Ca²⁺ synergy in BMDMs depends primarily on PLCβ3 with a small contribution by PLCβ2. BMDMs from WT, PLCβ3-deficient (PLCβ3^−/−), or double PLCβ2- and PLCβ3-deficient (PLCβ2/3^−/−) mice were assayed for their ability to reflect synergistic responses to C5a plus UDP. Intracellular Ca²⁺ responses were measured in Fura-2-loaded cells stimulated with C5a (0.75 nm), UDP (500 nm), or both ligands. Responses from WT (A) and PLCβ2/3^−/− (B) BMDMs are indicated by the line graphs, in which each line represents the average of 3–4 individual wells/assay. Data shown are from representative experiments of n = 18 with similar results. C, summary of synergy responses from WT, PLCβ3^−/−, and PLCβ2/3^−/− BMDMs. The predicted additive responses for dual-ligand stimulation were calculated for each assay from the measured single-ligand responses, and a synergy ratio was determined as the ratio of the observed/predicted additive dual-ligand responses, using the peak offset feature. Values shown are mean ± S.E. from n = 18–20 replicate assays/condition. D and E, single-ligand Ca²⁺ responses in PLCβ-deficient BMDMs. D, wild-type, PLCβ3^−/−, and PLCβ2/3^−/− cells were stimulated with C5a (10 nm) or UDP (10 μm), and the Ca²⁺ responses were measured. Shown are the results for peak offset responses. Results are mean ± S.E. from n = 3 assays. *, p < 0.05. E, expression of mPLCβ3 in PLCβ2/3^−/− BMDMs restores synergistic Ca²⁺ responses to levels observed in WT BMDMs. Single-cell Ca²⁺ assays were performed on WT or PLCβ2/3^−/− BMDMs transduced with retrovirus encoding YFP-FLAG or YFP-mPLCβ3. Cells were stimulated with C5a (0.75 nm), UDP (500 nm), or C5a + UDP, and the Ca²⁺ responses were measured by integration over 2.25 min. Responses by transduced cells were measured in multiple assays of each of three independent batches of infected BMDMs. Values shown are mean ± S.E. from n = 5–6 samples per condition. *, p < 0.05.

Expression of PLCβ isoforms in NIH-3T3 cells. Quantitative RT-PCR was used to measure PLCβ isoform mRNA levels. mRNA levels were compared with β-actin in each assay and normalized to PLCβ1 levels. Data are mean ± S.E. (error bars) from three assays.

Overexpression of PLCβ isoforms in NIH-3T3 cells perturbs single- and dual-ligand responses. NIH-3T3 cells stably expressing YFP-PLCβ isoforms or YFP control protein were assayed for Ca²⁺ responses to individual ligands (A) and C5a + UTP dual ligand (B). A, single-ligand concentrations were 100 μm UTP, 10 nm C5a, 500 nm LPA, and 500 nm S1P. Responses were normalized to YFP control lines using the peak offset measures of the responses. B, synergy between C5a (10 nm) and UTP (100 μm) was measured using the integrated responses and normalized to responses of control cells in each assay (dotted line, representing an average synergy ratio of 2.7). Values shown are mean ± S.E. (error bars) from n = 3 assays. *, p < 0.05.

Binding of both Gβγ and Gα_q contributes to the synergistic activation of PLCβ. NIH-3T3 cells were stably transfected with YFP or YFP-tagged PLCβ constructs including intact hPLCβ3, C-terminal mutant (hPLCβ3ΔCT), Y-box mutant (hPLCβ3ΔYB), and C-terminal mutant (hPLCβ3ΔYBΔCT). Cells were then subjected to RNAi using murine-specific anti-PLCβ3 or LacZ control siRNA. Synergy ratios were calculated for each cell line and treatment. Shown are results from n = 4–14 assays of 2–3 independently derived cell lines/target. Although intact hPLCβ3 slightly elevated synergy with RNAi of endogenous mPLCβ3, the C-terminal mutant had a reduced capacity, and the Y-box mutant had no activity. *, p < 0.05. NS, not significant; Error bars, S.E.

Schematic of PLCβ structure and design of G-protein binding site mutants. The theoretical PLCβ structural domain organization (NCBI) is shown along with sites of modification as described under “Experimental Procedures.” YFP was added as an N-terminal fusion protein, and point mutations targeting Gβγ and Gα_q binding sites, based on homology to PLCβ1, were created as X → Ala mutations (*). The arrows indicate the location of amino acids mutated, and the numbers indicate their position relative to the N terminus of hPLCβ3. The species of isoform used is indicated as human (h) and bovine (b). Also used but not shown are YFP-mPLCβ1, -2, -3, and -4 without mutation and mPLCb2 truncated at amino acid 840.

C5a in combination with UTP or UDP yields synergistic Ca²⁺ responses in NIH-3T3 cells. Intracellular Ca²⁺ levels were calculated for Fura-2-loaded NIH-3T3 cells stably expressing the human C5aR in kinetic assays in 96-well plates. After 40 s base-line readings, cells were stimulated with UDP (100 μm), UTP (100 μm), C5a (10 nm), or a combination of these ligands, and responses were monitored for 2.5 min. Shown are a combination of C5a + UDP (A) and a combination of C5a + UTP (B). Each line in the graphs represents the average of 4–5 individual wells/assay, and the error bars (S.E.) are shown for the dual-ligand line in each graph. Synergy was evaluated by comparing the experimentally observed dual-ligand responses to the predicted additive responses of the individual ligands. C, the responses were quantified by using peak offset measurements, the difference from base line to observed peak. The dual-ligand responses were compared with the predicted additive responses (sum of single-ligand responses) and a synergy ratio calculated as observed/predicted additive peak offsets. For the C5a + UTP experiment shown, the synergy ratio was 2, but the predicted additive level for C5a + UDP was too low for reliable estimation of the synergy ratio. Data shown are from a representative assay of n = 7 with similar results.

Knockdown of PLCβ3 in NIH-3T3 cells reduces C5a + UTP Ca²⁺ synergy. siRNA-mediated knockdown of PLCβ isoforms in NIH-3T3-C5aR cells preceded assays of C5a + UTP Ca²⁺ synergy. PLCβ isoform-specific siRNA versus LacZ control siRNA were applied to cells 72 h prior to assay. A, cells were stimulated with UTP (100 μm), C5a (10 nm) or UTP + C5a. Integrated measures of synergy were normalized to responses of control cells in each assay (dotted line, representing an average synergy ratio of 3.5). B, single-ligand responses following PLCβ isoform-specific RNAi. Peak offset values were calculated for each response. Results are mean ± S.E. (error bars) from n = 3–5 assays/condition from independent rounds of RNAi. *, p < 0.05.

Expression of human PLCβ3 can compensate for knockdown of endogenous murine PLCβ3. NIH-3T3 cells were stably transfected with YFP (A and B) or YFP-hPLCβ3 (C and D) and subjected to RNAi using murine-specific anti-PLCβ3 (B and D) or LacZ control (A and C) siRNA. Ca²⁺ assays were performed to assess synergy responses with C5a + UTP stimulations. E, responses were quantified for synergy ratio using peak offset measurements (1 unit = predicted additive response of each cell). Shown are results from n = 3 assays of 2 independently derived cell lines/target. Synergy was reduced by 0.4 units with RNAi against mPLCβ3 (*, p < 0.05) unless the cells were reconstituted with hPLCβ3. Error bars, S.E.