Tuning the Response of GPCR-Based Yeast Sensors Using Fluorescent Reporters.

Area of Science:

• Molecular Biology
• Biochemistry
• Pharmacology

Background:

• G protein-coupled receptors (GPCRs) are crucial cell surface proteins regulating diverse biological processes and are major drug targets.
• Existing high-throughput (HT) yeast-based GPCR sensors often suffer from low signal increases, limiting their utility.
• Development of robust GPCR assays is essential for identifying novel ligands and therapeutic agents.

Purpose of the Study:

• To identify an optimal fluorescent reporter to enhance signal dynamic range in yeast-based human GPCR (hGPCR) sensors.
• To streamline the generation of HT assays for biomedically important hGPCRs.
• To enable new applications for GPCR research, including point-of-care diagnostics.

Main Methods:

• Analysis of five fluorescent reporters for hGPCR-based sensors in yeast.
• Utilized a serotonin receptor 4 (HTR4)-based sensor as a testbed for reporter evaluation.
• Employed flow cytometry, fluorescence-activated cell sorting, and fluorescent plate readers for assay readout.

Main Results:

• YPet was identified as the optimal fluorescent reporter, significantly increasing the dynamic range of hGPCR sensors.
• Engineered sensors for HTR4, MC4R, S1PR2, HTR1A, Mel1A, and HTR1D demonstrated vastly improved signal increases.
• YPet's fast maturation reduced assay readout time from 4 hours to 30 minutes.

Conclusions:

• YPet is a superior fluorescent reporter for yeast-based hGPCR high-throughput assays.
• This optimization facilitates standardized generation of HT assays and enables ultrahigh-throughput single-cell experiments.
• The findings pave the way for GPCR deorphanization, designer ligand development, and point-of-care diagnostic tools.