Journal: eLife

Article Title: Discrete GPCR-triggered endocytic modes enable β-arrestins to flexibly regulate cell signaling

doi: 10.7554/eLife.81563

Figure Lengend Snippet: Direct NanoBiT luciferase complementation of β2-adrenergic receptor (β2AR)-LgBiT and SmBiT-tagged: Nb33 (a μOR receptor-specific nanobody, black), visual arrestin (pink), wild-type β-arrestin-2 (green), CT mutant (dark purple), CLB mutant (light purple), or double mutant (cyan) measured as an end point across a range of isoproterenol (Iso) concentrations ( a ) and kinetically ( b ) pre- and post-stimulation with 10 μM Iso. Dose–response curves were generated with three-parameter nonlinear fit ( R 2 = 0.94–0.99). ( c ) Endogenous β2AR cAMP response after stimulation with 10 μM Iso measured by a genetically encoded fluorescent cAMP biosensor, cADDis, and normalized to the response elicited by simultaneous treatment with 10 μM forskolin (Fsk) and 300 μM 3-isobutyl-1-methylxanthine (IBMX). ( d ) Area under the curve calculated from panel ( c ). All data are shown as mean ± standard error of the mean (SEM). from three independent experiments performed in βarr1/2 DKO HEK293 cells. Significance was determined by an ordinary one-way analysis of variance (ANOVA) (df = 4, F = 112.1) with Tukey’s multiple comparisons test. ns p ≥ 0.05, *p < 0.05, ** p < 0.01, ***p < 0.001. ( e ) Diagram of proposed model involving two differentially utilized allosteric paths from GPCRs through β-arrestins to promote endocytosis. Class A GPCRs (blue), exemplified by the β2AR, primarily utilize the CLB to drive endocytosis while Class B GPCRs, exemplified by V2R (magenta) and β2V2R (gold) utilize both determinants. Arrows represent the proposed allosteric paths linking the GPCR/β-arrestin interface to the β-arrestin/clathrin-coated pit (CCP) interface, explaining how the CLB-dependent endocytic mode is coupled to rapid desensitization of receptor signaling while the CT-dependent mode enables prolonged signaling. Figure 5—source data 1. Source data for results graphed in .

Article Snippet: The β2AR-3S was previously described ( ). β2AR-LgBiT was generated by PCR amplification from the LgBiT Expression Vector (Promega) and inserted into PCR linearized FLAG-tagged β2AR using InFusion HD. β-Arrestin-2–GFP and β-arrestin-2–mApple were previously described ( ; ). β-Arrestin-2-CCS–EGFP was previously described ( ).

Techniques: Luciferase, Mutagenesis, Generated

Journal: eLife

Article Title: Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

doi: 10.7554/eLife.43826

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: For LOVTRAP experiments, AsLOV2(V416L) and Zdk1 were amplified from Addgene plasmids #81036 and #81010 (Klaus Hahn), and β2AR was amplified from Addgene plasmid #104841 (Alice Ting).

