Optogenetic activation of heterotrimeric G-proteins by LOV2GIVe, a rationally engineered modular protein
- Department of Biochemistry, Boston University School of Medicine, United States
Figures
Figure 1 with 1 supplement
Design and optimization of LOV2GIV, an optogenetic activator of heterotrimeric G-proteins.
(A) Left, diagram depicting the design of the LOV2GIV fusion protein. The GBA motif of GIV (red) is fused to the C-terminus of the LOV2 domain (blue). Right, diagram depicting the design principle …
Figure 1—figure supplement 1
FA mutation in LOV2GIVe (L) impairs G-protein binding.
Top, diagram depicting the F1685A mutation of GIV’s GBA motinf in the context of LOV2GIVe. Bottom, approximately 20 µg of the indicated purified GST-fused constructs were immobilized on …
Figure 2 with 1 supplement
LOV2GIVe binds and activates Gαi3 in vitro in its lit conformation.
(A) LOV2GIVe binds with high affinity to Gαi3. Approximately 10 µg of the indicated purified GST-fused constructs were immobilized on glutathione-agarose beads and incubated with the indicated …
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Figure 2—source data 1
Numerical data used for panel A.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig2-data1-v2.xlsx
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Figure 2—source data 2
Numerical data used for panel C.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
LOV2GIVe (L) promotes GTP binding to Gαi3 in vitro.
Binding of radiolabeled GTPγS to purified His-Gαi3 was determined in the absence or presence 2 µM of purified GST-LOV2GIVe (L) (black), GST-LOV2GIVe (L + FA) (blue) or GST-LOV2GIVe (D) (red). …
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Figure 2—figure supplement 1—source data 1
Numerical data used to generate the graph.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 3 supplements
LOV2GIVe activates G-protein signaling in cells in its lit conformation.
(A) Left, diagram comparing key steps and components of the mating pheromone response pathway of Saccharomyces cerevisiae to those of an engineered, humanized strain used in the experiments shown in …
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Figure 3—source data 1
Numerical data used for panel A.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig3-data1-v2.xlsx
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Figure 3—source data 2
Numerical data used for panel B.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Carbachol dose-dependent G-protein activation and its blockade by atropine.
BRET was measured in HEK293T cells transfected with the components of the BRET biosensor described in Figure 3B after stimulation with the indicated concentrations of carbachol for 4 min in the …
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Figure 3—figure supplement 1—source data 1
Numerical data used to generate the graph.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig3-figsupp1-data1-v2.xlsx
Figure 3—figure supplement 2
FA mutation abolishes G-protein activation by mLOV2GIVe (L) in cells.
BRET was measured in HEK293T cells transfected with 2 µg of the indicated mLOV2GIVe plasmid constructs along with the components of the BRET biosensor described in Figure 3. Results in the graph on …
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Figure 3—figure supplement 2—source data 1
Numerical data used to generate the graph.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig3-figsupp2-data1-v2.xlsx
Figure 3—figure supplement 3
LOV2GIVe (L) expression enhances isoproterenol-induced cAMP levels in cells via Gi regulation.
(A) Changes in cAMP levels upon isoproterenol stimulation (100 nM) were measured in HEK293T cells transfected with the NLuc-EPAC-VV BRET biosensor and the indicated amounts of plasmids encoding …
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Figure 3—figure supplement 3—source data 1
Numerical data used for panel A.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig3-figsupp3-data1-v2.xlsx
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Figure 3—figure supplement 3—source data 2
Numerical data used for panel B.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig3-figsupp3-data2-v2.xlsx
Figure 4
mLOV2GIVe activates G-protein signaling in cells upon illumination.
(A) mLOV2GIVe activates G-proteins in mammalian cells upon illumination. Kinetic BRET measurements were carried out in HEK293T cells transfected with 2 µg of plasmids for the expression of mLOV2GIVe …
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Figure 4—source data 1
Numerical data used in panel A.
- https://cdn.elifesciences.org/articles/60155/elife-60155-fig4-data1-v2.xlsx
Author response image 1
Binding of N-terminally truncated Gαi1 (Gαi1ΔN) to LOV2GIVe is detectable by Ponceau S staining.
