A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

Abstract

G protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, G protein-independent signaling via the arrestin pathway has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either the G protein or arrestin-signaling pathways. Currently available screening techniques that measure arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure arrestin recruitment to any unmodified receptor.NanoLuc, an engineered luciferase from ophlorus gracilirostris (deep sea shrimp), is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. Here, we have identified a novel split site and have fused the N-terminal fragment to a membrane tether and the C-terminal fragment to the N-terminus of either β-arrestin 1 or 2. Upon receptor activation, arrestin is recruited to the plasma membrane in an agonist concentrationdependent manner and the two NanoLuc fragments complement to reconstitute functional luciferase, which allows quantification of recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification. The split NanoLuc arrestin recruitment assay has promise as a new generic assay for measuring arrestin recruitment to diverse GPCR types in heterologous or native cells.

Biochemical insights into structure and function of arrestins

“…The field of cell-based assays continues to evolve, bringing forth new ideas that combine the discussed strategies to improve signal quality and reduce artifacts. For instance, Pedersen et al have recently devised a membrane-tethered version of the NanoBiT assay, where a N-terminal fragment of the NLuc is anchored to a membrane tether and the C-terminal fragment to arr [109].…”

“…For instance, Pedersen et al . have recently devised a membrane‐tethered version of the NanoBiT assay, where a N‐terminal fragment of the NLuc is anchored to a membrane tether and the C‐terminal fragment to arr [109].…”