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. 2022 Aug 24;23(17):9590.
doi: 10.3390/ijms23179590.

Expression of the C-Terminal Domain of Phospholipase Cβ3 Inhibits Signaling via Gαq-Coupled Receptors and Transient Receptor Potential Channels

Gerald Thiel  1 , Oliver G Rössler  1
Affiliations

Expression of the C-Terminal Domain of Phospholipase Cβ3 Inhibits Signaling via Gαq-Coupled Receptors and Transient Receptor Potential Channels

Gerald Thiel et al. Int J Mol Sci. .

Abstract

Transient receptor potential (TRP) channels are cation channels that play a regulatory role in pain and thermosensation, insulin secretion, and neurotransmission. It has been proposed that activation of TRP channels requires phosphatidylinositol 4,5-bisphosphate, the major substrate for phospholipase C (PLC). We investigated whether inhibition of PLCβ has an impact on TRP channel signaling. A genetic approach was used to avoid off-target effects observed when using a pharmacological PLCβ inhibitor. In this study, we show that expression of PLCβ1ct and PLCβ3ct, truncated forms of PLCβ1 or PLCβ3 that contain the C-terminal membrane binding domains, almost completely blocked the signal transduction of a Gαq-coupled designer receptor, including the phosphorylation of ERK1/2. In contrast, expression of the helix-turn-helix motif (Hα1-Hα2) of the proximal C-terminal domain of PLCβ3 did not affect Gαq-coupled receptor signaling. PLCβ3ct expression impaired signaling of the TRP channels TRPM3 and TRPM8, stimulated with either prognenolone sulfate or icilin. Thus, the C-terminal domain of PLCβ3 interacts with plasma membrane targets, most likely phosphatidylinositol 4,5-bisphosphate, and in this way blocks the biological activation of TRPM3 and TRPM8, which require interaction with this phospholipid. PLCβ thus regulates TRPM3 and TRPM8 channels by masking phosphatidylinositol 4,5-bisphosphate with its C-terminal domain.

