FIG 2.

GIPR is internalized in a constitutive manner in the absence of GIP in adipocytes. (A) Epifluorescence images of adipocytes with HA-GIPR-GFP. GFP fluorescence is shown in the left panels, and Cy3 fluorescence is shown in the right panels. Cells were stained by indirect anti-HA immunofluorescence (Cy3) under nonpermeabilizing conditions to measure surface GIPR or with permeabilization to reveal total GIPR. Representative cells are shown. Bar, 10 μm. (B) GIPR trafficking is not altered within the range of expression of WT and E354Q GIPRs in 3T3-L1 cells. To establish that the level of GIPR expression achieved by electroporation does not lead to aberrant trafficking due to saturation of endocytosis and/or recycling, we determined, on an individual-cell basis, the steady-state amount of HA-GIPR-GFP on the plasma membrane in fixed cells by anti-HA epitope indirect immunofluorescence correlated to the total amount of HA-GIPR-GFP expressed in the cell. These data are from a representative experiment, and each point indicates the Cy3 and GFP fluorescence from a single cell. AU, arbitrary units. (C) Cartoon of the internalization experiment protocol. (D) Cells expressing HA-GIPR-GFP were incubated with anti-HA antibodies for the indicated times, fixed, and stained with saturating concentrations of anti-mouse Cy5 secondary antibodies to block the surface. Cells were refixed, permeabilized, and stained with anti-mouse Cy3-conjugated secondary antibodies to label intracellular anti-HA. Representative cells are shown. Bar, 10 μm. (E) Quantification of internalized anti-HA plotted as a function of incubation time. (F) HA-GIPR-GFP trafficking is not altered by incubation of living cells with anti-HA antibody. Adipocytes transiently expressing HA-GIPR-GFP were incubated at 37°C for 10 min in serum-free DMEM or in serum-free DMEM supplemented with anti-HA antibody. The cells were fixed, and the amount of HA-GIPR-GFP in the plasma membrane was determined by incubating the cells with anti-HA antibody, followed by incubation with Cy3-labeled goat anti-mouse secondary antibody. The ratio of the surface distribution to the total distribution of HA-GIPR-GFP was determined by quantitative fluorescence microscopy. ns, not significant. (G) Adipocytes expressing BTX-GIPR-GFP were incubated with BTX-Cy3 for the indicated times. Cells were fixed and imaged. Representative cells are shown. (H) Quantification of cells treated as described above for panel E. The Cy3/GFP fluorescence ratio per cell ratio is plotted as a function of incubation time. The data points are the averages ± standard errors of the means from 3 independent experiments. The data from each experiment were normalized to the Cy3/GFP value at the 30-min time point. Asterisks indicate the nucleus.