Differential mechanism for the cell surface sorting and agonist-promoted internalization of the alpha1B-adrenoceptor.
1. Alpha1B-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-N-terminus-antibody (1B-N1-C, (Hirasawa et al., 1996)) facilitated the quantification of cell surface alpha1B-adrenoceptor. Also, the cellular distribution of alpha1B-adrenoceptors was visually monitored by immunocytochemical confocal microscopy. 2. Utilizing this combined approach, we have examined the molecular mechanism for cellular trafficking of alpha1B-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 alpha1B-adrenoceptor inducible DDT1MF-2 cells for the sorting process and CHO cells stably expressing alpha1B-adrenoceptors for the agonist-promoted internalization. 3. We examined the effects of cytochalasin D and mycalolide B (actin depolymerization agents), demecolcine (a microtubule disrupting agent), brefeldin A (an inhibitor of vesicular transport and Golgi function), bafilomycin A1 (a specific 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 specifically blocked by brefeldin A, indicating that the two processes involve different components of the cellular endocytic machinery. 5. The experimental approach as exemplified in this study would provide a valuable system to study further the molecular mechanism(s) of cellular trafficking of G protein-coupled receptors.
Hirasawa
A
A
Department of Molecular, Cell Pharmacology, National Children's Medical Research Center, Tokyo, Japan.
Awaji
T
T
Sugawara
T
T
Tsujimoto
A
A
Tsujimoto
G
G
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis / Q000187:drug effects*; D005434:Flow Cytometry; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*
D058646:Adrenergic alpha-1 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D018340:Receptors, Adrenergic, alpha-1