Optogenetic activation of heterotrimeric Gi proteins by LOV2GIVe— a rationally engineered modular protein

Heterotrimeric G-proteins are signal transducers that mediate the action of many natural extracellular stimuli as well as of many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we engineered LOV2GIVe, a modular protein that allows the activation of Gi proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, i.e. metazoan opsins, which are light-activated GPCRs. To make LOV2GIVe, we fused a peptide derived from a non-GPCR protein that activates Gαi (but not Gαs, Gαq, or Gα12) to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity. GRAPHICAL SUMMARY


INTRODUCTION
Heterotrimeric G-proteins are critical transducers of signaling triggered by a large family of G-protein-coupled receptors (GPCRs). GPCRs are Guanine nucleotide Exchange Factors (GEFs) that activate G-proteins by promoting the exchange of GDP for GTP on the Gα-subunits (Gilman, 1987). This signaling axis regulates a myriad of (patho)physiological processes and also represents the target for >30% of drugs on the market (Sriram and Insel, 2018), which fuels the interest in developing tools to manipulate G-protein activity in cells with high precision.
Optogenetics, the use of genetically-encoded proteins to control biological processes with light (Warden et al., 2014) is well-suited for the non-invasive manipulation of signaling.
Metazoan opsins are light-activated GPCRs that have been repurposed as optogenetic tools (Airan et al., 2009;Bailes et al., 2012;Karunarathne et al., 2013;Oh et al., 2010;Siuda et al., 2015), albeit with limitations. For example, opsins tend to desensitize after activation due to receptor internalization and/or exhaustion of their chromophore, retinal (Airan et al., 2009;Bailes et al., 2012;Siuda et al., 2015). Exogenous supplementation of retinal, which is not always feasible, is required in many experimental settings because this chomophore is not readily synthesized by most mammalian cell types or by many organisms. Also, opsins are relatively large, post-translationally modified transmembrane proteins, which makes them inherently difficult to modify for optimization and/or customization for specific applications.
Here we leveraged the light-sensitive LOV2 domain of Avena sativa (Harper et al., 2003) to develop an optogenetic activator of heterotrimeric G-proteins not based on opsins. LOV2 uses ubiquitously abundant FMN as the chromophore and does not desensitize. It is small (~17 KDa) and expresses easily as a soluble globular protein in different systems (Lungu et al., 2012;Strickland et al., 2012;Zayner et al., 2013), making it experimentally tractable, easily customizable, and widely applicable. Our results provide the proof of principle for a versatile optogenetic activator of heterotrimeric G-proteins that does not rely on GPCR-like proteins.

Design and optimization of a photoswitchable G-protein activator
We envisioned the design of a genetically-encoded photoswitchable activator of heterotrimeric G-proteins by fusing a short sequence (~25 amino acids) from the protein GIV (a.k.a. Girdin) called the Gα-Binding-and-Activating (GBA) motif to the C-terminus of LOV2 (Fig.   1A, left). GBA motifs are evolutionarily conserved sequences found in various cytoplasmic, non-GPCR proteins that are sufficient to activate heterotrimeric G-protein signaling . We reasoned that the GBA motif would be "uncaged" and bind G-proteins when the C-terminal Jα-helix of LOV2 becomes disordered upon illumination (Harper et al., 2003) (Fig.   1A, right). We named this optogenetic construct LOV2GIV (pronounced "love-to-give"). We used two well-validated mutants that mimic either the dark (D) or the lit (L) conformation of LOV2 (Harper et al., 2004;Harper et al., 2003;Zimmerman et al., 2016) to facilitate LOV2GIV characterization (Fig. 1A, right).
Our first LOV2GIV prototype had a suboptimal dynamic range, as it bound the G-protein Gαi3 in the lit conformation only ~3 times more than in the dark conformation (Fig. 1B). Based on a structural homology model of LOV2GIV (Fig. 1C), we reasoned that it could be due to the relatively high accessibility of Gαi3-binding residues of the GIV GBA motif within the dark conformation of LOV2GIV. In agreement with this idea, fusion of the GBA motif at positions of the Jα helix more proximal to the core of the LOV2 domain tended to lower G-protein binding, including one variant ("e", henceforth referred to as LOV2GIVe) in which binding to the dark conformation was almost undetectable (Fig. 1D). Consistent with this, a structure homology model of LOV2GIVe showed that amino acids required for G-protein binding are less accessible than in the LOV2GIV prototype (Fig. 1E). In contrast, the lit conformation of LOV2GIVe displayed robust Gαi3 binding, thereby confirming an improved dynamic range compared to the LOV2GIV prototype (Fig. 1F).

