Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time

Abstract

Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requirement that the fusion proteins, and in particular the donor, need to be exogenously expressed. To address this, we have used CRISPR/Cas9-mediated homology-directed repair to generate protein-Nanoluciferase (Nluc) fusions under endogenous promotion, thus allowing investigation of proximity between the genome-edited protein and an exogenously expressed protein by BRET. Here we report BRET monitoring of GPCR-mediated β-arrestin2 recruitment and internalisation where the donor luciferase was under endogenous promotion, in live cells and in real time. We have investigated the utility of CRISPR/Cas9 genome editing to create genome-edited fusion proteins that can be used as BRET donors and propose that this strategy can be used to overcome the need for exogenous donor expression.

Figure 1

Monitoring β-arrestin2 recruitment to genome-edited CXCR4/Nluc using BRET. HEK293FT cells expressing genome-edited CXCR4 fused to Nluc (geCXCR4/Nluc) transiently transfected with cDNA coding for β-arrestin2/Venus (exβ-arr2/Venus) were used to (a) determine ligand-dependent CXCL12 (100 nM) recruitment of β-arrestin2 to CXCR4 in the absence or presence of the CXCR4 antagonist AMD3100 (10 µM). (b) Application of CXCL12 (1 pM–300 nM) resulted in a concentration-dependent recruitment of exβ-arr2/Venus to geCXCR4/Nluc. Modulation of CXCL12 (30 nM) mediated exβ-arr2/Venus recruitment to geCXCR4/Nluc by (c) additional co-expression of untagged bovine β-arr2 or (d) dominant-negative dynamin K44A. (e) Schematic representation of the BRET configuration. ‘BRET ratio (ligand-induced)’ was calculated as described in Methods. Points represent mean ± S.E.M. of three (c and d), four (a) or five (b) independent experiments. (b) Concentration-response curve fit was by non-linear regression and was used to calculate pEC₅₀, with points representing maximum response observed in a kinetic assay.

Figure 2

Investigating recruitment of genome-edited β-arrestin2 using BRET. (a) Schematic representation of the exogenously expressed GPCR fused to Venus (exGPCR/Venus) and β-arr2/Nluc BRET configuration. HEK293FT cells expressing genome-edited β-arrestin2 fused to Nluc (geβ-arr2/Nluc) transiently transfected with cDNA coding for (b,c) CXCR4 fused to Venus (exCXCR4/Venus; red circles) or (d,e) V2R fused to Venus (exV2R/Venus, blue circles) as well as HEK293FT cells transiently co-transfected to express exogenous β-arrestin2 fused to Nluc (exβ-arr2/Nluc, black squares) at near endogenous levels and (b,c) exCXCR4/Venus or (d,e) exV2R/Venus. (b,d) Kinetic profiles of β-arrestin2/Nluc recruitment initiated by addition of CXCL12 (30 nM) or AVP (100 nM) for CXCR4 and V2R respectively. Concentration-dependent recruitment of genome-edited or exogenous β-arrestin2/Nluc to (c) exCXCR4/Venus or (e) exV2R/Venus mediated by CXCL12 (10 pM–100 nM) or AVP (10 pM–100 nM) respectively. Inserts show geβ-arr2/Nluc recruitment to exV2R/Venus presented in (d,e). (f,g) Effect of overexpression of unlabelled exogenous β-arrestin2 (blue and red bars) on maximum recruitment response (f) and potency (g) in HEK293FT cells expressing geβ-arr2/Nluc transiently transfected with cDNA encoding exCXCR4/Venus (white and red bars) or exV2R/Venus (black and blue bars). (h) Schematic representation of the BRET configuration used in (f,g). ‘BRET ratio (ligand-induced)’ was calculated as described in Methods. Points and bars represent mean ± S.E.M. of three or four independent experiments. Statistical analysis by unpaired two-tailed t-test: ns, not significant, t-value = 1.144, df = 6; *p < 0.05, t-value = 3.610, df = 6; **p < 0.01, t-value = 6.105 df = 4; ***p < 0.001, t-value = 9.817 df = 4.