Techniques: Plasmid Preparation

Journal: eLife

Article Title: Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

doi: 10.7554/eLife.43826

Figure Lengend Snippet: ( A ) Schematic of NanoLuc fused to membrane-localized protein β2AR and asLOV2 in LOVTRAP. When asLOV2 is activated by NanoLuc (or blue light), mCherry-Zdk1 dissociates from the C-terminus of asLOV2. The asLOV2 contains a V416L mutation that slows its off kinetics (return to the dark state conformation), and consequently slows the rate of Zdk1 recapture (V416L reset t 1/2 =496 s). ( B ) Example immunofluorescence images of HEK293T cells expressing mCherry-Zdk1 and β2AR-NanoLuc-LOV. Left: mCherry expression is visible along the cell membrane in the dark. Right: following either furimazine for 1 min or blue light for 10 s, mCherry-Zdk1 relocalizes to the cytoplasm. The yellow line indicates pixels quantified for mCherry signal. Scale bars, 11 µm. ( C ) Mean normalized mCherry fluorescence measured across the cell membrane for the same cells before and after furimazine (top), or before and after blue light (bottom; n = 7 cells for both conditions). Dashed box indicates intracellular region quantified in panel D ). Data plotted as mean ±s.e.m. ( D ) The mean normalized cytosolic mCherry fluorescence increased following either furimazine or light exposure (Dark vs Furimazine: 0.38 ± 0.061 vs 0.82 ± 0.027, n = 7 cells, Wilcoxon’s signed-rank test, *p=0.016. Dark vs Light: 0.51 ± 0.035 vs 0.88 ± 0.025, n = 7 cells, Wilcoxon’s signed-rank test, *p=0.016). The Furimazine/Dark ratio of cytosolic mCherry fluorescence was comparable to the Light/Dark ratio (Furimazine/Dark: 2.75 ± 0.68, Light/Dark: 1.79 ± 0.12, n = 7 cells each group, Wilcoxon’s ranksum test: p=0.84). Data plotted as mean ±s.e.m. ( E ) Schematic of NanoLuc fused to extracellular N-terminus of β2AR-asLOV2 in LOVTRAP. When NanoLuc is expressed extracellularly, we do not expect to observe NanoLuc-asLOV activation with furimazine. As a result, mCherry-Zdk1 should remain at the membrane. ( F ) Example immunofluorescence images of HEK293T cells expressing mCherry-Zdk1 and NanoLuc-β2AR-LOV. Left: mCherry expression is visible along the cell membrane in the dark. Middle: following furimazine for 1 min, mCherry-Zdk1 remains along the cell membrane. Right: following 10 s of blue light, mCherry-Zdk1 relocalizes to the cytosol. The yellow line indicates pixels quantified for mCherry signal. Scale bars, 11 µm. ( G ) Mean normalized mCherry fluorescence measured across the cell membrane for the same cells in the dark, after furimazine, and then after blue light (n = 8 cells). Dashed box indicates intracellular region quantified in panel H ). Data plotted as mean ±s.e.m. ( H ) With the extracellular NanoLuc, the mean normalized cytosolic mCherry fluorescence did not increase following furimazine. However, subsequent blue light exposure resulted in an increase in the mean normalized cytosolic mCherry fluorescence (Dark: 0.56 ± 0.040; Furimazine: 0.53 ± 0.036; Light: 0.91 ± 0.025; n = 8 cells each group, 1-way ANOVA, F (2,21) =37.49, p = 1.18e-7; Tukey’s multiple comparison’s test, ***p < 0.001 Light compared to all other conditions). Data plotted as mean ± s.e.m. See also . 10.7554/eLife.43826.006 Figure 2—source data 1 Excel spreadsheet containing fluorescence intensity values used to generate panels C-D and G-H.

Article Snippet: For LOVTRAP experiments, AsLOV2(V416L) and Zdk1 were amplified from Addgene plasmids #81036 and #81010 (Klaus Hahn), and β2AR was amplified from Addgene plasmid #104841 (Alice Ting).

Techniques: Membrane, Mutagenesis, Immunofluorescence, Expressing, Fluorescence, Activation Assay

Journal: eLife

Article Title: Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

doi: 10.7554/eLife.43826

Figure Lengend Snippet: ( A ) HEK293T cells were transfected as in . Example sequential immunofluorescence images of mCherry-Zdk1 during baseline (Dark), 10 µM furimazine, and blue light for the intracellular β2AR-NanoLuc-asLOV2 construct. Left: mCherry expression is visible along the cell membrane in the dark. Middle: following furimazine for 1 min, mCherry-Zdk1 relocalizes to the cytosol. Right: subsequent blue light does not result in any additional mCherry-Zdk1 release to the cytosol. The yellow line indicates pixels quantified for mCherry signal. Scale bar, 11 µm. ( B ) Mean normalized mCherry fluorescence measured across the cell membrane for the same cells before and after furimazine and then blue light (n = 7 cells). Dashed box indicates intracellular region quantified in panel C ). Data plotted as mean ± s.e.m. ( C ) The mean normalized cytosolic mCherry fluorescence increased following furimazine exposure. There was no an additional increase in relative mCherry cytosolic fluorescence following subsequent light exposure (Dark: 0.48 ± 0.045; Furimazine: 0.92 ± 0.012; Light: 0.91 ± 0.026; n = 7 cells each group, 1-way ANOVA, F (2,18) =67.42, p = 4.36e-9; Tukey’s multiple comparison’s test for Furimazine vs Light, p = 0.93). ( D ) Even though the Extracellular NanoLuc construct did not activate LOVTRAP, NanoLuc bioluminescence measured in the Extracellular condition was greater than in the Intracellular condition (Intracellular: 104 × 10 4 ± 4.3 × 10 4 ; Extracellular: 53 × 10 4 ± 2.1 × 10 4 ; n = 4 replicates, Wilcoxon’s ranksum test, p = 0.03). 10.7554/eLife.43826.005 Figure 2—figure supplement 1—source data 1. Excel spreadsheet containing fluorescence intensity and luminescence values used to generate panels B-D.

