Novel Chemical Biology Methods Illuminate the Function of Single G Protein-Coupled Receptors

Abstract

The superfamily of G Protein-Coupled Receptors (GPCRs) is one of the largest in the human genome. GPCRs are targets for about a quarter of all prescription drugs. Recent progress in the structural biology of GPCRs renews the interest in rational drug design and discovery. Rational design of “functionally selective” drugs or ligands presents a particularly difficult problem. Agonist ligands frequently display functional selectivity where activated receptors are biased to either G protein- or arrestin-mediated downstream signaling pathways. However, functional characterization of GPCRs has largely been limited to cell-based high-throughput screening assays. The complexity of the cellular milieu introduces biological variables that make quantitative analysis of structure and dynamics of ligand-receptor complexes that underlie functional selectivity virtually impossible. We are developing single-molecule fluorescence methods to eliminate this biological noise and study such complexes in biochemically-defined systems.[1] Systematic introduction of signaling partners, such as G proteins and arrestins, enables us to study their allosteric effects on ligand-receptor interactions. Recent methodological advances enabled us to express GPCRs with genetically-encoded unnatural amino acids (uaas). We showed that bioorthogonal functional groups on these site-specifically introduced uaas might be used for bioorthogonal reactions. Among a broad spectrum of bioorthogonal reactions, strain-promoted azide-alkyne cycloaddition (SpAAC) with the uaa p-azido-phenylalanine and dibenzocyclooctyne (DIBO) reagents is specific and efficient and presents the best option to obtain stoichiometric receptor-fluorophore conjugates. We designed and built a fully-automatic, multi-color, single-molecule detection fluorescence microscope for long-timescale, multiplexed data acquisition using a multi-well tethered membrane “chip” system. Our convergent technology platform presents a new approach for single-molecule fluorescence studies of the kinetics and dynamics of GPCR signalosomes.[1] T. Huber and T.P. Sakmar. Chemical Biology Methods for Investigating G Protein-Coupled Receptor Signaling. Chem Biol. 18:1224-1237 (2014)