LOV2GIVe binds and activates G-proteins efficiently in vitro only in its lit conformation
Concentration-dependent binding curves revealed that the affinity of Gαi3 for the dark conformation of LOV2GIVe is orders of magnitude weaker than for the lit conformation ( Fig.   2A), which had an equilibrium dissociation constant (Kd) similar to that previously reported for the GIV-Gαi3 interaction . We also found that LOV2GIVe retains the same G-protein specificity as GIV. Much like GIV (Garcia-Marcos et al., 2010;Marivin et al., 2020), LOV2GIVe bound robustly only to members of the G i/o family among the 4 families of Gα proteins (G i/o , G s , G q/11 and G 12/13 ), and could discriminate within the G i/o family by binding to Gαi1, Gαi2 and Gαi3 but not to Gαo (Fig. 2B). Next, we assessed if LOV2GIVe can activate Gproteins in vitro, i.e. whether it retains GIV's GEF activity, by measuring steady-state GTPase activity as done previously for GIV and other related non-receptor GEFs (Garcia-Marcos et al., 2010;Maziarz et al., 2018). We found that LOV2GIVe in the lit, but not the dark, conformation led to a dose-dependent increase of Gαi3 activity (Fig. 2C), which was comparable to that previously shown for GIV (Garcia-Marcos et al., 2009). These findings indicate that LOV2GIVe recapitulates the G-protein activating properties of GIV in vitro, and that these are effectively suppressed for the dark conformation.

LOV2GIVe activates G-protein signaling in cells in its lit conformation
To investigate LOV2GIVe-dependent G-protein activation in cells, we initially used a humanized yeast-based system (Cismowski et al., 1999;DiGiacomo et al., 2020) in which the mating pheromone response pathway has been co-opted to measure activation of human Gαi3 using a gene reporter (LacZ, β-galactosidase activity) (Fig. 3A, left). When we expressed LOV2GIVe dark (D), lit (L) or wt in this strain, only very weak levels of β-galactosidase activity were detected (Fig. 3A, right). We reasoned that this could be due to the subcellular localization of the construct, presumed to be cytosolic in the absence of any targeting sequence, because we have previously shown that GIV requires membrane localization to efficiently activate G-proteins (Parag-Sharma et al., 2016). Expressing LOV2GIVe (L) fused to a membrane targeting sequence (mLOV2GIVe) led to a very strong induction of β-galactosidase activity, which was not recapitulated by expression of mLOV2GIVe (D) or wt (Fig. 3A, right).
LOV2GIVe-mediated activation levels (several hundred-fold over basal) are comparable to those previously reported for the endogenous pathway in response to GPCR activation by mating pheromone (Hoffman et al., 2002;Lambert et al., 2010). Next, we tested mLOV2GIVe in mammalian cells. Instead of using a downstream signaling readout as we did in yeast, we used a Bioluminescence Resonance Energy Transfer (BRET) biosensor that measures G-protein activity directly (Fig. 3B, left). Expression of mLOV2GIVe (L), but not (D), led to an increase of BRET proportional to the amount of plasmid transfected (Fig. 3B, right). At the highest amount tested, mLOV2GIVe (L) caused a BRET increase comparable to that observed upon agonist stimulation of the M4 muscarinic receptor, a Gi-activating GPCR. Introducing a mutation that precludes G-protein activation by GIV's GBA motif (F1685A (FA), (Garcia-Marcos et al., 2009)), in mLOV2GIVe (L) also abolished its ability to induce a BRET increase ( Fig. 3-figure   supplement 1). These results indicate that the lit conformation of LOV2GIVe activates Gprotein signaling in different cell types.

LOV2GIVe activates G-protein signaling in cells upon illumination
Next, we tested whether LOV2GIVe can trigger G-protein activation in cells in response to light. For this, we used two complementary experimental systems. The first one consisted of measuring G-protein activity directly with the mammalian cell BRET biosensor described above upon illumination with a pulse of blue light. We found that, in HEK293T cells expressing mLOV2GIVe wt, a single short light pulse resulted in a spike of G-protein activation that decayed at a rate similar to that reported for the transition from lit to dark conformation of LOV2 (T 1/2 ~1 min) (Fig. 4A). This response was not recapitulated by a mLOV2GIVe construct bearing the GEF-deficient FA mutation (Fig. 4A). For the second system, we turned to a spot growth assay with the humanized yeast stain described above. In this system, conditional histidine prototrophy controlled by FUS1 promoter is used as a signaling readout downstream of Gprotein activation under conditions of homogenous and continued illumination (Fig. 4B, left).
Yeast cells expressing mLOV2GIVe wt grew in the absence of histidine only when exposed to blue light (Fig. 4B, right). This effect was specifically caused by light-dependent activation of mLOV2GIVe wt because cells expressing the light-insensitive mLOV2GIVe (D) construct failed to grow under the same illumination conditions, whereas mLOV2GIVe (L) grew the same regardless of illumination conditions (Fig. 4B, right). Taken together, these results show that mLOV2GIVe activates G-protein signaling in cells upon blue light illumination.

