BRET-Based Ligand Binding at Recombinant and Endogenous Dopamine D2 Receptors

Dopamine D2 receptor (D2R) regulates diverse biological processes and is an important therapeutic target, underscoring the need for robust methods to quantify ligand binding at this receptor. Bioluminescence resonance energy transfer (BRET)-based ligand binding assays have emerged as a powerful platform for quantifying binding in living cells, but their application to D2R has been limited by the availability of suitable fluorescent tracers. Here, we developed three far-red fluorescent antagonists (CELT-240, CELT-383, and CELT-385) as BRET tracers for D2R. All three compounds produced specific, saturable D2R binding with nanomolar affinity. Further characterization of CELT-385 demonstrated its utility for kinetic and competition binding measurements and revealed high affinity binding at the closely related dopamine D3 receptor (D3R). We also extended BRET-based ligand binding beyond genetically-modified receptors using bystander BRET, enabling measurements at unmodified recombinant and endogenously expressed D2R. Together, these findings provide new tools for quantitative D2R/D3R ligand binding and a template for extending BRET-based ligand binding to endogenously expressed GPCRs.