Agonist-dependent internalization of β2AR-Gαs. A. HEK293 cells stably expressing β2AR or β2AR-Gαs were not treated (NT) or treated with isoproterenol for 5, 20 or 60 minutes to induce receptor internalization. The cells were then fixed in 4% formaldehyde and sequentially immuno-stained with anti-Flag and Alexa-Fluor 594 conjugated anti-mouse IgG. The disappearance of the fluorescence from the plasma membrane was monitored with an epifluorescence microscope. Scale bars, 20 μm. B. Immunofluorescence showing the recruitment of both receptors into endocytic vesicles after 60 minutes of agonist treatment. In this case, cells were permeabilized with 0.2% NP-40 before the immunofluorescence reaction. Nuclei were stained with Hoechst 33258 (1 μg/ml). Scale bars, 10 μm. C. β2AR-Gαsand wild type β2AR expressing cells were cultured on 96 multiwell plates and treated with isoproterenol for the indicated times (abscissae). Fixed cells were immuno-stained with Flag-antibody followed by Alexa-Fluor 488-conjugated anti-mouse IgG. Nuclei were stained with Hoechst 33258 (1 μg/ml). Fluorescence was measured by a multi-label counter. Each point represents the mean of 4 different experiments performed in triplicate. Data are expressed as ratios of the relative fluorescence measured after immuno-staining with the Alexa-Fluor 488-labeled secondary antibody (RFUFitc) and following Hoechst staining of the same monolayers (RFUHoechst), as described in Methods. Data were fitted to an exponential decay equation of the form: y = a1 exp (-k t) + a0, where t is time in min, a1 and a0 are decaying and not decaying components of fluorescence, respectively, and k, the time constant, represents the inverse of the half-time (t1/2) of the disappearance of immunoreactivity from the cell surface.
Di Certo et al. BMC Cell Biology 2008 9:56 doi:10.1186/1471-2121-9-56