Article Snippet: For LOVTRAP experiments, AsLOV2(V416L) and Zdk1 were amplified from Addgene plasmids #81036 and #81010 (Klaus Hahn), and β2AR was amplified from Addgene plasmid #104841 (Alice Ting).

Techniques: Transfection, Immunofluorescence, Construct, Expressing, Membrane, Fluorescence

Journal: eLife

Article Title: Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

doi: 10.7554/eLife.43826

Figure Lengend Snippet: ( A ) Schematic of NanoLuc fused to arrestin-TEVp, co-expressed with GPCR-LOV-TEVcs-Gal4 and UAS-Citrine. In the dark and in the absence of the GPCR’s agonist, the TEVcs is caged by the LOV domain and the NanoLuc-arrestin-TEVp is not in proximity to the TEVcs. In the presence of both furimazine and agonist, NanoLuc-arrestin-TEVp is recruited to the GPCR, allowing NanoLuc to uncage eLOV and the TEVp to cleave the TEVcs. The released Gal4 then drives transcription of a UAS reporter gene. ( B ) Example immunofluorescence images of UAS-Citrine expression in HEK293T cells ~ 8 hr following 10 µM furimazine and agonist (20 min 100 µM isoetharine for β2AR, 15 min 10 µM vasopressin for AVPR2, 2 hr 10 µM dopamine for DRD1). Control conditions without agonist and/or without furimazine had low Citrine expression. Scale bars, 100 µm. ( C ) HEK293T cells were transfected with β2AR SPARK2 components as in panel A). SPARK2 activation (Citrine reporter) and SPARK2 expression (HA tag on NanoLuc-arrestin-TEVp) were imaged ~8 hr after 15 min of exposure to 10 µM furimazine or light and 10 µM isoetharine. ( D ) All HA-positive cells were quantified by their Citrine to HA fluorescence intensity ratio. Background fold changes in Furimazine/-Iso and Light/-Iso signal are displayed in red, while ±Ligand signal ratios are displayed in black. There was no additional background signal in the Furimazine/-Iso condition compared to the Dark/-Iso condition (1.0-fold), whereas the Light/-Iso condition had a significant 1.8-fold increase in background compared to the Dark/-Iso condition. There was a 2.2-fold ± Iso signal ratio with furimazine, and a 1.9-fold ± Iso signal ratio with light (n = 10 FOVs each group, two-way ANOVA, interaction F (2,54) =25.49, p = 1.61e-8; Tukey’s multiple comparison’s test, *p < 0.001). Data plotted as mean ± s.e.m. ( E ) Top: Schematic illustrating potential for background TEVcs cleavage in the absence of a PPI using a luciferase-mediated single ‘AND’ gate (NanoLuc directly fused to N-terminus of LOV domain). Bottom: Schematic illustrating negligible background TEVcs cleavage in the absence of a PPI using the luciferase-mediated nested ‘AND’ gate in SPARK2 (NanoLuc fused to arrestin-TEVp). ( F ) HEK293T cells were transfected either with a modified β2AR-NanoLuc-LOV direct fusion SPARK construct and Arrestin-TEVp (top) or β2AR SPARK2 components (bottom). SPARK activation (Citrine reporter) and SPARK expression (HA tag on Arrestin-TEVp or NanoLuc-arrestin-TEVp) were imaged ~8 hr after continuous exposure to 10 µM furimazine and 10 µM isoetharine. Scale bars, 100 µm. ( G ) Data were quantified as in panel D). In the modified fusion SPARK (single ‘AND’ gate), there was not a robust ±Iso signal ratio with furimazine (1.5-fold, not significant, n.s.), and a 2.8-fold background increase due to furimazine alone (n = 10 FOVs each group, two-way ANOVA, interaction F (2,54) =14.11, p = 1.18e-5; Tukey’s multiple comparison’s test, *p < 0.001). In SPARK2 (nested ‘AND’ gate), there was a 2.9-fold ± Iso signal ratio with furimazine, and no significant background due to furimazine alone (1.0-fold, n.s.; n = 10 FOVs each group, two-way ANOVA, interaction F (2,54) =9.82, p = 2.0e-4; Tukey’s multiple comparison’s test, *p < 0.001). Data plotted as mean ± s.e.m. ( H ) HEK293T cells transfected as in panel F) were exposed to blue light for 5 min, with or without 10 µM isoetharine. The fusion and SPARK2 conditions resulted in similar ±Iso signal ratios with light, 2.5- and 2.4-fold, respectively (Data analyzed in two-way ANOVA with data from panel (G). Fusion: n = 10 FOVs each group, two-way ANOVA, interaction F (2,54) =14.11, p = 1.18e-5; SPARK2: n=10 FOVs each group, two-way ANOVA, interaction F (2,54) = 9.82, p = 2.0e-4; Tukey’s multiple comparison’s test, *p < 0.01). Data plotted as mean ± s.e.m. See also . 10.7554/eLife.43826.010 Figure 3—source data 1. Excel spreadsheet containing fluorescence ratio intensity values used to generate panels D, G, and H.

