Development of a V5-tag-directed nanobody and its implementation as an intracellular biosensor of GPCR signaling

Abstract

Protein-protein interactions (PPIs) form the foundation of any cell signaling network. Considering that PPIs are highly dynamic processes, cellular assays are often essential for their study because they closely mimic the biological complexities of cellular environments. However, incongruity may be observed across different PPI assays when investigating a protein partner of interest; these discrepancies can be partially attributed to the fusion of different large functional moieties, such as fluorescent proteins or enzymes, which can yield disparate perturbations to the protein's stability, subcellular localization, and interaction partners depending on the given cellular assay. Owing to their smaller size, epitope tags may exhibit a diminished susceptibility to instigate such perturbations. However, while they have been widely used for detecting or manipulating proteins in vitro, epitope tags lack the in vivo traceability and functionality needed for intracellular biosensors. Herein, we develop NbV5, an intracellular nanobody binding the V5-tag, which is suitable for use in cellular assays commonly used to study PPIs such as BRET, NanoBiT, and Tango. The NbV5:V5 tag system has been applied to interrogate G protein-coupled receptor signaling, specifically by replacing larger functional moieties attached to the protein interactors, such as fluorescent or luminescent proteins (∼30 kDa), by the significantly smaller V5-tag peptide (1.4 kDa), and for microscopy imaging which is successfully detected by NbV5-based biosensors. Therefore, the NbV5:V5 tag system presents itself as a versatile tool for live-cell imaging and a befitting adaptation to existing cellular assays dedicated to probing PPIs.

Keywords: G protein-coupled receptor (GPCR); bioluminescence resonance energy transfer (BRET); crystal structure; nanobody); nanoluciferase binary technology (NanoBiT); protease-dependent reporter assay (Tango); protein-protein interaction; single-domain antibody (sdAb.

Figure 1

Overview of the selection of a synthetic nanobody interacting with the V5-tag.A, sequence of the V5-tag. B, schematic of the phage-display panning for the enrichment of nanobodies interacting with the V5-peptide tag. C, schematic of the yeast two-hybrid screening (Y2H) for the enrichment of nanobodies interacting with the V5-peptide tag in an intracellular environment (intrabody).

Figure 2

The first generation of the anti-V5 nanobody (NbA1) only recognizes the C-terminal–positioned V5-tag.A, schematic of the protease-dependent cell-based assay (TANGO) used to assay the anti-V5 nanobodies. The original NbA1 clone selected from the Y2H screening was fused with the TEV219 protease and cloned into a eukaryotic expression vector. The initial assessment revealed that the nanobody was not well expressed in HEK293 and codon optimization (NbA1(C.O)) increased its expression and consequently its functional activity. The μ-OR-TANGO (B) and AT₁R-TANGO (C) were cotransfected with β-arrestin2 carrying a C- or N-terminal V5-peptide tag along with either the NbA1-TEV219 or NbA1(C.O)-TEV219 fusion protein the receptor stimulated with dose-response agonists, DAMGO for μ-OR and angiotensin II (AngII) for AT₁R. The original NbA1 clone only recognizes the C-terminally tagged β-arrestin2 (β-arrestin2-V5). Dose-response curves were built using XY analysis for nonlinear regression curve and the 3-parameters dose-response stimulation function from GraphPad Prism. Baseline corrected curves were built using the “Remove baseline and column math” function (Value-Baseline/Baseline). Wells in the absence of ligand were used as the baseline for each condition. All error bars represent SD of three or four technical replicates. Data presented are representative of three biological replicates. μ-OR, mu-opioid receptor; AT1R, angiotensin II receptor type 1; HEK293, human embryonic kidney 293; TEV, tobacco etch virus; Y2H, yeast two-hybrid.

Figure 3

Structure of the NbA1 bound to the V5 peptide.A, overview of the NbA1:V5 peptide complex shown as cartoon representation. NbA1 is colored in blue with CDRs 1 to 3 colored in red, orange, and yellow, respectively. Three residues from the CDR2 were not resolved in the refined structure. B, close-up view of the polar interactions within the complex including water-bridged interaction. The V5-peptide (green) is shown as stick representation with the N terminal on the left. Interacting residues from the paratope are labeled in blue, while the peptide labels are green and marked with a prime symbol. H-bonds are shown as yellow dashed lines and water molecules as red spheres. C, the NbA1:V5 peptide complex shown as surface representation and colored using the color_h script based on the Eisenberg hydrophobicity scale ⁶⁴. D, sequence of NbA1 with CDRs 1 to 3 colored in red, orange, and yellow, respectively. The square highlights the RQG tripeptide that is disordered in the CDR2 loop. E, the μ-OR-TANGO and AT₁R-TANGO were cotransfected with β-arrestin2 carrying a C- or N-terminal V5-peptide tag along with either the NbV5-TEV219 or codon-optimized NbA1(C.O)-TEV219 fusion protein the receptor stimulated with dose-response agonists, DAMGO for μ-OR and angiotensin II (AngII) for AT₁R. The NbV5 recognizes the N- or C-terminally V5-tagged β-arrestin2 with similar logistic parameters (potency and efficacy), while the original NbA1 clone only recognizes the C-terminally tagged β-arrestin2 (β-arrestin2-V5). Dose-response curves were built using XY analysis for nonlinear regression curve and the 3-parameters dose-response stimulation function from GraphPad Prism. Baseline correction was performed using the “Remove baseline and column math” function (Value-Baseline/Baseline). Wells in the absence of ligand were used as the baseline for each condition. All error bars represent SD of three or four technical replicates. Data presented are representative of three biological replicates. μ-OR, mu-opioid receptor; AT1R, angiotensin II receptor type 1; CDR, complementarity-determining region; TEV, tobacco etch virus.