Conclusions and future perspectives
Here we have presented proof-of-principle evidence for a photoswitchable G-protein activator that does not rely on opsins, i.e., light-activated GPCRs. This tool is based on a modular design that combines the properties of the light-sensitive LOV2 domain with a motif present in a non-GPCR activator of G-proteins. We propose that the versatility of the LOV2GIVe design could help overcome some limitations of currently available optogenetic tools that are based on GPCR-like proteins. For example, our results indicate that LOV2GIVe requires targeting to membranes where the substrate G-protein localizes (Fig. 3A), a feature that could be leveraged in the future to target it to different membranous organelles to study how activation of G-proteins in discrete subcellular compartments triggers different signaling events (Stoeber et al., 2018;Tsvetanova et al., 2015). Our results in yeast also support that LOV2GIVe can activate G-protein signaling ( Fig. 4C) even in cell types where there is not sufficient synthesis of retinal to support opsin-based activation (Scott et al., 2019). A limitation of LOV2GIVe is that it only acts on one subset of heterotrimeric G-proteins, those containing Gαi subunits.
Nevertheless, potential applications are still broad, as Gi proteins control processes as diverse as inhibitory neuromodulation, opioid action, or heart rate, among many others.

Reagents and Antibodies
Unless otherwise indicated, all chemical reagents were obtained from Sigma or Fisher Scientific. Fluorescein di-β-D-galactopyranoside (FDG) was from Marker Gene Technologies, and the protein inhibitor mixture was from Sigma (catalog no. S8830). Leupeptin, pepstatin, and aprotinin were from Gold Biotechnology. All restriction endonucleases and E. coli strain BL21(DE3) were from Thermo Scientific. E. coli strain DH5α was purchased from New England Biolabs. Pfu ultra DNA polymerase used for site-directed mutagenesis was purchased from Agilent. [γ-32 P]GTP was from Perkin Elmer. Mouse monoclonal antibodies raised against αtubulin (T6074), FLAG tag (F1804) or His tag (H1029) were from Sigma. Mouse monoclonal antibody raised against hemagglutinin (HA) tag (clone 12CA5, #11583816001) was obtained from Roche. Mouse monoclonal antibody raised against MYC tag (9B11, #2276) was from Cell Signaling. Rabbit polyclonal antibody raised against Gαs (C-18, sc-383) was purchased from Santa Cruz Biotechnology. Goat anti-rabbit Alexa Fluor 680 (A21077) and goat anti-mouse IRDye 800 (#926-32210) secondary antibodies were from Life technologies and LiCor, respectively.

