Functional immobilization of a ligand-activated G-protein-coupled receptor

Abstract

G-protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. They are the largest family of receptors in the human genome and constitute the largest class of targets for drug discovery. To facilitate studies of GPCR activation and interactions with other proteins, we developed a simple method to immobilize a functional, detergent-solubilized GPCR on gold and glass surfaces. The β2 adrenergic receptor (β2AR), a prototypical GPCR, was purified and labeled with a reporter fluorophore at a conformationally sensitive site. The detergent-soluble fluorescent β2AR was immobilized through its amino-terminal FLAG epitope on a surface layered with biotinylated bovine serum albumin, avidin, and biotinylated M1 antibody. Agonist activation of the β2AR was monitored in real time by fluorescence microscopy. This approach will make it possible to study conformational dynamics of single immobilized receptors and to generate arrays of functional GPCRs for novel high-throughput screening strategies.