FIGURE 8.
hβ1AR N-terminal cleavage is a regulated process. A, receptor activation by increasing concentrations of the βAR-agonist ISO. Stably transfected HEK293i cells were induced, labeled with [35S]methionine/cysteine, and chased for 240 min, as described in the legend to Fig, 3. ISO at the concentrations indicated was added to the chase medium 45 min after the beginning of the chase. GM6001 (10 μm) and CGP-20712 (10 μm) were added 15 min prior to ISO. Cellular membranes were prepared using a buffer containing EDTA and 1,10-phenanthroline, and DDM-solubilized receptors were purified with the anti-FLAG M2 antibody (Ab) and analyzed by SDS-PAGE and fluorography. Changes in the relative amounts of the full-length mature receptor (a closed circle) and the larger cleaved fragment (a closed square), as revealed by densitometric scanning of fluorograms, are shown in B. The data are expressed as (Fl/(Fl + Fr)) × 100 and (Fr/(Fl + Fr)) × 100 for the full-length receptor and cleaved fragment, respectively, where Fl = full-length receptor, and Fr = receptor fragment. C shows relative changes in the total amount of receptor. The values were normalized to that in the nontreated cells, which was set to 100%. The data shown represent the means ± S.E. from three independent experiments and were analyzed by GraphPad Prism using the repeated measures two-way analysis of variance followed by Bonferroni's post-test to compare the ISO-treated samples with the nontreated controls. The post-test did not reveal any significant differences for the data in C, although the analysis of variance performed before normalization revealed significant differences between the samples (p = 0.0081). D, time-dependent receptor activation by ISO. E, receptor activation by dobutamine. F, activation of protein kinase C and adenylyl cyclase by PMA and forskolin, respectively. Stably transfected HEK293i cells were induced and labeled with [35S]methionine/cysteine as described for A. ISO (10 μm) was added to the chase medium 45 min after the beginning of the chase or not, and the cells were harvested 30, 60, 120, or 240 min later, as indicated (D). Dobutamine (DOB) (10 μm) was added 45 min after the beginning of the chase, and the cells were harvested 195 min later (E), Bisindolylmaleimide I (Bis I; 10 μm) was added 30 min after the beginning of the chase, and PMA (0.5 μm) and forskolin (15 μm) 15 min later, and the cells were harvested after a further 195 min (F). DDM-solubilized receptors were analyzed as described for A. Symbols are as in Fig. 1. ***, p < 0.001; *, p < 0.05. IP, immunoprecipitation.