Gβγ engages PLCβ3 at multiple sites to reorient and facilitate its activation
- James Tarpo Jr. and Margaret Tarpo Department of Chemistry, Purdue University, United States
- Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, United States
- Department of Biological Sciences, Purdue University, United States
- Center for Clinical and Translational Research, Abigail Wexner Research Institute at Nationwide Children’s Hospital, United States
- Graduate School of Pharmaceutical Sciences, Tohoku University, Japan
- Graduate School of Pharmaceutical Sciences, Kyoto University, Japan
- Molecular, Cellular and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Germany
Figures
Figure 1 with 5 supplements
Cryo-EM reconstruction of the Gβγ–PLCβ3 Δ892-PHcys complex.
(A) Domain diagram of human PLCβ3, with numbers above corresponding to domain boundaries. PLCβ3 is regulated by the X–Y linker (hot pink), proximal C-terminal domain (pCTD, cyan), and distal CTD (purple). The CTDs are connected by the unconserved CTD linker. (B) Cryo-EM density map and (C) structure of the 4 Å Gβγ–Δ892-PHcys complex crosslinked with BMOE, with PLCβ3 colored as in 1 A, Gβ in blue, and Gγ in red.
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Figure 1—source data 1
Annotated SDS-PAGE gels described in Figure 1.
- https://cdn.elifesciences.org/articles/110382/elife-110382-fig1-data1-v1.zip
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Figure 1—source data 2
Original, unannotated SDS-PAGE gels.
- https://cdn.elifesciences.org/articles/110382/elife-110382-fig1-data2-v1.zip
Figure 1—figure supplement 1
Isolation of a Crosslinked and Functional Gβγ–PLCβ3 complex.
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Figure 1—figure supplement 1—source data 1
Annotated, uncropped SDS-PAGE gels for those shown in Figure 1—figure supplement 1B.
- https://cdn.elifesciences.org/articles/110382/elife-110382-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Unannotated, uncropped SDS-PAGE gels corresponding to those shown in Figure 1—figure supplement 1b.
- https://cdn.elifesciences.org/articles/110382/elife-110382-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
Cryo-EM densities of Gβγ–PLCβ3 Δ892-PHcys complexes.
Figure 1—figure supplement 3
Contoured cryo-EM density maps of the Gβγ–PLCβ3 Δ892-PHcys complexes.
Figure 1—figure supplement 4
Cryo-EM data workflow and resolution analysis of the BMOE-crosslinked Gβγ–PLCβ3 Δ892-PHcys complex.
Figure 1—figure supplement 5
Cryo-EM data workflow and resolution analysis of the BMPEG-crosslinked Gβγ–PLCβ3 Δ892-PHcys complex.
Figure 2 with 4 supplements
Functional analysis of Gβγ–PLCβ3 interfaces: IP accumulation.
Comparison of the (A) Gβγ–Δ892-PHcys interface observed in this study, and the previously reported (B) Gβγ–PLCβ3 PH and (C) Gβγ–PLCβ3 EF hand interfaces (PDB ID: 8EMW) (Falzone and MacKinnon, 2023). Proteins are colored as in Figure 1. Residues in PLCβ3 and Gβγ shown as balls and sticks were mutated, and their basal and G protein-dependent activities quantified in a cell-based [3H]-IPx accumulation assay. Basal and Gαq-dependent activation was used as a control to confirm the PLCβ3 variants were properly folded. Changes in activity are not due to differences in expression (Figure 2—figure supplement 4). The activities of the PLCβ3 mutants in the (D) BMOE-crosslinked Gβγ–Δ892-PHcys interface and (E) Gβγ–PLCβ3 PH/EF hand interfaces were measured. Mutations to the Gβ1 subunit were similarly assessed for (F) the crosslinked Gβγ–Δ892-PHcys interface and (G) the Gβγ–PH/EF hand interfaces. All assays were performed in triplicate from at least three independent transfections, and data shown are mean ± SEM. Data in D and E were analyzed using a two-way ANOVA followed by Dunnett’s post-hoc multiple comparisons test, comparing the basal activity of each PLCβ3 variant to its activation Gβγ or Gαq. ***, p<0.0005, **, p<0.005, *, p<0.05. Data in F and G were analyzed using a one-way ANOVA, followed by Dunnett’s post-hoc multiple comparisons test, comparing the Gβγ-stimulated activity of each PLCβ3 mutant to that of wild-type PLCβ3. ***, p<0.0005, **, p<0.005, *, p<0.05.
