Antagonist-mediated recycling of internalized β2AR andβ2AR-Gαs. A. Cells stably expressing the β2AR or the fusion protein β2AR-Gαs 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).
Di Certo et al. BMC Cell Biology 2008 9:56 doi:10.1186/1471-2121-9-56