1 a 1B -adrenoceptors are localized at a steady state in the plasma membrane in untreated cells, and internalize to intracellular vesicles when exposed to agonist. Flow cytometry analysis with an anti-Nterminus-antibody (1B-N1-C, ) facilitated the quanti®cation of cell surface a 1B -adrenoceptor. Also, the cellular distribution of a 1B -adrenoceptors was visually monitored by immunocytochemical confocal microscopy. 2 Utilizing this combined approach, we have examined the molecular mechanism for cellular tracking of a 1B -adrenoceptors, including the process of sorting of the synthesized receptor protein to the cell surface, and the agonist-induced internalization. The two processes were separately examined by using a 1B -adrenoceptor inducible DDT 1 MF-2 cells for the sorting process and CHO cells stably expressing a 1B -adrenoceptors for the agonist-promoted internalization. 3 We examined the eects of cytochalasin D and mycalolide B (actin depolymerization agents), demecolcine (a microtubule disrupting agent), brefeldin A (an inhibitor of vesicular transport and Golgi function), ba®lomycin A1 (a speci®c inhibitor of the vacuolar proton pump) or hyperosmotic sucrose treatment (that may inhibit clathrin-mediated endocytosis) on these processes. 4 We found that the agonist-promoted internalization was blocked by cytochalasin D and mycalolide B, while the cell surface sorting process was speci®cally blocked by brefeldin A, indicating that the two processes involve dierent components of the cellular endocytic machinery. 5 The experimental approach as exempli®ed in this study would provide a valuable system to study further the molecular mechanism(s) of cellular tracking of G protein-coupled receptors.
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