Figure 2—figure supplement 1
Gβγ–effector enzyme complexes.
Figure 2—figure supplement 2
PLCβ binding surfaces are conserved in human Gβ1–4 isoforms.
Figure 2—figure supplement 3
Gβγ binding residues are not well conserved across the human PLCβ isoforms.
Figure 2—figure supplement 4
Expression of PLCβ3 and Gβγ mutants.
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Figure 2—figure supplement 4—source data 1
Uncropped and annotated western blots corresponding to those shown in Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/110382/elife-110382-fig2-figsupp4-data1-v1.zip
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Figure 2—figure supplement 4—source data 2
Uncropped, unannotated western blots corresponding to those in Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/110382/elife-110382-fig2-figsupp4-data2-v1.zip
Figure 3 with 2 supplements
Functional analysis of Gβγ–PLCβ3 interfaces: interaction with G proteins and PI(4,5)P2 hydrolysis.
(A) BRET between membrane-anchored HiBit-PLCβ3-CAAX and Venus-Gβγ increases after activation of AT1 with angiotensin II (AngII; 1 μM). Signals are blocked by membrane-tethered GRK3ct (GRK3ct) and enhanced by membrane-tethered GRK2RH, which sequester free Gβγ and Gαq-GTP, respectively. Traces are the average of twenty replicates from five independent experiments. (B) AngII-induced changes in BRET between HiBit-PLCβ3-CAAX and Venus-Gβγ (ΔBRET) for sixteen mutants across the three Gβγ–PLCβ3 interfaces; most mutants showed significant changes (denoted by an asterisk) in their interactions with Venus-Gβγ compared to wild-type HiBit-PLCβ3-CAAX. (C) In ΔPLC cells, expression of HiBit-PLCβ3 (without LgBit) reconstitutes AngII-induced PI(4,5)P2 hydrolysis, as indicated by bystander BRET between Nluc-PH and mem-link-Venus (mem-Venus). Traces are the average of sixteen replicates from four independent experiments. (D) AngII-induced PI(4,5)P2 hydrolysis (ΔBRET) for the same HiBit-PLCβ3 mutants as panel B; most mutants showed significant changes in PI(4,5)P2 hydrolysis (denoted by asterisks). (E) In HEK cells, BRET between HiBit-PLCβ3 and Gαq-Venus increases after activation of AT1. Traces are the average of twelve replicates from three independent experiments. (F) BRET between the same HiBit-PLCβ3 mutants as panel B and Gαq-Venus; none of the mutants showed significant changes compared to wild-type HiBit-PLCβ3. For B, D, and F, all mutants were compared to wild-type HiBit-PLCβ3-CAAX or HiBit-PLCβ3 using one-way ANOVA with Dunnett’s post-hoc comparisons; data points represent averages from independent experiments (n=3–11) performed in quadruplicate. For all mutants, * p<0.05 and individual p values are given in Supplementary file 1b-e.
Figure 3—figure supplement 1
Gαq binding promotes Gβγ binding to PLCβ3.
Figure 3—figure supplement 2
PLCβ-mediated PI(4,5)P2 hydrolysis is facilitated by Gβγ derived from both Gq and Gi/o heterotrimers.
Figure 4
Gβγ facilitation of PLCβ3 activation does not reflect membrane recruitment.
(A) Activation of AT1 induces translocation of HiBit-PLCβ3 to the plasma membrane as indicated by bystander BRET between HiBit-PLCβ3 and mem-Venus. Traces are the average of twelve replicates from three independent experiments. (B) BRET between the same HiBit-PLCβ3 mutants as Figure 3 and mem-Venus; none of the mutants showed a significant defect in membrane translocation compared to wild-type HiBit-PLCβ3. Individual p values are given Supplementary file 1f. (C) In ΔPLC cells, expression of HiBit-PLCβ3-CAAX reconstitutes AngII-induced PI(4,5)P2 hydrolysis, indicated by bystander BRET between Nluc-PH and mem-Venus. Signals are inhibited by membrane-tethered GRK3ct, which sequesters free Gβγ. Traces are the average of twenty-eight replicates from seven independent experiments. (D) PLCβ3-CAAX variants shown to be defective with respect to Gβγ binding are also defective with respect to PI(4,5)P2 hydrolysis. For B, all mutants were compared to wild-type HiBit-PLCβ3, and for D all mutants were compared to +GRK3 ct RQ (a mutant defective in binding Gαq·GTP), using one-way ANOVA with Dunnett’s post-hoc comparisons; data points represent averages from independent experiments (n=3 or 7) performed in quadruplicate. For all mutants, * P<0.05 and individual p values are given in Supplementary file 1f and g.
