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

TL;DR

This paper shows how using a specific fluorescent protein improves yeast-based sensors for studying human GPCRs, making them more effective and faster for drug discovery.

Contribution

The study identifies YPet as the optimal fluorescent reporter for enhancing the performance of GPCR-based yeast sensors.

Findings

YPet increases the dynamic range and signal strength of multiple hGPCR-based sensors.

YPet enables the construction of a functional HTR1D-based sensor previously difficult to develop.

The use of YPet reduces the time to readout from 4 hours to 30 minutes.

Abstract

G protein-coupled receptors (GPCRs) recognize ligands
on the cell
surface, initiating intracellular signaling pathways that control
a variety of biological processes, from neurotransmission and hormone
regulation to light detection and smell. As entryways into these pathways,
GPCRs are key pharmacological targets, with 30% of FDA-approved drugs
targeting them. High-throughput GPCR-based sensors in yeast are proven
platforms for the identification of novel GPCR ligands. Most human
GPCRs (hGPCRs), however, led to small increases in the signal after
activation, hindering the development of high-throughput (HT) assays.
To streamline the generation of HT assays for biomedically important
hGPCRs, here we analyze five fluorescent reporters in the context
of hGPCR-based sensors. Using the serotonin receptor 4 (HTR4)-based
sensor as a testbed, we identify YPet, a yellow fluorescent protein
previously evolved for improved intracellular fluorescence, as the
optimal fluorescent reporter when using flow cytometry, fluorescence-activated
cell sorting, or a fluorescent plate reader. YPet increases the dynamic
range of hGPCR-based sensors in general, enabling the engineering
of HTR4-, MC4R- S1PR2-, HTR1A-, and Mel1A-based sensors with vastly
higher increases in signal than previously engineered sensors. YPet
even allowed the construction of a functional HTR1D-based sensor,
a sensor that had been difficult for the field to construct. Finally,
the fast maturation of YPet reduces the time to readout from 4 h to
30 min, unlocking point-of-care diagnostic applications previously
inaccessible to hGPCR-based sensors in yeast. Looking ahead, the
identification of YPet as the optimal fluorescent reporter for yeast
hGPCR-based sensors opens the door to the standardized generation
of hGPCR high-throughput assays in this host, and sets the stage for
ultrahigh-throughput single-cell experiments toward the identification
of new ligands for known GPCRs, GPCR deorphanization, and GPCR engineering
to bind designer ligands.

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