Engineering, applications, and future perspectives of GPCR‐based genetically encoded fluorescent indicators for neuromodulators

Abstract

This review explores the evolving landscape of G‐protein‐coupled receptor (GPCR)‐based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand‐binding properties, structural insights, and spectral diversity.

A Bright Future? A Perspective on Class C GPCR Based Genetically Encoded Biosensors

“…In recent years, several biosensors based on GPCR class A and class B have been developed. − Examples include dopamine, ,,− fluid sheer stress, norepinephrine and epinephrine, , somatostatin, cholecystokinin, corticotropin releasing factor, neuropeptides, , neurotensin, nociceptin/orphanin-FQ, vasoactive intestinal peptide ghrelin, GLP-1, urocortin, parathyroid-hormone-related-peptide, orexin and hypocretin orexin-A and orexin-B, oxytoci, , substance P 27 serotonin, − and histamine . An overview of existing biosensors can be found for example in Kubitschke et al (2024) and Rohner et al (2024). , …”

“…The design of a single wavelength genetically encoded biosensor is performed in several steps (for detailed review, see refs and ). We briefly illustrate the principle of design and optimization.…”