Plasmid Constructs
The parental LOV2GIV sequence was obtained as a synthetic gene fragment from GeneScript and subsequently amplified by PCR with extensions at the 5' and 3' ends that made it compatible with a ligation independent cloning (LIC) system (Stols et al., 2002). For the bacterial expression of GST-LOV2GIV constructs, we inserted the LOV2GIV sequence into the pLIC-GST plasmid kindly provided by J. Sondek (UNC-Chapel Hill) (Cabrita et al., 2006). Two Information. For the yeast expression of LOV2GIVe constructs, we inserted the LOV2GIVe sequence into two different versions of a previously described pLIC-YES2 plasmid (Coleman et al., 2016). Both versions contain a MYC-tag sequence cloned upstream of the LIC cassette between the HindIII and KpnI sites, but in one of the two plasmids it was preceded by a sequence encoding the first 9 amino acids of Saccharomyces cerevisiae Gpa1, a previously validated membrane-targeting sequence (Parag-Sharma et al., 2016;Song et al., 1996). For the mammalian expression of LOV2GIVe constructs, we inserted the LOV2GIVe sequence into a modified pLIC-myc plasmid ( (Cabrita et al., 2006), kindly provided by J. Sondek (UNC-Chapel Hill, NC)), in which a sequence encoding the first 11 amino acids of Lyn, a previously validated membrane-targeting sequence (Inoue et al., 2005;Parag-Sharma et al., 2016), was inserted in the AflII/KpnI sites upstream of the MYC-tag. The resulting sequence preceding the first amino acid of LOV2GIVe is MGCIKSKGKDSGTELGSMEQKLISEEDLGILYFQSNA (bold= Lyn11, underline= MYC-tag). Cloning of the pET28b-Gαi3 and pET28b-Gαo plasmids for the bacterial expression of rat His-Gαi3 or rat His-Gαo (isoform A), respectively, have been described previously (Garcia-Marcos et al., 2010;Garcia-Marcos et al., 2009). Plasmid pLIC-Gαi1(int.6xHis) for the bacterial expression of rat Gαi1 with an internal hexahistidine tag at position 120 was generated by PCR amplification from pQE6-Gαi1(int.6xHis) ( (Dessauer et al., 1998), kindly provided by Carmen Dessauer, University of Texas, Houston) and insertion at NdeI/BglIIsites of the pLIC-His plasmid (Stols et al., 2002). The plasmid for the bacterial expression of rat His-Gαi2 (pET28b-Gαi2) was generated by inserting the Gαi2 sequence into the NdeI/EcoRI of pET28b. Plasmids for expression of FLAG-Gαi3 (rat, p3XFLAG-CMV10-Gαi3, N-terminal 3XFLAG tag) or Gαs (human, pcDNA3.1(+)-Gαs) in mammalian cells were described previously (Beas et al., 2012;Garcia-Marcos et al., 2009). Plasmids for the expression of Gαq-HA (mouse, pcDNA3-Gαq-HA, internally tagged) or Gα12-MYC (mouse, pcDNA3.1-Gα12-MYC, internally tagged) in mammalian cells were kindly provided by P. Wedegaertner (Thomas Jefferson University) (Wedegaertner et al., 1993)  The plasmid for the expression of M4R was obtained from the cDNA Resource Center at Bloomsburg University (pcDNA3.1-3xHA-M4R, cat# MAR040TN00).

Steady-state GTPase Assay
This assay was performed as described previously

Yeast Strains and Manipulations
The previously described (Cismowski et al., 1999) Saccharomyces cerevisiae strain  (Cismowski et al., 2002;Cismowski et al., 1999;Maziarz et al., 2018). Plasmid transformations were carried out using the lithium acetate method. CY7967 was first transformed with a centromeric plasmid (CEN TRP) encoding the LacZ gene under the control of the FUS1 promoter (FUS1p), which is activated by the pheromone response pathway. The FUS1p::LacZ-expressing strain was transformed with pLIC-YES2 plasmids (2µm, URA) encoding each of the LOV2GIV constructs described in "Plasmid Constructs". Double transformants were selected in synthetic defined (SD)-TRP-URA media.
Individual colonies were inoculated into 3 ml of SDGalactose-TRP-URA and incubated overnight at 30°C to induce the expression of the proteins of interest under the control of the galactoseinducible promoter of pLIC-YES2. This starting culture was used to inoculate 20 ml of SDGalactose-TRP-URA at 0.3 OD600. Exponentially growing cells (~0.7-0.8 OD600, 4-5 hours) were pelleted to prepare samples for "β-galactosidase Activity Assay" and "Yeast Spot Growth Assay" described below and for preparing samples for immunobloting as previously described Maziarz et al., 2018). Briefly, pellets corresponding to 5 OD600 were washed once with PBS + 0.1% BSA and resuspended in 150 µl of lysis buffer (10 mM Tris-HCl, pH 8.0, 10% (w:v) trichloroacetic acid (TCA), 25 mM NH 4 OAc, 1 mM EDTA). 100 µl of glass beads were added to each tube and vortexed at 4°C for 5 min. Lysates were separated from glass beads by poking a hole in the bottom of the tubes followed by centrifugation onto a new set of tubes. The process was repeated after the addition of 50 µl of lysis buffer to wash the glass beads. Proteins were precipitated by centrifugation (10 min, 20,000 g) and resuspended in 60 µl of solubilization buffer (0.1 M Tris-HCl, pH 11.0, 3% SDS). Samples were boiled for 5 min, centrifuged (1 min, 20,000 g) and 50 µl of the supernatant transferred to new tubes containing 12.5 µl of Laemmli sample buffer and boiled for 5 min.
Enzymatic activity was calculated as arbitrary fluorescent units (a.f.u.) per minute (min).

