A modular click ligand-directed approach to label endogenous aminergic GPCRs in live cells

Abstract

New technologies based on luminescence have been essential to monitor the organization, signaling, trafficking or ligand binding of G Protein-Coupled Receptors (GPCRs), but they rely on the overexpression of genetically modified receptors. As more and more studies indicate the importance of studying native receptors in their natural environment, it is essential to develop approaches allowing the specific labeling of native receptors. Here we report an innovative ligand directed approach to specifically label residues of native GPCRs upon ligand binding. To this end, we developed a ligand-directed toolbox based on a novel approach that uses molecular modules to build fluorescent ligand-directed probes that can label an archetypical aminergic GPCR (D1R). Our probes can be readily prepared before the labeling reaction from two molecular modules: an activated electrophilic linker which includes a fluorescent dye and a GPCR ligand that may include nucleophilic groups. Thanks to a fast and specific click reaction, the nucleophilic ligand can barely react with the activated linker before it is bound to the native target GPCR and the labeling reaction occurs. Subsequently, the ligand unbinds the GPCR pocket, leaving the receptor fluorescently labeled and fully functional. This novel labeling approach allowed us to label both D1 receptor in transfected cells and native receptors in neuronal cell lines. This approach will pave the way to develop new reagents and assays to monitor endogenous GPCRs distribution, trafficking, activity or binding properties in their native environment.

Ligand-Directed Labeling of the Adenosine A1 Receptor in Living Cells

“…18 The ligand-directed compound could selectively label endogenous A 2A AR, thereby enabling their detection in human monocyte-derived macrophages by FACS and visualization on a human breast cancer cell line using confocal microscopy. The utility of ligand-directed technology for labeling membrane receptors in living cells has also been described for a small number of other GPCRs: the bradykinin B2 receptor 19 with biotin and the μ opioid 20 and dopamine D1 21 and cannabinoid CB 2 22 receptors with a fluorescent group. In the present study, we describe the design and application of a ligand-directed chemistry technology that allows selective labeling of endogenous A 1 ARs with functional probes and monitors its localization and dynamics in living cells.…”