Imaging GPCR internalization using near-infrared Nebraska red-based reagents.

摘要: Internalization of G protein-coupled receptor (GPCRs) represents a nearly universal pathway for downregulation. Imaging this process provides means the identification pharmaceutical agents as well potential ligands orphan receptors. However, there is need further development near-infrared (NIR) probes capable monitoring internalization in order to enable multiplexing with existing green fluorescent GPCR activity assays. Our laboratory has recently described series fluorophores which phosphinate functionality inserted at bridging position xanthene scaffold. These fluorophores, termed Nebraska Red (NR) dyes, provide attractive reagents imaging protein localization. Herein, we disclose NR-based HaloTag membrane proteins on living cells. new are utilized image pools human orexin type 2 receptor, an established target treatment insomnia. We demonstrate ability fetal bovine serum (FBS) noncovalently associate spirolactonized NR probe, enabling no-wash 45-fold enhancement fluorescence. Furthermore, characterize utility real-time upon agonist stimulation. assays identify modulators