Optical tools for visualizing and controlling human GLP-1 receptor activation with high spatiotemporal resolution

Abstract

The glucagon-like peptide-1 receptor (GLP1R) is a broadly expressed target of peptide hormones with essential roles in energy and glucose homeostasis, as well as of the blockbuster weight-loss drugs semaglutide and liraglutide. Despite its large clinical relevance, tools to investigate the precise activation dynamics of this receptor with high spatiotemporal resolution are limited. Here we introduce a novel genetically-encoded sensor based on the engineering of a circularly-permuted green fluorescent protein into the human GLP1R, named GLPLight1. We demonstrate that fluorescence signal from GLPLight1 accurately reports the expected receptor conformational activation in response to pharmacological ligands with high sensitivity (max ΔF/F0 = 528%) and temporal resolution (τON = 4.7 sec). We further demonstrated that GLPLight1 shows comparable responses to GLP-1 derivatives as observed for the native receptor. Using GLPLight1, we established an all-optical assay to characterize a novel photocaged GLP-1 derivative (photo-GLP1) and to demonstrate optical control of GLP1R activation. Thus, the new all-optical toolkit introduced here enhances our ability to study GLP1R activation with high spatiotemporal resolution.

Probing PAC1 receptor activation across species with an engineered sensor

“…Our previous work on the development of another class-B1 sensor based on the GLP1 receptor, led us to discover that the kinetics of the sensor’s response can be used to infer the occupancy of the ECD by an antagonist peptide 24 . Given that all class-B1 GPCRs share a similar ECD high- affinity ligand binding mechanism, we performed similar experiments to determine whether PAClight1 P78A could also be used in a similar manner.…”

“…Onephoton fluorescence excitation (λem = 560nm) and emission (λexc = 470nm) spectra were determined on a Tecan M200 Pro plate reader at 37°C. 24 200V for the forward scatter detector, to 400V for the side scatter detector, and to 350V for the FITC channel fluorescence detector. Excitation was performed at 488nm, while emission was directed through a 502LP mirror and a 530/30 band pass filter.…”

“…Recently, genetically-encoded GPCR-based sensors have been developed that enable the direct optical detection of GPCR activation by agonist ligands with high sensitivity and spatiotemporal resolution [10][11][12][13][14][15][16][17][18] . While these tools hold great potential for drug development, pharmacology and neuroscience applications, only a few of them are built from class-B1 peptide-sensing GPCRs 17,19 . Thus, the development of new biosensors based on peptide-sensing GPCRs would greatly benefit the community and serve as a powerful resource for drug screening and life sciences.…”

Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release

“…> 420 nm). 46,69,70,71,72 In these cases, use of 405 nm as the isosbestic channel for these sensors may lead to confounding results or difficulty in interpretation in some contexts, such as regions where N/OFQ release in response to a given behavior is relatively low, or during behaviors that are highly subject to motion artefact. Since it can be cost and time prohibitive for research groups to add an alternative recording parameter to existing photometry setups, we demonstrated that we were still able to detect meaningful changes in NOPLight fluorescence when using the 405 nm wavelength in some of our fibre photometry recordings.…”

Nonperturbative Fluorogenic Labeling of Immunophilins Enables the Wash-free Detection of Immunosuppressants

“…With most optical technologies for therapeutic drug monitoring relying on antibody-based assays, , there are few examples of fluorescent probes to detect immunosuppressive drugs in biosamples. − In this regard, the site-specific labeling of peptides and proteins with fluorogenic dyes can be an effective strategy to generate turn-on probes for real-time analyte detection. − Thus, we envisioned that the synthesis of fluorogenic reporters based on whole immunophilins may represent a modular and versatile approach to detect immunosuppressive drugs in real time. Our group and others have reported fluorogenic probes to measure immune cell activity − by flanking enzyme-responsive substrates with fluorophore-quencher pairs − and by embedding environmentally sensitive BODIPY amino acids within targeted peptide sequences .…”