Article Snippet: For LOVTRAP experiments, AsLOV2(V416L) and Zdk1 were amplified from Addgene plasmids #81036 and #81010 (Klaus Hahn), and β2AR was amplified from Addgene plasmid #104841 (Alice Ting).

Techniques: Immunofluorescence, Expressing, Control, Transfection, Activation Assay, Fluorescence, Luciferase, Modification, Construct

Journal: eLife

Article Title: Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

doi: 10.7554/eLife.43826

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with either β2AR SPARK1 (arrestin-TEVp) or SPARK2 (NanoLuc-arrestin-TEVp) and UAS-Citrine. The same transmembrane construct was used in both conditions (β2AR-eLOV-TEVcs-Gal4). SPARK1/2 activation (Citrine reporter) and SPARK1/2 expression (HA tag on arrestin-TEVp or NanoLuc-arrestin-TEVp) were imaged ~8 hr after 15 min of exposure to 10 µM furimazine or blue light, and 10 µM isoetharine. Scale bars, 100 µm. ( B ) All HA-positive cells were quantified by their Citrine to HA fluorescence intensity ratio. Background fold changes in Furimazine/-Iso and Light/-Iso signal are displayed in red, while ±Ligand signal ratios are displayed in black. With SPARK1, there was a 2.8-fold ± Iso signal ratio with light, but a significant 2.2-fold background increase due to light alone (n=5 FOVs each group, two-way ANOVA, interaction F (1,16) =41.85, p = 7.74e-6; Tukey's multiple comparison's test, *p < 0.05). With SPARK2, there was a 4.4-fold ± Iso signal ratio with furimazine, and no significant background due to furimazine alone (1.1-fold, n.s.). As expected, SPARK2 with light-gating had a lower 2.1-fold ± Iso signal ratio, and a significant 2.3-fold background increase due to light alone (n = 5 FOVs each group, two-way ANOVA, interaction F (2,24) =36.74, p = 4.96e-8; Tukey’s multiple comparison’s test, *p < 0.01). Data plotted as mean ± s.e.m. ( C ) All individual cells analyzed in the SPARK2 experiment in plotted according to their mean HA and Citrine intensities. 10.7554/eLife.43826.009 Figure 3—figure supplement 1—source data 1 Excel spreadsheet containing fluorescence intensity ratio values used to generate panel B.

Article Snippet: For LOVTRAP experiments, AsLOV2(V416L) and Zdk1 were amplified from Addgene plasmids #81036 and #81010 (Klaus Hahn), and β2AR was amplified from Addgene plasmid #104841 (Alice Ting).

Techniques: Transfection, Construct, Activation Assay, Expressing, Fluorescence, Comparison

