Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCgamma2 in B lymphocytes

Abstract

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 Cgamma2 (PLCgamma2)-deficient cells. Association between receptor-activated TRPC3 Ca2+ channels and PLCgamma2, which cooperate in potentiating Ca2+ responses, was demonstrated by co-immunoprecipitation. PLCgamma2-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 PLCgamma2 mutants demonstrated that Ca2+ entry evoked a C2 domain-mediated PLCgamma2 translocation towards the plasma membrane in a lipase-independent manner to activate PLCgamma2. Strikingly, Ca2+ entry-activated PLCgamma2 maintained Ca2+ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca2+ entry with PLCgamma2 translocation and activation controls the amplification and co-ordination of receptor signalling.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 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.

Fig. 6. Functional and physical association of PLCγ2 with the TRPC3 Ca²⁺ channel promotes receptor-evoked TRPC3 currents and Ca²⁺ responses. (A) RT–PCR analysis of RNA expression of TRPC homologues in DT40 cells. (B) Average time courses of Ca²⁺ responses upon stimulation of MRs in HEK cells expressing TRPC3 and/or PLCγ2 (WT or LD) (n = 32–38). (C) Peak MR-activated [Ca²⁺]_i rises in HEK cells expressing respective TRPC1–C7 channels alone or along with PLCγ2. (D) Left, representative traces of ionic currents recorded from cells expressing TRPC3 alone or plus PLCγ2 at a holding potential of –60 mV. Right, peak and sustained (30 s after reaching peak) amplitudes of the MR-activated TRPC3 currents (n = 9–10). (E) Co-immunoprecipitation of TRPC3 and PLCγ2 via SH3. Immunoprecipitates (IP) with anti-GFP polyclonal antibody from TRPC3/GFP- and TRPC3/PLCγ2–YFP-expressing HEK cells (upper) or GFP/PLCγ2- and TRPC3–GFP/PLCγ2-expressing HEK cells (lower) were separated on 8% SDS-PAGE, and blotted with anti-PLCγ2 or anti-TRPC3 antibody, respectively, in western blot analysis (WB). (F) IP with the anti-GFP antibody from LD/TRPC3-, minΔC2/TRPC3- and ΔSH3/TRPC3-expressing HEK cells. Anti-TRPC3 antibody was used to assess co-immunoprecipitation in WB. All the PLCγ2 constructs were tagged with YFP. (G) IP with the anti-GFP antibody from TRPC3-GFP-expressing WT and TRPC3–GFP-expressing PLCγ2^– cells with (+) or without (–) 5 min BCR stimulation (anti-IgM). Co-immunoprecipitation was assessed using anti-PLCγ2 antibody in WB.

Fig. 7. PLCγ2 requires its lipase activity and C2 domain to elicit BCR-evoked Ca²⁺ oscillation. (A–D) Representative time courses of BCR-induced Ca²⁺ responses in WT and PLCγ2^– cells in Ca²⁺-containing (A–C and E–H) (n = 16–72) or Ca²⁺-free (D) (n = 50) solution (long time course, 60 min). PLCγ2 constructs were the same as those in Figure 4.

Fig. 8. Requirement of Ca²⁺ influx-dependent PLCγ2 activation for sustained BCR-induced activation of ERK in DT40. (A) WT or (B) PLCγ2^– cells were stimulated with anti-IgM (1 µg/ml) for indicated time in the presence or absence of extracellular Ca²⁺. Treatment with BIM (10 µM) was started 10 min before BCR stimulation. PMA (50 nM) was applied to anti-IgM in PLCγ2^– cells (B). (C and D) Average time courses of ERK activation evoked by TG (2 µM) in WT (C) and PLCγ2^– (D) cells. The ERK activities were assessed by the quantification of its phosphorylation using phospho-specific antibody.

Fig. 9. Model for amplification of Ca²⁺ signalling by PLCγ2 in B lymphocytes. BCR stimulation induces activation of PLCγ2 via phosphorylation by Syk tyrosine kinase, and the activated PLCγ2 hydrolyses PIP₂ to produce IP₃. IP₃ leads to Ca²⁺ release from ER and Ca²⁺ influx via TRPC3-containing receptor-activated Ca²⁺ channels (RACCs), which may correspond to SOCs activated through interaction with IP₃R or to DG-activated Ca²⁺ channels. Ca²⁺ influx via RACC promotes lipase-independent PLCγ2 translocation from the cytosol to the plasma membrane (PM) by acting on the C2 domain. The targeted PLCγ2 is activated to hydrolyse PIP₂ and produce IP₃ and DG, which in turn induces Ca²⁺ release from ER and Ca²⁺ influx via Ca²⁺ entry channels such as TRPC3. PLCγ2 may also positively regulate TRPC3 channels via direct interaction.