Optical tools for visualizing and controlling human GLP-1 receptor activation with high spatiotemporal resolution
- Institute of Pharmacology and Toxicology, University of Zürich, Switzerland
- Department of Chemistry, University of Zürich, Switzerland
- Neuroscience Center Zurich, University and ETH Zürich, Switzerland
Figures
Figure 1 with 3 supplements
Development and optical properties of GLPLight1.
(a) Structural model of GLPLight1 obtained using Alphafold (Mirdita et al., 2022). The human glucagon-like peptide-1 receptor (GLP1R) is shown in gold, cpGFP in green, and residue targets of …
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Figure 1—source data 1
Development and optical properties of GLPLight1.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Screening process for the development of GLPLight1.
(a) Maximal fluorescence response to 10 μM glucagon-like peptide-1 (GLP-1) of the prototype GLP-1 sensor and the N-terminus deletion variants (16 or 23 first amino acids). (b) Maximal fluorescence …
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Figure 1—figure supplement 1—source data 1
Screening process for the development of GLPLight1.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Development of the control sensor GLPLight-ctr.
(a) Maximal response of GLPLight1 binding pocket variants to 1 μM glucagon-like peptide-1 (GLP-1) (7–37). (b) Structural model of GLPLight-ctr shown from the side (left) or top (right) perspective. …
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Figure 1—figure supplement 2—source data 1
Development of the control sensor GLPLight-ctr.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
Intracellular signaling characterization of glucagon-like peptide-1 receptor (GLP1R) and GLPLight1.
Normalized timelapse recording of agonist-induced miniG protein or β-arrestin recruitment in GLP1R or GLPLight1 expressing HEK293T cells along with quantification of the mean signal between …
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Figure 1—figure supplement 3—source data 1
Intracellular signaling characterization of glucagon-like peptide-1 receptor (GLP1R) and GLPLight1.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig1-figsupp3-data1-v1.xlsx
Figure 2 with 1 supplement
Pharmacological characterization of GLPLight1.
(a) Absolute ΔF/F0 responses of GLPLight1 expressing HEK293T cells to various glucagon-like peptide-1 (GLP-1) agonists, antagonist, or to other class B1 neuropeptide ligands applied at 1 μM final …
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Figure 2—source data 1
Pharmacological characterization of GLPLight1.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Detecting endogenous glucagon-like peptide-1 (GLP-1) release from enterocrine cells using GLPLight1.
(a) Representative fluorescence and brightfield images of HEK293T co-transfected with GLPLight1 and cytosolic mKate2 cultured overnight in the absence (top row) or presence (bottom row) of GLUTag …
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Figure 2—figure supplement 1—source data 1
Detecting endogenous glucagon-like peptide-1 (GLP-1) release from enterocrine cells using GLPLight1.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 2 supplements
All-optical visualization and control of human glucagon-like peptide-1 receptor (GLP1R) activation.
(a) Schematic representation of the N-terminal chemical caging strategy used to generate photocaged glucagon-like peptide-1 derivative (photo-GLP1). The peptide product (native GLP-1) after optical …
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Figure 3—source data 1
All-optical visualization and control of human glucagon-like peptide-1 receptor (GLP1R) activation.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Biochemical characterization of photocaged glucagon-like peptide-1 derivative (photo-GLP1).
(a) Scheme illustrating the uncaging reaction of photo-GLP1 when exposed to UV light at 370–405 nm, producing WT GLP-1. (b) LCMS chromatographic traces of (i) pure WT GLP-1, (ii) pure photo-GLP1, …
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Figure 3—figure supplement 1—source data 1
Biochemical characterization of photocaged glucagon-like peptide-1 derivative (photo-GLP1).
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig3-figsupp1-data1-v1.xlsx
Figure 3—figure supplement 2
Further characterization of photocaged glucagon-like peptide-1 derivative (photo-GLP1) uncaging.
(a) Fluorescence response (ΔF/F0) of GLPLight1-expressing HEK293T cells after bolus addition of 10 μM photo-GLP1. The time of addition is represented by the blue rectangle above; n=16 cells. (b) …
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Figure 3—figure supplement 2—source data 1
Further characterization of photocaged glucagon-like peptide-1 derivative (photo-GLP1) uncaging.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig3-figsupp2-data1-v1.xlsx
Figure 4 with 1 supplement
Effect of photocaged glucagon-like peptide-1 derivative (photo-GLP1) uncaging on intracellular signaling.