Figure 3

Determining luciferase and assay sensitivity by Z′ factor calculation. HEK293FT cells expressing genome-edited β-arrestin2 fused to Nluc (a,b, geβ-arr2/Nluc) or Rluc8 (c,d, geβ-arr2/Rluc8) were transiently transfected with cDNA coding for V2R fused to Venus (exV2R/Venus) and used to calculate Z′ factors. Cells seeded at 25,000 cells/well (25kc/w; a,c) or 50,000 cells/well (50kc/w; b,d) were treated with vehicle (black symbols) or AVP (1 µM; blue triangles, green squares). Points represent raw BRET ratio of individual wells ~25 minutes following vehicle or AVP addition. Dashed and dotted lines indicate 3 standard deviations (SD) from the mean of the AVP-treated and vehicle-treated data sets respectively. Z′ factors are calculated as described in Methods. Data shown are representative of three independent experiments.

Figure 4

Kinetic profiling of the internalisation and trafficking of genome-edited CXCR4. HEK293FT cells expressing genome-edited CXCR4 fused to Nluc (geCXCR4/Nluc) transiently transfected with cDNA encoding one of the subcellular markers (a,c) Rab 1, 4 and 5 (b,c) Rab 7, 9, and 11, or the plasma membrane marker K-ras (d) fused to Venus in the absence (a–d) or presence (c,d) of co-expressed dominant-negative dynamin K44A. Internalisation and trafficking was induced by CXCL12 (30 nM) added when indicated. (e) Schematic representation of the NanoBRET configuration used in the trafficking assay. (f) HEK293FT cells expressing genome-edited CXCR4 fused to Nluc (geCXCR4/Nluc) transiently transfected with cDNA coding for Venus/K-ras were used to determine the effect of receptor priming by CXCL12 (30 pM) on internalisation induced by CXCL12 (30 nM). (g) Schematic representation of the NanoBRET multiplex configuration. (h) HEK293FT cells expressing genome-edited CXCR4 fused to Nluc (geCXCR4/Nluc) transiently co-transfected with cDNA coding for HaloTag/K-ras and Rab4/Venus were used to study genome-edited CXCR4/Nluc receptor internalisation and trafficking induced by CXCL12 (30 nM) in the same cell using a BRET multiplex assay. ‘BRET ratio (ligand-induced)’ was calculated as described in Methods. Arrows indicate the delay in internalisation following ligand addition. Points represent mean ± S.E.M. of three (a,b), four (h) or five (d,f) independent experiments. Bars (c) represent maximum response ± S.E.M. of three independent experiments observed in kinetic time-courses, with empty vector controls obtained from (a,b). Statistical analysis by a two-way ANOVA with a Sidak’s post-test for multiple comparisons. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Demonstration of GPCR-HIT BRET assay using genome-edited CXCR4. HEK293FT cells expressing genome-edited CXCR4 fused to Nluc (geCXCR4/Nluc) transiently transfected with cDNA coding for β-arrestin2/Venus and (a) β₂-adrenoceptors or (b) CXCR7 were used to carry out BRET assays using the GPCR-HIT configuration. (a) Cells were stimulated with CXCL12 (30 nM, black squares), isoprenaline (100 µM, green upward triangles) or both ligands simultaneously (blue downward triangles) or (b) CXCL12 (30 nM, black squares), the CXCR7-specific ligand CXCL11 (30 nM, green upward triangles) or both ligands simultaneously (blue downward triangles). (b) For comparison, responses mediated by CXCL12 (30 nM, red diamonds) in HEK293FT cells expressing genome-edited CXCR4 fused to Nluc (geCXCR4/Nluc) transiently transfected with only cDNA coding for β-arrestin2/Venus is shown. (c) Schematic representation of the GPCR-HIT configuration using NanoBRET. ‘BRET ratio (ligand-induced)’ was calculated as described in Methods. Points represent mean BRET ratio ± S.E.M.; bars represent maximum corrected BRET ratio ± S.E.M. observed in kinetic time courses. Ligand added at approximately 8 minutes. Data generated from three independent experiments.