Nanobodies as sensors of GPCR activation and signaling

Abstract

Nanobodies have emerged as useful tools to study G protein-coupled receptor (GPCR) structure, dynamic, and subcellular localization. Initially, several nanobodies have been developed as chaperones to facilitate GPCR crystallization. To explore their potential as biosensors to monitor receptor activation and dynamics, we here described protocols to characterize nanobody's interaction with GPCRs and their application as probes for protein identification and visualization on the cellular level. We also introduced a chimeric approach to enable a kappa-opioid receptor derived nanobody to bind to other GPCRs, including orphan GPCRs whose endogenous ligand or intracellular transducers are unknown. This approach provides a reporter assay to identify tool molecules to study the function of orphan GPCRs.

Keywords: BRET; Biosensor; Chimera; GPCR activation; Nanobody.