FIGURE 1.

The LHR and B2AR traffic to distinct compartments in the endocytic pathway. A, HEK 293 cells stably expressing FLAG-tagged LHR or B2AR were labeled with fluorescently tagged anti-FLAG antibodies and imaged live with confocal microscopy before and after agonist treatment. The LHR was stimulated with 10 nm LH, and the B2AR was stimulated with 10 μm isoproterenol. The frames shown were taken from a time-lapse series of supplemental Movies S1 and S2. B, the size of the LHR or B2AR containing endosomes was assessed by measuring the diameter of 10 endosomes at each time point stated across three to four movies. Data represent mean ± S.E. C, cells expressing either the LHR or B2AR were fed with anti-FLAG antibody and treated with the agonist for 10 min. Cells were fixed, permeabilized, and stained with an anti-EEA1 antibody and imaged with confocal microscopy. The images shown are representative of 16 cells. D, numbers of LHRs or B2ARs containing endosomes positive for EEA1 following either 10 or 30 min of agonist stimulation were quantified, and the percentage was calculated. Data are mean + S.E.; n = 16 cells, 298 and 295 endosomes respectively for each receptor. E and F, cells were treated as in C and D except that cells were transfected with the PI3P marker 2xFYVE-GFP F, and the number of receptor-containing endosomes positive for 2xFYVE-GFP was quantified (n = 24 and 22 cells, respectively; LHR and B2AR, ∼980 and 500 endosomes, respectively). The arrows represent examples of colocalization. Scale bars = 5 μm. ***, p < 0.001. See also supplemental Movies S1 and S2.