Bioluminescence Resonance Energy Transfer (BRET)-based G Protein Activation Assay-
BRET experiments were conducted as described previously (Maziarz et al., 2018) . HEK293T cells (ATCC®, CRL-3216) were seeded on 6-well plates (~400,000 cells/well) coated with gelatin and after one day transfected using the calcium phosphate method with plasmids encoding for the following constructs (DNA amounts in parenthesis): Venus(155-239)-Gβ 1 (VC-Gβ 1 ) (0.2 μg), Venus(1-155)-Gγ 2 (VN-Gγ 2 ) (0.2 μg) and Gαi3 (1 μg) mas-GRK3ct-Nluc (0.2 μg) along with mLOV2GIVe constructs in the amounts indicated in the corresponding figure legends. Approximately 16-24 hours after transfection, cells were washed and gently scraped in room temperature PBS, centrifuged (5 min at 550g) and resuspended in assay buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.37 mM NaH 2 PO 4 , 20 mM HEPES pH 7.4, 0.1% glucose) at a concentration of 1 million cells/ml. 25,000-50,000 cells were added to a white opaque 96-well plate (Opti-Plate, Perkin Elmer) and mixed with the nanoluciferase substrate Nano-Glo (Promega cat# N1120, final dilution 1:200) for 2 min before measuring luminescence signals in a POLARstar OMEGA plate reader (BMG Labtech) at 28 ºC. Luminescence was measured at 460 ± 40 nm and 535 ± 10 nm, and BRET was calculated as the ratio between the emission intensity at 535 ± 10 nm divided by the emission intensity at 460 ± 40 nm. For the activation of M4R in Fig. 3B, cells were exposed to 100 µM carbachol for 4 min prior to measuring BRET. For measurements shown in Fig. 3B and Fig. 4B, BRET data are presented as the difference from cells not expressing LOV2GIVe constructs. For kinetic BRET measurements shown in Fig. 4A, luminescence signals were measured every 0.24 seconds for the duration of the experiment. The illumination pulse was achieved by switching from the luminescence read mode to the fluorescence read mode of the plate reader, with the following settings: 485 ± 6 nm filter, 200 flashes (~1.5 s). After the pulse of illumination, measurements were returned to luminescence mode with the same settings as prior to illumination. BRET data was corrected by subtracting the BRET signal baseline (average of 30 seconds pre-light pulse) and then subjected to a smoothening function (second order, four neighbors) in GraphPad for presentation. At the end of some BRET experiments, a separate aliquot of the same pool of cells used for the luminescence measurements was centrifuged for 1 min at 14,000 g and pellets stored at -20 °C. To prepare lysates for immunoblotting, pellets were resuspended in lysis buffer (20 mM HEPES, pH 7.2, 5 mM Mg(CH3COO) 2 , 125 mM K(CH3COO), 0.4% Triton X-100, 1 mM DTT, and protease inhibitor mixture). After clearing by centrifugation at 14,000 g at 4 °C for 10 min, protein concentration was determined by Bradford and samples boiled in Laemmli sample buffer for 5 min before following the procedures described in "Immunoblotting".

Yeast Spot Growth Assay
Cells bearing LOV2GIVe constructs growing exponentially in SDGalactose media were pelleted as described above ("Yeast Strains and Manipulations"), and resuspended at equal densities. Equal volumes of each strain were spotted on agar plates in 4 identical sets. Two of the sets were seeded on SDGalactose-TRP-URA plates with histidine and the other two sets were seeded on SDGalactose-TRP-URA-HIS (supplemented with 5 mM 3-amino-1,2,4-triazole).
From each one of the two pairs of sets, one of the plates was exposed to a homemade array of blue LED strips positioned approximately 12 cm above the plates (~2,000 Lux as determined by a Trendbox Digital Light Meter HS1010A) whereas the other one was incubated side by side under the same light but tightly wrapped in aluminum foil. Plates were incubated simultaneously under these conditions for 4 days at 30 °C and then imaged using an Epson flatbed scanner.

