FIGURE 10.

Distinct GPCRs localized to small endosomes also require GIPC for a sustained MAPK signaling profile. A, the size of FSHR- and B1AR-containing endosomes following agonist-induced internalization. Cells were treated with either FSH (10 nm) or isoproterenol (10 μm) following treatment with fluorescently labeled FLAG (FSHR) or HA (B1AR) antibodies. Endosome size was assessed by measuring the diameter of 10 endosomes at each time point stated across three movies. Data represent mean ± S.E. The data from Fig. 1B of LHR and B2AR endosome diameters are shown for comparison. B and C, representative immunoblot analyses of ligand-induced FSHR (B) and B1AR (C) ERK 1/2 phosphorylation treated with either control or GIPC siRNA (SiGIPC). Cells were treated with either FSH (10 nm) or isoproterenol (10 μm) for the indicated time points. D, cells expressing B1AR were pre-treated with pertussis toxin (PTX) (200 ng/ml, 18 h) with or without cotreatment of GIPC siRNA. Cells were then treated with isoproterenol (10 μm) for the indicated time points. Phosphorylation of ERK 1/2 was determined by Western blotting, and total ERK 1/2 was used as a loading control. A representative immunoblot analysis is shown.