area to the plasma membrane of the WT and the PLCγ2− cells. Within 4 min, but not within 2 min, stimulation of BCR efficiently concentrated PLCγ2 near the plasma membrane in the presence of extracellular Ca2+ (Figure 5A). This PLCγ2 translocation was abolished by removing Ca2+, using Ba2+ for Ca2+, using ΔC2 and minΔC2 in the WT and the PLCγ2− cells (Figure 5A and B; Table I). ΔPH also suppressed translocation. The EF and SH3 mutants showed significant translocation (Table I). Importantly, even in the absence of extracellular Ca2+, expression of membrane-bound PLCγ2 (mPLCγ2) (Ishiai et al., 1999) in the PLCγ2− cells promoted sustained [IP3]i and [Ca2+]i increases (Figure 4D and E). Thus, C2 mediates the Ca2+ entry-induced transportation of PLCγ2 for subsequent activation. Furthermore, in the WT cells, but not in the PLCγ2− cells, the LD mutant of PLCγ2 showed significant BCR-induced plasma membrane localization inhibited by the PLC blocker, U73122 (Figure 5C). This suggests that transfected PLCγ2 does not require its own lipase activity for Ca2+ entry-induced translocation to the plasma membrane when endogenous lipase-active PLCγ2 is present. Figure 5.Ca2+ influx induces translocation of PLCγ2 via the C2 domain. (A) Confocal fluorescence images indicating BCR-induced changes of subcellular localization of YFP-tagged PLCγ2 in PLCγ2− cells in Ca2+-containing or Ca2+-free solution (n = 5). Right, representative fluorescent changes of PLCγ2–EYFP distributed in 1 μm widths peripheral regions (Mem.) and in the rest the cytosolic areas (Cyt.). (B) Confocal images indicating changes of localization of PLCγ2 constructs (WT, LD, ΔC2 or ΔPH) in WT and PLCγ2− cells by 5 min BCR stimulation (n = 5). (C) PLC inhibitor U73122 blocks BCR-induced localization changes of LD in WT cells. Treatment with U73122 (3 μM) or its inactive analogue U73343 was started 20 min prior to BCR stimulation. Lower left, average time courses of BCR-induced F/F0 changes of R9-PHIP3-D106 in WT cells expressing GFP or LD, or pretreated with U73122 or U73343. Lower right, maximal [IP3]i elevation upon BCR stimulation. Download figure Download PowerPoint Functional and physical association of PLCγ2 with TRPC3 Among the TRPC channels responsible for receptor-activated Ca2+ entry (Montell et al., 2002), expression of TRPC1, C3, C4, C5 and C7 was identified in DT40 cells (Figure 6A). The respective TRPC channels were tested for functional coupling with PLCγ2 by co-expressing PLCγ2 in HEK cells. Upon stimulation of the endogenous muscarinic acetylcholine receptors (MRs) with CCh, TRPC3, C5, C6 and C7 alone promoted increased [Ca2+]i (Figure 6C), while co-expression of PLCγ2 exerted further potentiation only in the TRPC3-expressing cells (Figure 6B and C). LD elicited the intact potentiation of peak Ca2+ responses but failed to sustain Ca2+ response. In the patch-clamp recording, PLCγ2 significantly sustained CCh-activated TRPC3 currents (Figure 6D). Upon MR stimulation, PLCγ2–YFP fluorescence near the plasma membrane showed increases that indicate translocation in TRPC3-expressing cells (47.8 ± 0.7 to 52.3 ± 0.3%, P < 0.05). Furthermore, immumoprecipitation analysis revealed a physical association between PLCγ2 and TRPC3 (Figure 6E), which was impaired by ΔSH3, but not by LD or minΔC2 (Figure 6F), in the HEK cells. In the DT40 cells, co-immunoprecipitation of TRPC3 with PLCγ2 enhanced by BCR stimulation was observed (Figure 6G). These results suggest that receptor-activated TRPC3 channels functionally and physically interact with PLCγ2 via SH3 to enhance Ca2+ response and to maintain their own channel activity.