Introduction
G protein-coupled receptors (GPCRs) are membrane-spanning proteins that transmit extracellular stimuli to the inside of a cell. Activation by stimulating molecules (e.g. neurotransmitter, hormone or chemokine) starts a signaling cascade that results in a cellular response. An example is the chemokine CXCL12 that activates the receptor CXCR4, resulting in G protein activation and recruitment of β-arrestin2. Subsequently, the receptor is internalized for either recycling to the plasma membrane or lysosomal breakdown.
GPCRs are important drug targets requiring receptor-protein interaction and trafficking studies to reveal how they function. Bioluminescence resonance energy transfer (BRET) is a versatile tool to study such interactions and trafficking. However it is limited by the ectopic expression of labelled interaction partners. CRISPR/Cas9 genome editing overcomes the limitation by enabling endogenous expression of luciferase-labelled proteins.
Assay Principle
BRET uses a luciferase that in the presence of a substrate produces blue light. If a suitable fluorophore is in very close proximity and appropriate orientation, less light is emitted and resonance energy is transferred. Thus, interaction of fluorophore- and luciferase-labelled proteins can be monitored by relating fluorophore emission to luciferase signal.
The CRISPR/Cas9 genome editing method incises dsDNA at a precise target site. In the presence of a donor DNA template, homology-directed repair results in the insertion of the donor sequence.
CRISPR/Cas9 enabled insertion of the DNA coding for nanoluciferase (Nluc) into the endogenous genomic locus of CXCR4 of HEK293FT cells. The resulting CXCR4/Nluc fusion protein acts as a BRET donor and overcomes the need for exogenous donor expression.
Materials & Methods
- White 96-well plate (Greiner)
- CLARIOstar® and LUMIstar® (BMG LABTECH)
- CXCL12 (Preprotech), AMD3100 (Sigma-Aldrich), Isoproterenol (Sigma-Aldrich)
- Furimazine (Promega)
Experimental procedure
For detailed descriptions please refer to White et al. (2017). Briefly, CRISPR/Cas9 genome-edited HEK293FT cells expressing CXCR4/Nluc were transfected with cDNA coding for the protein with acceptor-fluorophore using Fugene®6 (Promega). 48 h after transfection cells were incubated with the luciferase substrate furimazine and filtered light emissions were analyzed on a CLARIOstar or LUMIstar Omega.
Settings stable through experiments | ||
Luminescence, top optic, plate mode | ||
Measurement interval time 1 s | ||
Incubation at 37 °C | ||
Optic settings (Filters or monochromator and [gains]) | ||
Fig. 1 | Fig. 2 | Fig. 3 |
CLARIOstar Filters Nluc 450-60 (3400) Venus 570-100 (2800) |
CLARIOstar Monochromator Nluc 450-60 (3600) Venus 550-60 (3600) HaloTag 660-100 (3200) |
LUMIstar Filters Nluc 475-30 (3200) Venus 535-30 (3600) |
Number of cycles / Cycle time |
||
40 / 33 s | 30 / 49 s | 60 / 31 s |
Results & Discussion
The suitability of genome-edited cells expressing CXCR4/Nluc to report on receptor interaction was studied by combining it with exogenously expressed β-arrestin- 2/Venus (Fig. 1A). Upon activation of CXCR4 with its ligand CXCL12, β-arrestin2 was recruited as reported by an increase in the BRET ratio (Fig. 1B, purple). The CXCR4 antagonist AMD3100 inhibited CXCL12-induced recruitment of β-arrestin2 as seen by the reduction in energy transfer between CXCR4/Nluc and β-arrestin2/Venus (Fig. 1B, orange).
The GPCR Heteromer Investigation Technology (GPCR-HIT, Dimerix) reports on GPCR heteromer formation by the recruitment of an interacting protein specifically to the heteromer complex. Cells expressing CXCR4/Nluc were transiently transfected with cDNA coding for β2-adrenoceptor as well as the interacting protein β-arrestin2/Venus. Treating the cells with CXCL12 resulted in the expected recruitment of β-arrestin2/Venus to genome-edited CXCR4/Nluc, as did treatment with the β2-adrenoceptor agonist isoprenaline, indicating the close proximity of β2-adrenoceptor to CXCR4/Nluc. Applying both agonists resulted in a greater than additive BRET signal, again suggestive of heteromer formation.
Conclusion
The novel CRISPR/Cas9 technique successfully fused the Nluc BRET donor to endogenously-expressed CXCR4. Luminescence generated by the resulting protein-luciferase fusion was sufficient to monitor receptor-protein interactions as well as trafficking. The multiplex internalization assay depends on two acceptor fluorophores whose selective detection was rendered possible by the CLARIOstar’s monochromator.
References
- White CW et al. (2017) Sci Rep. 7:3187. doi: 10.1038/s41598-017-03486-2