Figure 5
G protein–PLCβ3 complexes at the membrane.
(A) The crosslinked Gβγ–PLCβ3 complex is compatible with membrane localization, but not lipase activity. PLCβ3 is shown as a surface, and Gβγ in ribbon. Proteins are colored as in Figure 1A. (B) The liposome-tethered Gβγ–PLCβ3 complex would allow the PLCβ3 active site to interact with the membrane. (C) The crosslinked and (D) liposome-tethered complexes are compatible with Gαq·GTP binding and activation via displacement of the Hα2' helix (cyan) and engagement of the dCTD (dark gray). In both models, the dCTD binds the membrane through electrostatic interactions.
Tables
Table 1
Cryo-EM data collection, refinement, and validation statistics.
| Gβγ-PLCβ3 BMOE (4.1 Å) | Gβγ-PLCβ3-BMOE (7.0 Å) | Gβγ-PLCβ3 BMPEG | |
|---|---|---|---|
| Data collection and processing | |||
| Grids Vitrification Method Microscope Magnification | Cu Quantifoil FEI Vitrobot Titan Krios 81000 | Cu Quantifoil FEI Vitrobot Titan Krios 81000 | Cu Quantifoil FEI Vitrobot Titan Krios 81000 |
| Voltage (kV) Detector | 300 K3 | 300 K3 | 300 K3 |
| Electron exposure (e–/Å2) | 53.69 | 53.69 | 53.69 |
| Defocus range (μm) | 0.5–2.0 | 0.5–2.0 | 0.5–2.0 |
| Pixel size (Å) Frames | 0.539 40 | 0.539 40 | 0.539 40 |
| Symmetry Micrographs | C1 | C1 | C1 |
| Initial particle images (no.) | 1,248,953 | 1,248,953 | 579,743 |
| Final particle images (no.) | 92,777 | 65,306 | 142,175 |
| Map resolution (Å) FSC threshold | 4.06 0.143 | 6.77 0.143 | 4.40 0.143 |
| Refinement | |||
| Initial model used (PDB code) | 4GNK (PLCβ3) 1GP2 (Gβ1γ2) | 4GNK (PLCβ3) 1GP2 (Gβ1γ2) | 4GNK (PLCβ3) 1GP2 (Gβ1γ2) |
| Model resolution (Å) FSC threshold | 4.06 0.143 | 6.77 0.143 | 4.40 0.143 |
| Model resolution range (Å) | 3.4–7.0 | 7–10.7 | 4–10.7 |
| Model composition Non-hydrogen atoms Protein residues Ligands | 8,913 1,125 8 | 8,896 1,125 0 | 8,967 1134 0 |
| B factors (Å2) Protein Ligand | 74.40 79.08 | 45.30 0 | 55.51 0 |
| R.m.s. deviations Bond lengths (Å) Bond angles (°) | 0.003 0.474 | 0.002 0.542 | 0.003 0.519 |
| Validation MolProbity score Clashscore Poor rotamers (%) | 1.97 6.37 1.64 | 2.61 11.34 4.71 | 2.98 40.75 3.35 |
| Ramachandran plot Favored (%) Allowed (%) Disallowed (%) | 92.9 6.92 0.18 | 90.75 9.25 0 | 91.80 8.02 0.18 |
Additional files
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Supplementary file 1
Supplemental tables reporting protein expression as quantified by luminescence and BRET measurements.
(a) Expression of HiBit-PLCβ3 variants as indicated by LgBit-complemented luminescence in intact cells. (b) Expression of HiBit-PLCβ3-CAAX variants as indicated by LgBit-complemented luminescence in intact cells. (c) Angiotensin II-induced BRET between HiBit-PLCβ3-CAAX variants and Venus-Gβγ. (d) Angiotensin II-induced PI(4,5)P2 hydrolysis mediated by HiBit-PLCβ3 variants. (e) Angiotensin II-induced BRET between HiBit-PLCβ3 variants and Gαq-Venus. (f) Angiotensin II-induced BRET between HiBit-PLCβ3 variants and mem-Venus.
- https://cdn.elifesciences.org/articles/110382/elife-110382-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/110382/elife-110382-mdarchecklist1-v1.pdf
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Gβγ engages PLCβ3 at multiple sites to reorient and facilitate its activation
eLife 15:RP110382.
https://doi.org/10.7554/eLife.110382.3
