Figure 1

Trafficking of yeast Ste2 in human HEK293 cells in response to α-factor: analysis by confocal microscopy. HEK cells were transiently transfected to express either Ste2-GFP or β₂AR-GFP. Unstimulated cells display receptors that are largely localized to the cell membrane (white arrows denote cell membrane-localized receptors). Cells expressing yeast Ste2-GFP and challenged with α-factor (10 μM, upper panel) as well as those expressing β₂AR-GFP and challenged with beta-adrenergic agonist isoproterenol (Iso, 10 μM; lower panel) for 0.5 hr displayed frank internalization (yellow arrowheads denote internalized receptors). Agonist ligands were then washed from the media and the cells treated the appropriate antagonist ligand. Possible recycling of internalized receptors back to the cell membrane was followed for 3 hours. For cells expressing Ste2-GFP, the a-factor antagonist (des-Trp, Ala-3 analog of a-factor, 10 μM) was added after wash-out and the cells were monitored at 0.5 (panel c), 1 (panel d), 2 (panel e), and 3 (panel f) hour time periods after wash-out of agonist. For cells expressing β₂AR-GFP, the β-adrenergic antagonist propranolol (10 μM) was added after wash-out of isoproterenol and the cells were monitored at 0.5 (panel i), 1 (panel j), 2 (panel k), and 3 (panel l) hour time periods post wash-out of agonist. The images displayed are from a single experiment, representative of more than five replicate, separate experiments. Bar equals 10 μm.