Approximately 20 µg of the indicated purified GST or GST-LOV2GVe (L) were immobilized on glutathione-agarose beads and incubated with 3 µg (~300 nM) of purified Gαi3ΔN. Resin-bound proteins were …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3) | Invitrogen | Cat# C600003 | |
| Strain, strain background (Escherichia coli) | DH5alpha | New England Biolabs | Cat# C2987I | |
| Genetic reagent (Saccharomyces cerevisiae) | CY7967 [MATα GPA1(1–41)-Gαi3 far1Δ fus1p-HIS3 can1 ste14:trp1:LYS2 ste3Δ lys2 ura3 leu2 trp1 his3] | Cismowski et al., 1999 | Provided by James Broach (Penn State University) | |
| Cell line (Homo sapiens) | HEK293T cells | ATCC | CRL3216 | |
| Antibody | α-tubulin (mouse monoclonal) | Sigma | T6074 | Immunoblotting Dilution (1: 2,500) |
| Antibody | FLAG tag (mouse monoclonal) | Sigma | F1804 | Immunoblotting Dilution (1: 2,000) |
| Antibody | His-tag (mouse monoclonal) | Sigma | H1029 | Immunoblotting Dilution (1: 2,500) |
| Antibody | Hemagglutinin (HA) tag (clone 12CA5) (mouse monoclonal) | Roche | Cat# 11583816001 | Immunoblotting Dilution (1: 1,000) |
| Antibody | MYC-tag (9B11) (mouse monoclonal) | Cell Signaling | Cat# 2276 | Immunoblotting Dilution (1: 1,000) |
| Antibody | Gαs (C-18) (rabbit polyclonal) | Santa Cruz Biotechnology | Cat# sc-383 | Immunoblotting Dilution (1: 500) |
| Antibody | Goat anti-rabbit Alexa Fluor 680 (goat polyclonal) | Life Technologies | Cat# A21077 | Immunoblotting Dilution (1:10,000) |
| Antibody | Goat anti-mouse IRDye 800 (goat polyclonal) | LiCor | Cat# 926–32210 | Immunoblotting Dilution (1:10,000) |
| Recombinant DNA reagent | pLIC-GST (plasmid) | Cabrita et al., 2006 | Provided by John Sondek (University of North Carolina- Chapel Hill) | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIV (plasmid) | This paper | ||
| Recombinant DNA reagent | pLIC-GST-LOV2GIV (L) (plasmid) | This paper | Contains the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIV (D) (plasmid) | This paper | Contains the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIVa (D) (plasmid) | This paper | Contains the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIVb (D) (plasmid) | This paper | Contains the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIVc (D) (plasmid) | This paper | Contains the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIVd (D) (plasmid) | This paper | Contains the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIVe (D) (plasmid) | This paper | Contains the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIVe (L) (plasmid) | This paper | Contains the lit-mimicking mutation I539E | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIVe (D) (plasmid) | This paper | Contains the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-GST-LOV2GIVe (L + FA) (plasmid) | This paper | Contains the lit-mimicking mutation I539E and the GEF-deficient mutation F1685A | |
| Recombinant DNA reagent | pET28b-Gαi3 (plasmid) | Garcia-Marcos et al., 2009 | For the bacterial expression of rat His-Gαi3 | |
| Recombinant DNA reagent | pET28b-Gαo (plasmid) | Garcia-Marcos et al., 2010 | For the bacterial expression of rat His-Gαo (isoform A) | |
| Recombinant DNA reagent | pLIC-His (plasmid) | Stols et al., 2002 | ||
| Recombinant DNA reagent | pLIC-His- Gαi1(int.6xHis) (plasmid) | This paper | For the bacterial expression of rat Gαi1 with an internal His-tag | |
| Recombinant DNA reagent | pET28b-Gαi2 (plasmid) | This paper | For the bacterial expression of rat His-Gαi2 | |
| Recombinant DNA reagent | p3XFLAG-CMV10-Gαi3 (plasmid) | Garcia-Marcos et al., 2009 | For the mammalian expression of rat FLAG-Gαi3 | |
| Recombinant DNA reagent | pcDNA3.1(+)-Gαs (plasmid) | Beas et al., 2012 | For the mammalian expression of human Gαs | |
| Recombinant DNA reagent | pcDNA3-Gαq-HA (plasmid) | Wedegaertner et al., 1993 | For the mammalian expression of mouse Gαq with an internal HA tag. Provided by P. Wedegaertner (Thomas Jefferson University) | |
| Recombinant DNA reagent | pcDNA3.1-Gα12-MYC (plasmid) | Ritchie et al., 2013 | For the mammalian expression of mouse Gα12 with an internal MYC-tag. Provided by T. Meigs (University of North Carolina, Asheville) | |
| Recombinant DNA reagent | pLIC-YES2 (plasmid) | Coleman et al., 2016 | ||
| Recombinant DNA reagent | pLIC-YES2-N9Gpa1 (plasmid) | Coleman et al., 2016 | ||
| Recombinant DNA reagent | pLIC-YES2-LOV2GIVe wt (plasmid) | This paper | For the yeast expression of cytosolic LOV2GIVe | |