Immunoblotting
Proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were blocked with 5% (w:vol) non-fat dry milk and sequentially incubated with primary and secondary antibodies. For protein-protein binding experiments with GST-fused proteins, PVDF membranes were stained with Ponceau S and scanned before blocking. The primary antibodies used were the following: MYC (1:1,000), His (1:2,500), FLAG (1:2,000), α-tubulin (1:2,500), HA (1:1,000) and Gαs (1:500). The secondary antibodies were goat anti-rabbit Alexa Fluor 680 (1:10,000) and goat anti-mouse IRDye 800 (1:10,000). Infrared imaging of immunoblots was performed using an Odyssey Infrared Imaging System (Li-Cor Biosciences). Images were processed using the ImageJ software (NIH) and assembled for presentation using Photoshop and Illustrator softwares (Adobe). . Right, diagram depicting the design principle of the photoswitchable G-protein activator LOV2GIV. In the dark conformation (D, which is mimicked by the LOV2 C450S mutant), the C-terminus of LOV2 forms an α-helix (Jα) and the GBA motif is not readily accessible for G-proteins. In the lit conformation (L, which is mimicked by the LOV2 mutant I539E), the C-terminus of LOV2 is more disordered and the GBA motif becomes accessible for G-proteins, which in turn are activated upon binding to the GBA motif. (B) LOV2GIV (L) binds Gαi3 better than LOV2GIV (D). Approximately 20 µg of the indicated purified GST-fused constructs were immobilized on glutathione-agarose beads and incubated with 3 µg (~300 nM) of purified His-Gαi3. Resin-bound proteins were eluted, separated by SDS-PAGE, and analyzed by Ponceau S-staining and immunoblotting (IB) with the indicated antibodies. Input= 10% of the total amount of His-Gαi3 added in each binding reaction. (C) Ribbon representation of a LOV2GIV structure homology model generated using the I-TASSER server. On the left, the model is colored blue for LOV2 and red for GIV, whereas on the right it is colored according to solvent accessibility. Selected GIV residues known to be important for G-protein binding (L1682, F1685, L1686) Kalogriopoulos et al., 2019)   Approximately 20 µg of the indicated purified GST-fused constructs were immobilized on glutathione-agarose beads and incubated with the lysates of HEK293T cells expressing the indicated G-proteins (FLAG-Gαi3, Gαs, Gαq-HA and Gα12-MYC on the left panels) or purified His-tagged proteins (3 µg, ~300 nM of His-Gαi1, His-Gαi2, His-Gαi3 and His-Gαo on the right panels). One representative experiment of at least three is shown. (C) LOV2GIVe (L), but not LOV2GIVe (D), increases Gαi3 activity in vitro. Steady-state GTPase activity of purified His-Gαi3 was determined in the presence of increasing amounts (0-2 µM) of purified GST-LOV2GIVe (L) (black) or GST-LOV2GIVe (D) (red) by measuring the production of [ 32 P]Pi at 15 min. Results are the mean ± S.D. of n=2. In the engineered strain, no pheromone responsive GPCR (Ste3) is expressed, the yeast G-protein Gpa1 is replaced by human Gαi3, and downstream signaling does not lead to growth arrest but promotes the activation of a reporter gene (LacZ) under the control of the pheromonesensitive, G-protein-dependent promoter of FUS1 (FUS1p). Right, membrane-anchored LOV2GIVe (mLOV2GIVe), but not its untargeted parental version, leads to strong G-protein activation only in the lit conformation. Yeast strains expressing the indicated LOV2GIVe constructs ((D), (L) or wt) or an empty vector (-) were lysed to determine β-galactosidase activity using a fluorogenic substrate (mean ± S.E.M., n=3) and to prepare samples for immunoblotting (one experiment representative of 3 is shown). (B) Left, diagrams showing the principle for the G-protein activity biosensor used in this panel. Upon action of a GPCR (top) or mLOV2GIVe (bottom), G-protein activation leads to the release of YFP-tagged Gβγ from Gαi, which then can associate with Nluc-tagged GRK3ct and results in the subsequent increase in BRET. Right, mLOV2GIVe (L) but not mLOV2GIVe (D), leads to increased G-protein activation similar to a GPCR as determined by BRET. BRET was measured in HEK293T cells transfected with the indicated amounts of mLOV2GIVe plasmid constructs along with the components of the BRET biosensor and the GPCR M4R. M4R was stimulated with 100 µM carbachol. Results in the graph on the top are expressed as difference in BRET (∆BRET) relative to cells not expressing mLOV2GIVe (mean ± S.E.M., n=3). One representative immunoblot from cell lysates made after the BRET measurements is shown on the bottom. . 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 Fig. 3. Results in the graph on the top are expressed as difference in BRET (∆BRET) relative to cells not expressing mLOV2GIVe (mean ± S.E.M., n=5). One representative immunoblot from cell lysates made after the BRET measurements is shown on the bottom.