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. 2021 Sep 6;12:735876. doi: 10.3389/fphar.2021.735876

FIGURE 4.

FIGURE 4

Intramolecular FRET sensor imaging of EG as a 5-HT1B agonist. (A,B) Flp-In™ T-REx™ 293 cells induced to express mGluR5-VSV-G-5-HT1B-FlAsH-CFP shown in FRET imaging. CP-94253 (A) or EG (B) at three concentrations (10−10, 10−8 and 10−6 M) added at the indicated times and removed after 15 s. FRET signal was monitored over 60 s. (C) Changes in normalized FRET signals due to ligand addition and effect of 10−6 M CP-94253 defined as 100% and vehicle as 0%. 5-HT1B receptor antagonist SB 224289 did not modulate mGluR5-VSV-G-5-HT1B-FlAsH-CFP FRET signals, but co-addition with CP-94253 or with EG blocked agonists’ effects. Studies were quantified, and data are represented as means ± S.E., n = 6. (D) Flp-In™ T-REx™ 293 cells induced to express mGluR5-VSV-G-5-HT1B-FlAsH-CFP imaged to detect CFP following addition of CP-94253 or EG (10−7 M). CP-94253 or EG-induced internalization, reducing the cell surface mGluR5-VSV-G-5-HT1B-FlAsH-CFP in a time-dependent fashion. Representative examples of n = 3 independent experiments are shown. FRET: fluorescence resonance energy transfer; FlAsH: fluorescein arsenical hairpin binder via tetra-cysteine; CFP: cyan fluorescent protein; VSV: vesicular stomatitis virus; EG: emodin-8-O-β-d-glucopyranoside; CP-94253: 4-{5-propoxypyrrolo [3,2-b]pyridin-3-yl}-1.2,3,6-tetrahydropyridine; SB 224289 [4-[2-methyl-4-(5-methyl-1,2,4-oxadiazol-3-yl)phenyl]phenyl]-(1′-methylspiro [6,7-dihydro-2H-furo [2,3-f]indole-3.4′-piperidine]-5-yl) methanone; 5-HT1B: serotonin receptor 1B; mGluR5: metabotropic glutamate receptor 5; FlAsH: fluorescein arsenical hairpin binder via tetra-cysteine.