Journal: Nature Communications
Article Title: Targeting GLP-1 receptor trafficking to improve agonist efficacy
Figure Lengend Snippet: Binding kinetics influence GLP-1R recycling. a Dissociation curve indicating FRET between FITC-agonist complexed with surface SNAP-GLP-1R, after inhibition of internalization, using NaN 3 and 2-deoxyglucose 9 (Supplementary Fig. 9 ), 30 min agonist exposure, washout, and exendin(9-39) blockade, n = 4. Unmodified (non-FITC) agonist b residence time (1/ k off ), c association rate constant ( k on ), and d affinity, measured by TR-FRET in competition with exendin-4-FITC, with internalization inhibitors as above, and calculated using competitive kinetic method 42 , n = 5, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4. e Confocal fluorescence indicating co-localization of exendin-4-FITC or exendin-phe1-FITC with SNAP-GLP-1R (labeled with SNAP-Surface-549) after 60 min agonist exposure in MIN6B1-SNAP-GLP-1R cells, representative images from n = 2 experiments; scale bars, 8 μm. Individual red and green channels shown in Supplementary Fig. 12 . f Schematic illustrating endosomal binding protocol. SA-Tb streptavidin-terbium cryptate. g Real-time FRET measurement of FITC-agonist complexed with internalized SNAP-GLP-1R after 30 min agonist exposure, washout, exendin(9-39) blockade, and cleavage of SNAP-biotin from surface SNAP-GLP-1R with MesNa, n = 5. Exendin-4 h residence time and i association rate constant ± 10 μM BETP, measured by TR-FRET in competition with exendin-4-FITC, n = 4, paired t -test. Exendin-4-induced j internalization (30 min), and k recycling (60 min) ± 3 μM BETP, n = 4, paired t -test. l Prolonged insulin secretion with exendin-4 ± 3 µM BETP in INS-1 832/3 cells, 16 h, n = 5, paired t -test for E max assessed by four-parameter fit. Exendin-4 m cAMP, and n β-arrestin-2 responses, in PathHunter CHO-GLP-1R cells ± 3 μM BETP, n = 3. Agonists applied at 100 nM, except where indicated, and performed in CHO-SNAP-GLP-1R cells, except where indicated. * p
Article Snippet: Antibodies were primaries rabbit anti-β-arrestin-1/2 (D24H9, Cell Signaling, 1/1000), rabbit anti-SNAP tag (New England Biolabs, 1/500), mouse anti-α-tubulin (T5168, Sigma, 1/1000), rabbit anti-β-actin (4970, Cell Signaling, 1/1000), and mouse anti-GAPDH (6C5, Merck, 1/10,000); and IgG-HRP secondaries (Santa Cruz Biotechnology).
Techniques: Binding Assay, Inhibition, Blocking Assay, Fluorescence, Labeling