Nile Red-Based GPCR Ligands as Ultrasensitive Probes of the Local Lipid Microenvironment of the Receptor
Preprints are manuscripts made publicly available before they have been submitted for formal peer review and publication. They might contain new research findings or data. Preprints can be a draft or final version of an author's research but must not have been accepted for publication at the time of submission.
The local lipid microenvironment of transmembrane receptors is an essential factor in GPCR signaling. However, tools are currently missing for studying endogenously expressed GPCRs in primary cells and tissues. Here we introduce fluorescent environment-sensitive GPCR ligands for probing the microenvironment of the receptor in living cells using fluorescence microscopy under no-wash conditions. We designed and synthesized antagonist ligands of the oxytocin receptor (OTR) by conjugating a high-affinity non-peptidic OTR ligand PF-3274167 to the environment-sensitive fluorescent dye Nile Red. The length of the polar PEG spacer between the pharmacophore and the fluorophore was adjusted to lower the non-specific interactions of the probe while preserving a strong fluorogenic response. We demonstrated that the new probes embed into the lipid bilayer in the vicinity of the receptor and convey the information about the local polarity and the lipid order via the wavelength-shifting emission of the Nile Red fluorophore.