Keywords: ERK1/2; Gαq-coupled designer receptor; TRPM3; TRPM8; m-3M3FBS; phospholipase Cβ.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of the C-terminal domain of phospholipase Cβ3 blocks signaling of Rαq, a Gαq-coupled designer receptor. (A) Multi-domain structure of phospholipase Cβ3 and the truncated mutant PLCβ3ct. PLCβ3 contains a pleckstrin homology (PH) domain, four tandem EF-hand repeats, the catalytic triose phosphate isomerase (TIM) barrel domain, the C2 domain, the helix-turn-helix motif (Hα1—Hα2), and a C-terminal domain (CTD). (B) Expression of PLCβ3ct mutant in HEK293 cells following infection of the cells with an PLCβ3ct-encoding lentivirus. Cells were infected with a lentivirus encoding either PLCβ3ct or β-galactosidase (mock). The Western blot was developed with an antibody against the FLAG-tag. kDa, molecular mass markers. (C) Modular structure of Rαq. The two-point mutations essential to change the M3 muscarinic acetylcholine receptor into a Gαq-coupled, CNO-sensitive designer receptor, are indicated. (D) Provirus, depicting the Coll.luc reporter gene, used as a sensor to measure AP-1 activity. (E) HEK293 cells containing a chromatin-integrated Coll.luc reporter gene were infected with a lentivirus encoding the designer receptor Rαq. In addition, cells were infected with a lentivirus encoding either PLCβ3ct or β-galactosidase (mock). Serum-starved cells were stimulated with CNO (1 μM) for 24 h. Cell extracts were prepared, and luciferase activities and protein concentrations were determined. Luciferase activity was normalized to the protein concentration. Data shown are means +/− SD of four experiments performed in quadruplicate (*** p < 0.001).
Figure 2
Figure 2
Expression of the C-terminal domain of phospholipase Cβ3 blocks the phosphorylation of extracellular signal-induced protein kinase following stimulation of the Rαq receptor. HEK293 cells expressing the Rαq designer receptor were infected with a lentivirus encoding either PLCβ3ct or β-galactosidase (mock). Cells were serum-starved for 24 h and then stimulated with CNO (1 μM). Cells were harvested at different times. Whole cell extracts were prepared and subjected to Western blot analysis. The blots were incubated with a monoclonal antibody directed against the phosphorylated active form of ERK1/2. As loading control, an antibody detecting ERK1/2 was used.
Figure 3
Figure 3
A fusion protein containing the helix-turn-helix (Hα1—Hα2) motif of PLCβ3 does not function as a dominant negative of Gαq-coupled receptor signaling. (A) Modular structure of HTH-CFP-CaaX. The FLAG-tagged fusion protein consists of the Hα1—Hα2 domain of PLCβ3, CFP, and the C-terminal domain of Rac1. (B) Expression of HTH-CFP-CaaX in HEK293 cells following infection with lentivirus encoding HTH-CFP-Caax. As a control, a virus encoding β-galactosidase (mock) was used for infection. The Western blot was developed with an antibody against the FLAG-tag. kDa, molecular mass markers. (C) HEK293 cells containing a chromatin-integrated Coll.luc reporter gene were infected with a lentivirus encoding the designer receptor Rαq. In addition, cells were infected with a lentivirus encoding either HTH-CFP-CaaX or β-galactosidase (mock). Cells were stimulated, harvested, and analyzed as described in the legend to Figure 1 (n = 4). Expression of HTH-CFP-CaaX did not significantly impair Gαq-coupled receptor signaling.
Figure 4
Figure 4
Expression of the C-terminal domain of phospholipase Cβ1 blocks signaling Rαq, a Gαq-coupled designer receptor. (A) Multi-domain structure of phospholipase Cβ1 and the truncated mutant PLCβ1ct. PLCβ1 has a similar domain structure as depicted for PLCβ3 in Figure 1. (B) Expression verification of PLCβ1ct in HEK293 cells following infection with an PLCβ1ct-encoding lentivirus. Cells were infected with a lentivirus encoding either PLCβ3ct or β-galactosidase (mock). The Western blot was developed with an antibody against the FLAG-tag. kDa, molecular mass markers. (C) HEK293 cells containing a chromatin-integrated Coll.luc reporter gene were infected with a lentivirus encoding the designer receptor Rαq. Cells were infected with a lentivirus encoding either PLCβ1ct or β-galactosidase (mock). Serum-starved cells were stimulated with CNO (1 μM) for 24 h. Cells were harvested and analyzed as described in the legend to Figure 1 (n = 3; *** p < 0.001).
Figure 5
Figure 5
Expression of the C-terminal domain of phospholipase Cβ3 blocks icilin-induced signaling via TRPM8. (A) Modular structure of TRPM8. (B) HEK293-TRPM8 cells containing a chromatin-integrated Coll.luc reporter gene were infected with a lentivirus encoding either PLCβ3ct or β-galactosidase (mock). Serum-starved cells were stimulated with icilin (1 μM) for 24 h. Cells were harvested and analyzed as described in the legend to Figure 1 (n = 3; *** p < 0.001).
Figure 6
Figure 6
Expression of the C-terminal domain of phospholipase Cβ3 blocks pregnenolone sulfate-induced signaling via TRPM3. (A) Modular structure of TRPM3. (B) T-REx-TRPM3 cells containing a chromatin-integrated Coll.luc reporter gene were infected with a lentivirus encoding either PLCβ3ct or β-galactosidase (mock). Cells were serum-starved for 24 h in the presence of tetracycline (1 μg/mL) to induce TRPM3 expression. Serum-starved cells were stimulated with icilin (1 μM) for 24 h. Cells were harvested and analyzed as described in the legend to Figure 1 (n = 4; ** p < 0.01).
Figure 7
Figure 7
The compound m-3M3FBS blocks intracellular signaling mediated by TRPM8 and TRPM3. (A) Chemical structure of m-3M3FBS. (B) HEK293 cells containing the Coll.luc reporter gene were incubated with different concentrations of m-3M3FBS for 24 h. Cells were harvested and analyzed as described in the legend to Figure 1 (n = 3). (C) HEK293-M8 cells were infected with a recombinant lentivirus containing the Coll.luc reporter gene. Cells were serum-starved for 24 h, preincubated for 3 h with m-3M3FBS (5 μM), and then stimulated with icilin (1 μM). Cells were harvested and analyzed as described in the legend to Figure 1 (n = 3; *** p < 0.001). (D) T-REx-TRPM3 cells were infected with a recombinant lentivirus containing the collagenase promoter/luciferase reporter gene (Coll.luc). Cells were serum-starved for 24 h in the presence of tetracycline (1 μg/mL) to induce TRPM3 expression. Cells were preincubated for 3 h with m-3M3FBS (5 μM) and then stimulated with pregnenolone sulfate (PregS, 20 μM) for 24 h in the presence of the inhibitor. Cells were harvested and analyzed as described in the legend to Figure 1 (n = 3; *** p < 0.001).
Figure 8
Figure 8
The compound m-3M3FBS fails to block B-Raf signaling. (A) Signaling pathway connecting TRPM8 and TRPM3 stimulation with AP-1-induced gene transcription. The activation point of the fusion protein ∆B-Raf:ER is indicated. (B) Signaling pathway connecting TRPM8 and TRPM3 stimulation with AP-1-induced gene transcription. The activation point of the fusion protein ∆B-Raf:ER is indicated. (C) HEK293-∆B-Raf:ER cells were infected with a recombinant lentivirus containing Coll.luc reporter gene. The cells were incubated in medium containing 0.05% serum for 24 h. Stimulation was performed with 4OHT (100 nM) for 24 h. Cells were harvested and analyzed as described in the legend to Figure 1 (n = 3; n.s., not significant).
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