(a) Representative images of HEK293T cells used in (c,d). Human glucagon-like peptide-1 receptor (hmGLP1R) expression was visualized using an Alexa-647-conjugated anti-FLAG antibody. The uncaging …
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Figure 4—source data 1
Effect of photocaged glucagon-like peptide-1 derivative (photo-GLP1) uncaging on intracellular signaling.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Control experiment for intracellular signaling characterization.
(a) Timelapse imaging of G-Flamp1 response in HEK293T cells co-expressing glucagon-like peptide-1 receptor (GLP1R) after addition of HBSS buffer (used for imaging and dilution of the ligands). The …
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Figure 4—figure supplement 1—source data 1
Control experiment for intracellular signaling characterization.
- https://cdn.elifesciences.org/articles/86628/elife-86628-fig4-figsupp1-data1-v1.xlsx
Tables
Table 1
Titration parameters of alanine scanned variants of glucagon-like peptide-1 (GLP-1) peptide.
The Emax and pEC50 values were derived from the four-parameter non-linear fit for each peptide and the EC50 shift by comparison against WT GLP-1 peptide measured alongside.
| Entry | GLP-1 variant | Emax (% WT GLP-1) | pEC50 (M) | Fold-reduction EC50 vs. WT GLP-1 |
|---|---|---|---|---|
| a | H1A | 56±4 | 5.89±0.05 | ≈ 63 |
| b | E3A | 96±3 | 6.10±0.03 | ≈ 37 |
| c | G4A | 14±3 | 6.00±0.13 | ≈ 48 |
| d | T5A | 100±4 | 6.73±0.04 | ≈ 9 |
| n/a | WT GLP-1 | 100 | 7.69±0.04 | 0 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (human) | GLP1R | Integrated DNA Technologies | ||
| Strain, strain background (Escherichia coli) | NEB 10-beta Competent E. coli | NEB | C3019 | |
| Commercial assay or kit | NEBuilder HiFi | NEB | E2621 | |
| Recombinant DNA reagent | Pfu-Ultra II Fusion | Agilent | 600387 | |
| Cell line (Mus musculus) | GLUTag enterocrine cell line | Daniel J Drucker (Univ. of Toronto) | ||
| Cell line (Homo sapiens) | Human Embryonic Kidney (HEK293T) | ATCC | CRL-3216 | |
| Antibody | Anti-FLAG-Alexa-647 | M1 (Sigma-Aldrich) In-house conjugated to Alexa-647 (Miriam Stoeber, University of Geneva) | F3040 | IF(1:1000) |
| Chemical compound | Fmoc- and side chain-protected L-amino acids | Bachem AG | ||
| Chemical compound | Piperidine | Chemie Brunschwig AG | 110-89-4 | 99% |
| Chemical compound | O-(7-Azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) | Bachem AG | 148893-10-1 | |
| Chemical compound | (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP) | Advanced ChemTech CreoSalus | 156311-83-0 | |
| Chemical compound | N,N-Diisopropylethylamine (DIPEA) | Sigma-Aldrich Chemie GmbH | 7087-68-5 | |
| Chemical compound | Trifluoroacetic acid (TFA) | Sigma-Aldrich Chemie GmbH | 76-05-1 | For HPLC, ≥99.0% |
| Chemical compound | Triisopropylsilane (TIPS) | Sigma-Aldrich Chemie GmbH | 6485-79-6 | 98% |
| Chemical compound | 3,6-Dioxa-1,8-octane-dithiol (DODT) | Sigma-Aldrich Chemie GmbH | 14970-87-7 | 95% |
| Chemical compound | α-Methyl-5-methoxy-2-nitro-4-(2-propyn-1-yloxy)benzyl alcohol | Sigma-Aldrich Chemie GmbH | 1255792-05-2 | |
| Chemical compound | N,N'-Disuccinimidyl carbonate (DSC) | Sigma-Aldrich Chemie GmbH | 74124-79-1 | |
| Other | NovaPEG Rink Amide resins (0.41 mmol/g and 0.20 mmol/g loading) | Sigma-Aldrich Chemie GmbH | ||
| Other | AldraAmine trapping packets (volume 1000–4000 mL) | Sigma-Aldrich Chemie GmbH |
Additional files
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Source data 1
Synthesis and Characterization of Building Blocks and Peptides.
- https://cdn.elifesciences.org/articles/86628/elife-86628-data1-v1.pdf
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MDAR checklist
- https://cdn.elifesciences.org/articles/86628/elife-86628-mdarchecklist1-v1.docx
