Investigation of Receptor Heteromers Using NanoBRET Ligand Binding

Abstract

Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the β2 adrenergic receptor (AT1-β2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).

Luminescence Energy Transfer–Based Screening and Target Engagement Approaches for Chemical Biology and Drug Discovery

“…NanoBRET has been used to monitor interactions between various protein partners in different cellular compartments with examples including membrane proteins (e.g., GPCRs 20,21 and EGFR/GRB2 22 ), signaling protein kinases (e.g., KRAS/BRAF 23 ), or transcription regulators (cMyc/MAX 24 ). Typically, luciferases have been introduced through the exogenous expression of a fusion protein.…”

Profiling novel pharmacology of receptor complexes using Receptor-HIT

“…To do this, we adapted our NanoBRET ligand binding assay [52], which enables monitoring and quantification of binding of fluorescently labelled ligands to Nanoluciferase-(Nluc)-labelled receptors, in live cells and in real time. We recently published this Receptor-HIT ligand binding assay using two established heteromers [53], the heteromer between the AT 1 receptor and the β 2 adrenergic receptor (β 2 AR) [20,54,55], and between the AT 1 receptor and angiotensin II type 2 (AT 2 ) receptor [18,[56][57][58]. For the AT 1 -β 2 AR heteromer [53], we first monitored binding of the BODIPY-630/650 tagged antagonist propranolol (BODIPY-propranolol) to Nluc/β 2 AR (Figure 2a) and observed saturable binding that was displaced by unlabelled propranolol (Figure 2b), enabling generation of a specific binding curve (Figure 2c).…”

“…We recently published this Receptor-HIT ligand binding assay using two established heteromers [53], the heteromer between the AT 1 receptor and the β 2 adrenergic receptor (β 2 AR) [20,54,55], and between the AT 1 receptor and angiotensin II type 2 (AT 2 ) receptor [18,[56][57][58]. For the AT 1 -β 2 AR heteromer [53], we first monitored binding of the BODIPY-630/650 tagged antagonist propranolol (BODIPY-propranolol) to Nluc/β 2 AR (Figure 2a) and observed saturable binding that was displaced by unlabelled propranolol (Figure 2b), enabling generation of a specific binding curve (Figure 2c). We tested binding of BODIPY-propranolol to Nluc/AT 1 (Figure 2d), but did not observe any specific binding that could be displaced by the AT 1 antagonist olmesartan (Figure 2e,f ).…”

“…Data are presented as the raw BRET ratio ( b , e , h ) or specific binding ( c , f , i ), mean ± SEM of three ( e , f ), four ( b , c ) or eight ( h , i ) independent experiments. Figure reproduced from [ 53 ].…”

“…If this results in modulation of the proximity signal between the tag on the receptor and the tag on the interacting biomolecule, this indicates heteromerisation of the two receptors. Figure reproduced from [ 53 ].…”