NanoBiT Complementation to Monitor Agonist-Induced Adenosine A1 Receptor Internalization

Receptor internalization in response to prolonged agonist treatment is an important regulator of G protein-coupled receptor (GPCR) function. The adenosine A₁ receptor (A₁AR) is one of the Adenosine Receptor family of GPCRs, and evidence for its agonist-induced internalization is equivocal. The recently developed NanoBiT technology uses split NanoLuc Luciferase to monitor changes in protein interactions. We have modified the human A₁AR on the N-terminus with the small high-affinity HiBiT tag. In the presence of the large NanoLuc subunit (LgBiT), complementation occurs, reconstituting a full-length functional NanoLuc Luciferase. Here, we have used complemented luminescence to monitor the internalization of the A₁AR in living HEK293 cells. Agonist treatment resulted in a robust decrease in cell-surface luminescence, indicating an increase in A₁AR internalization. These responses were inhibited by the A₁AR-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), with an antagonist affinity that closely matched that measured using ligand binding with a fluorescent A₁ receptor antagonist (CA200645). The agonist potencies for inducing A₁AR internalization were very similar to the affinities previously determined by ligand binding, suggesting little or no amplification of the internalization response. By complementing the HiBiT tag to exogenous purified LgBiT, it was also possible to perform NanoBRET ligand-binding experiments using HiBiT-A₁AR. This study demonstrates the use of NanoBiT technology to monitor internalization of the A₁AR and offers the potential to combine these experiments with NanoBRET ligand-binding assays.