Journal: eLife

Article Title: Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

doi: 10.7554/eLife.43826

Figure Lengend Snippet: ( A ) Schematic of the general timeline for SPARK2 assays using an orthogonal luciferase reporter such as FLuc. For simplicity, only the luciferases and LOV domain are illustrated (unfilled luciferases indicate no luminescence; filled luciferases indicate luciferin substrate-mediated luminescence). ( B ) HEK293T were transfected with β2AR SPARK2 components and UAS-FLuc.~8 hr after SPARK2 labeling with 10 µM furimazine/blue light and 10 µM isoetharine, the UAS-FLuc luminescence recorded using a platereader (n = 4 replicates each condition). The highest ±Iso signal ratio was achieved using 20 min exposure to furimazine (11.6-fold). 5 min of blue light resulted in a ± Iso signal ratio of 6.4-fold, whereas 20 min of blue light or 5 min of furamizine resulted in similar ±Iso signal ratios of 4.2- and 4.6-fold, respectively. ( C–D ) HEK293T cells were transfected with either β2AR or DRD1 SPARK2 components and UAS-FLuc. Cells were exposed to 10 µM furimazine and 5 µM each of 92 different drugs for 1 hr (BioMol FDA-approved compound library). In addition, we performed two DMSO vehicle measurements ( A1–A2 ), and two positive control spike-ins (isoetharine in A3 and dopamine in A4). ~8 hr later, FLuc luminescence was measured on a platereader. The Log 2 fold change in FLuc luminescence is plotted corresponding to each compound’s position in the 96-well plate. Data plotted as the average of two biological replicates. Compound list and positions in . ( E ) Sorted heatmap of fold changes in FLuc luminescence for β2AR and DRD1 SPARK2 for all 94 compounds (excluding DMSO replicates). Example compound hits for each receptor are labeled. Heatmap thresholded from 1 to 2.5 for visualization, mean fold-changes for selected hits listed in parentheses. Fold-changes for all compounds listed in . 10.7554/eLife.43826.014 Figure 4—source data 1. Excel spreadsheet containing luminescence values used to generate panel B. 10.7554/eLife.43826.015 Figure 4—source data 2. Excel spreadsheet containing SPARK2 drug screen compounds and mean fold-changes (n = 2 biological replicates) to generate panels C-E.

Article Snippet: For LOVTRAP experiments, AsLOV2(V416L) and Zdk1 were amplified from Addgene plasmids #81036 and #81010 (Klaus Hahn), and β2AR was amplified from Addgene plasmid #104841 (Alice Ting).

Techniques: Luciferase, Transfection, Labeling, Drug discovery, Positive Control

Journal: eLife

Article Title: Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

doi: 10.7554/eLife.43826

Figure Lengend Snippet: ( A ) Schematic for testing whether NanoLuc activation of eLOV is proximity-dependent. NanoLuc was fused either to arrestin-TEVp (left, Complexed), or expressed at the membrane as CD4-NanoLuc (right, Nearby). ( B ) HEK293T cells were transfected with constructs in Panel A ), with either the Complexed or Nearby (membrane-bound NanoLuc) configurations of β2AR SPARK2. The UAS-FLuc reporter luminescence was measured ~8 hr after 20 min of exposure to 10 µM furimazine and 10 µM isoetharine (n = 4 replicates each condition). The Complexed condition resulted in a ± Iso signal ratio of 9.0-fold, and the Nearby condition resulted in a ± Iso signal ratio of 3.4-fold. ( C ) Even though the Nearby NanoLuc construct did not activate LOV as efficiently as the Complexed NanoLuc construct, NanoLuc bioluminescence measured in the Nearby condition was greater than in the Complexed condition (Complexed: 4.55e6 ±1.46e5, Nearby: 7.55e6 ±3.47e4; n = 5 technical replicates each condition; Student’s t-test, t 8 = −20.01, p=4.05e-8). ( D ) FLuc reporter luminescence measurements following 10 min of blue light with and without 10 µM isoetharine were similar between the Complexed and Nearby conditions (n = 4 replicates each condition). 10.7554/eLife.43826.013 Figure 4—figure supplement 1—source data 1. Excel spreadsheet containing luminescence values used to generate panels B-D.

Article Snippet: For LOVTRAP experiments, AsLOV2(V416L) and Zdk1 were amplified from Addgene plasmids #81036 and #81010 (Klaus Hahn), and β2AR was amplified from Addgene plasmid #104841 (Alice Ting).

Techniques: Activation Assay, Membrane, Transfection, Construct

Journal: Cell

Article Title: Multidimensional tracking of GPCR signaling via peroxidase-catalyzed proximity labeling

doi: 10.1016/j.cell.2017.03.028

Figure Lengend Snippet: (A) Internalization kinetics of β2AR (class A GPCR) are different from those of AT1R (class B GPCR). β-arrestin is recruited with the peak at 180 s, but clathrin recruitment is delayed to 600 s. Also, Rab5 enrichment peak is at 600 s, indicating arrestin-receptor complex formation and endosomal entry do not occur simultaneously. Late endosomal marker Rab7 and lysosomal marker LAMP1 enrichment peaks appear afterward.

Article Snippet: Each well was transfected with 20 ng each of a pcDNA5 encoding β2AR-APEX/β2AR wild-type and CRE-SEAP reporter plasmid (BD Biosciences).

Techniques: Marker