Fig. 2

GLP-1R agonist trafficking in MIN6B1-SNAP-GLP-1R cells. a Schematic for GLP-1R internalization and recycling measurements by FACS after labeling with cleavable SNAP-Surface probe. b Agonist-induced GLP-1R internalization in MIN6B1-SNAP-GLP-1R cells, n = 3, two-way randomized block ANOVA with Dunnett’s test vs. exendin-4. c GLP-1R recycling in MIN6B1-SNAP-GLP-1R cells 30 min after an initial 15 min agonist pulse to induce internalization; recycling measured in the presence of exendin(9-39) to block further endocytosis, n = 5 (exendin-4) or 3 (exendin-phe1 and -asp3), one-way ANOVA with Dunnett’s test vs. exendin-4. d Confocal images indicating GLP-1R internalization in MIN6B1-SNAP-GLP-1R cells, 30 min agonist incubation after SNAP-Surface-488 labeling, representative image from n = 3 experiments; scale bars, 8 μm. e Immunofluorescence showing increased co-localization of exendin-phe1-FITC vs. exendin-4-FITC and exendin-asp3-FITC with Rab11-positive recycling endosomes after 60 min agonist exposure, representative image from n = 2 experiments; scale bars, 4 μm. Individual red and green channels shown in Supplementary Fig. 12. f Representative electron micrographs showing subcellular localization of SNAP-GLP-1R (labeled with cleavable SNAP-Surface-biotin plus streptavidin-10 nm gold, arrows), 60 min agonist exposure; scale bars, 0.1 μm; larger area micrographs shown in Supplementary Fig. 5a. g Gold-particle quantification from f; n = 3 experiments, paired t-tests. PM plasma membrane, EE early endosome, MVB LM multivesicular body limiting membrane, MVB ILV multivesicular body intraluminal vesicle, Tub/RE tubular/recycling endosome, LE late endosome. h Immunoblots showing SNAP-GLP-1R (~73 kDa) levels in MIN6B1-SNAP-GLP-1R cells after 4 and 16 h agonist exposure, representative of n = 3 experiments. A smaller band possibly corresponding to deletion of GLP-1R C-terminal domain is detected under all conditions analyzed. i Confocal images demonstrating agonist-induced surface GLP-1R downregulation in MIN6B1-SNAP-GLP-1R cells; surface receptor labeled after 16 h agonist treatment; scale bar, 100 μm. j Quantification of experiments from i, five images analyzed per condition from n = 3 coverslips, mean cellular fluorescence indicated, one-way ANOVA with Dunnett’s test vs. exendin-4. k As for j, but quantified by FACS in separate experiments, results normalized to vehicle control, n = 4, one-way randomized block ANOVA with Dunnett’s test vs. exendin-4. Agonists applied at 100 nM. *p < 0.05, ***p < 0.001, by statistical test indicated above. Error bars indicate SEM