Figure 4

NbV5 as a versatile nanobody-based biosensor: application in protease-dependent cell-based assay (TANGO). The μ-OR-TANGO (A) and AT₁R-TANGO (B) were cotransfected with β-arrestin1 or β-arrestin2 carrying a C- or N-terminal V5-peptide tag along with the NbV5-TEV219 fusion protein. Dose-response agonist treatments demonstrate the recruitment of β-arrestin in all configurations tested. C, the μ-OR-TANGO and AT₁R-TANGO were cotransfected with β-arrestin2-TEV219 and stimulated with agonists showing that EC50 obtained using the original TANGO is similar to the NbV5-adapted TANGO. Dose-response curves were built using XY analysis for nonlinear regression curve and the 3-parameters dose-response stimulation function from GraphPad Prism. Baseline corrected curves were constructed using the “Remove baseline and column math” function (Value-Baseline/Baseline). Wells in the absence of ligand were used as the baseline for each condition. All error bars represent SD of three or four technical replicates. Data presented are representative of three biological replicates. μ-OR, mu-opioid receptor; AT1R, angiotensin II receptor type 1; TEV, tobacco etch virus.

Figure 5

NbV5 as a versatile nanobody-based biosensor: application in Bioluminescence Resonance Energy Transfer (BRET²).A, schematic of the BRET² cell-based assay used to evaluate the NbV5. The recruitment of the β-arrestin–NbV5-GFP2 complex to the receptor fused to RLuc8 allows the energy transfer between the Renilla reniformis Luciferase mutant (RLuc8) and the GFP mutant GFP2 in the presence of the RLuc8 substrate Coelenterazine 400a. B and C, NbV5-based detection of β-arrestin1 and β-arrestin2 recruitment at the AT₁R-RLuc8. N- and C-terminally V5-tagged β-arrestins were tested as well as both N- and C-terminally GFP2-tagged NbV5. Dose-response curve treatment with angiotensin II reveals equivalent recruitment of β-arrestin1 and 2 at the AT₁R. D, similar results were obtained with the nanobody that recognizes the synthetic ALFA-tag (NbALFA). Dose-response curves were built using XY analysis for nonlinear regression curve and the 3-parameters dose-response stimulation function. All error bars represent SD of three biological replicates with two technical replicates (n = 6). AT1R, angiotensin II receptor type 1.

Figure 6

NbV5 as a versatile nanobody-based biosensor: application in Nanoluciferase Binary Technology.A, schematic of the NanoBiT cell-based assay used to evaluate the NbV5. NbV5-based detection of β-arrestin1 and β-arrestin2 recruitment at the AT₁R-SmBiT (B–D) and μ-OR-SmBiT (E–G) was assayed. N- and C-terminally V5-tagged β-arrestin was tested as well as both N- and C-terminally LgBiT-tagged NbV5. D and G, live kinetic trace at 1 μM agonist are shown in (D) for the AT1R and (G) for the μ-OR. The trace represents the mean of a quadruplicate experiment. H, schematic of the NanoBiT cell-based assay used to assess the internal V5-tag. I, functional recognition of an internal localized V5-tag was also assayed by NanoBiT by incorporating the V5-tag at position 92 of the G⍺oA (G⍺oA-iV5) and measuring the dissociation from the Gβ3 and SmBiT-Gɣ2 dimer upon μ-OR activation with DAMGO. B, C, E, F, and I, dose-response curves were built using XY analysis for nonlinear regression curve and the 3-parameters dose-response stimulation function from GraphPad Prism. Baseline correction was performed using the “Remove baseline and column math” function, calculated as Value-Baseline/Baseline (B–G) or Value-Baseline (I). Wells in the absence of ligand were used as the baseline for each condition. All error bars represent SD of three or four technical replicates. Data presented are representative of three biological replicates. μ-OR, mu-opioid receptor; AT1R, angiotensin II receptor type 1; LgBiT, Large BiT; NanoBiT, Nanoluciferase Binary Technology; SmBiT, Small BiT.

Figure 7

NbV5-based detection of V5-tagged proteins by cell imaging. Fluorescence imaging of HT1080 cells expressing NbV5-eGFP and ɣ-Actin-V5 or pcDNA3.1+ as a negative control. In the absence of ɣ-Actin-V5, NbV5-eGFP (green) is diffused throughout the cell, while in the presence of ɣ-Actin-V5, NbV5-eGFP is enriched in actin-rich protrusions and structures. Cells were costained with the F-actin probe Alexa Fluor 568 phalloidin (red) and the blue DNA stain Hoechst. The overlay is shown on the top panel. Images are representative of 25 cells from three independent experiments. Scale bars represent 25 μm.