Figure 3

Antagonist-mediated recycling of internalized β ₂ AR andβ ₂ AR-Gα _s . A. Cells stably expressing the β₂AR or the fusion protein β₂AR-Gα_s were treated (panels b, b', c, c') or not (panels a, a') with the agonist isoproterenol to induce complete receptor internalization. The agonist-mediated endocytosis was stopped by the addition of the antagonist propranolol for 60 minutes (panels c,c'). Fixed cells were then processed by fluorescence microscopy for membrane immuno-localization of Flag-tagged receptors, as described in Methods. Scale bars, 20 μm. B. Cells expressing wild type or fusion protein receptor were incubated with 1 μM isoproterenol for 5 hours to induce maximal internalization. Reversal of internalization was initiated by adding 1 μM propranolol, and cells were fixed and subjected to immuno-staining with Flag-antibody and Alexa Fluor 488-conjugated anti mouse IgG, at the indicated times, to detect the recovery of the cell surface receptors. Data (means of 3 experiments) are reported as the percent of the control (not stimulated) receptor immunoreactivity. The same experiment was performed in the absence (control) or presence of cycloheximide (CHX).