| Recombinant DNA reagent | pLIC-YES2-LOV2GIVe (L) (plasmid) | This paper | For the yeast expression of cytosolic LOV2GIVe bearing the lit-mimicking mutation I539E | |
| Recombinant DNA reagent | pLIC-YES2-LOV2GIVe (D) (plasmid) | This paper | For the yeast expression of cytosolic LOV2GIVe bearing the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-YES2- N9Gpa1-LOV2GIVe wt (plasmid) | This paper | For the yeast expression of membrane-anchored (m)LOV2GIVe | |
| Recombinant DNA reagent | pLIC-YES2- N9Gpa1-LOV2GIVe (L) (plasmid) | This paper | For the yeast expression of membrane-anchored (m)LOV2GIVe bearing the lit-mimicking mutation I539E | |
| recombinant DNA reagent | pLIC-YES2- N9Gpa1-LOV2GIVe (D) (plasmid) | This paper | For the yeast expression of membrane-anchored (m)LOV2GIVe bearing the dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-myc (plasmid) | Cabrita et al., 2006 | Provided by John Sondek (University of North Carolina- Chapel Hill) | |
| Recombinant DNA reagent | pLIC-lyn11-myc (plasmid) | This paper | ||
| Recombinant DNA reagent | pLIC-lyn11-myc-LOV2GIVe (plasmid) | This paper | For the mammalian expression of mLOV2GIVe | |
| Recombinant DNA reagent | pLIC-lyn11-myc-LOV2GIVe (L) (plasmid) | This paper | For the mammalian expression of mLOV2GIVe bearing the lit-mimicking mutation I539E | |
| Recombinant DNA reagent | pLIC-lyn11-myc-LOV2GIVe (D) (plasmid) | This paper | For the mammalian expression of mLOV2GIVe dark-mimicking mutation C450S | |
| Recombinant DNA reagent | pLIC-lyn11-myc-LOV2GIVe (FA) (plasmid) | This paper | For the mammalian expression of mLOV2GIVe GEF-deficient mutation F1685A | |
| Recombinant DNA reagent | pLIC-lyn11-myc-LOV2GIVe (L) (FA) (plasmid) | This paper | For the mammalian expression of mLOV2GIVe lit-mimicking mutation I539E and the GEF-deficient mutation F1685A | |
| Recombinant DNA reagent | pcDNA3.1-Venus(155-239)-Gβ1 (plasmid) | Hollins et al., 2009 | For the mammalian expression of Gβ1 tagged with a fragment of Venus (VC-Gβ1). Provided by N. Lambert (Augusta University, GA) | |
| Recombinant DNA reagent | pcDNA3.1-Venus(1-155)-Gγ2 (plasmid) | Hollins et al., 2009 | For the mammalian expression of Gγ2 tagged with a fragment of Venus (VN-Gγ2). Provided by N. Lambert (Augusta University, GA) | |
| Recombinant DNA reagent | pcDNA3.1-Gβ1 (plasmid) | Hollins et al., 2009 | For the mammalian expression of untagged Gβ1. Provided by N. Lambert (Augusta University, GA) | |
| Recombinant DNA reagent | pcDNA3.1-Gγ2 (plasmid) | Hollins et al., 2009 | For the mammalian expression of untagged Gγ2. Provided by N. Lambert (Augusta University, GA) | |
| Recombinant DNA reagent | pcDNA3-Gαi3 (plasmid) | Garcia-Marcos et al., 2010 | For the mammalian expression of rat Gαi3 | |
| Recombinant DNA reagent | pcDNA3.1-masGRK3ct-Nluc (plasmid) | Masuho et al., 2015 | Provided by K. Martemyanov (Scripps Research Institute, FL) | |
| Recombinant DNA reagent | pcDNA3.1-Nluc-EPAC-VV (plasmid) | Masuho et al., 2015 | Provided by K. Martemyanov (Scripps Research Institute, FL) | |
| Recombinant DNA reagent | pcDNA3.1-3xHA-M4R (plasmid) | cDNA Resource Center at Bloomsburg University | Cat# MAR040TN00 | |
| Commercial assay or kit | QuikChange II Site-Directed Mutagenesis Kit | Agilent | Cat# #200523 | |
| Chemical compound, drug | NanoGlo Luciferase Assay System | Promega | Cat# N1120 | |
| Chemical compound, drug | Carbachol | Acros Organics | Cat# AC-10824 | |
| Chemical compound, drug | Atropine | Alfa Aesar | Cat# A10236 | |
| Chemical compound, drug | DL-isoproterenol hydrochloride | Alfa Aesar | Cat# J61788 | |
| Chemical compound, drug | [γ-32P]GTP | Perkin Elmer | NEG004Z250UC | |
| Chemical compound, drug | [32S]GTPγS | Perkin Elmer | NEG030H250UC | |
| Chemical compound, drug | Fluorescein di-β-D-galactopyranoside (FDG) | Marker Gene Technologies | Cat# M0250 | |
| Software, algorithm | I-TASSER | Yang et al., 2015 | https://zhanglab.ccmb.med.umich.edu/I-TASSER/ | Homology modeling server |
| Software, algorithm | ICM version 3.8–3 | Molsoft LLC., San Diego, CA | Protein structure visualization software |
Additional files
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Supplementary file 1
Sequences of LOV2GIV constructs.
- https://cdn.elifesciences.org/articles/60155/elife-60155-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60155/elife-60155-transrepform-v2.pdf
