Skip to main content
. 2021 Sep 6;12:735876. doi: 10.3389/fphar.2021.735876

FIGURE 3.

FIGURE 3

5-HT1B FRET sensor construction. (A) Primary human 5-HT1B-receptor amino acid sequence. FlAsH motif replaces FRET sensor sequence (underlined in red). (B) N terminus modification of human 5-HT1B receptor by adding a leader sequence from mGluR5 followed by the VSV-G peptide sequence, while CFP was added at C terminus. FlAsH motif sequence FLNCCPGCCMEP introduced into third intracellular loop-linking transmembrane domains V and VI. Energy transfer from CFP to FlAsH and subsequent output is illustrated. (C) Stable cell lines harboring inducible 5-HT1B FRET sensor, VSV-G-mGluR5-5-HT1B, and corresponding vector constructs induced by 10 ng ml−1 doxycycline for 12 h, followed by 12 h of serum starvation quiescence, and 5 min stimulation by graded concentrations (10−9 to 10−6 M) of the 5-HT1B selective agonist CP-94253, using FBS as the positive control and deionized water as negative control in vector construct cells. Samples were then subjected to western blotting. The expression of 5-HT1B was determined by its fusion protein tag using VSV antibody. Phosphorylation of ERK 1/2 MAP kinases was detected by a phospho-ERK1/2-specific antibody (anti-p-ERK), and the anti-ERK1/2 antibody was used to assess the protein loading equivalence. (D) 5-HT1B FRET sensor was cloned into the inducible Flp-In™ T-REx™ locus of Flp-In™ T-REx™ 293 cells, and expression was induced by doxycycline. Imaging of CFP (left), labeled FlAsH (middle), and light field (right) demonstrated effective delivery of this sensor to the cell surface. FRET: fluorescence resonance energy transfer; VSV: vesicular stomatitis virus; FlAsH: fluorescein arsenical hairpin binder via tetra-cysteine; ERK: extracellular regulated protein kinases; 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; 5-HT1B: serotonin receptor 1B; FBS: fetal bovine serum; DIC: differential interference contrast; mGluR5: metabotropic glutamate receptor 5.