|Budget Amount *help
¥3,800,000 (Direct Cost : ¥3,800,000)
Fiscal Year 1999 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1998 : ¥3,100,000 (Direct Cost : ¥3,100,000)
The study of G protein-coupled receptor (GPCR) signal transduction and behavior in living cells is technically difficult because of a lack of useful biological reagents. We showed that a fully functional α1B-adrenoceptor (α1B-AR) tagged with the Green Fluorescent Protein can be used to determine the molecular mechanism of internalization in living cells. Utilizing this approach, we have examined the molecular mechanism for cellular trafficking of α1B-AR, 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 α1B-AR inducible DDTIMF-2 cells for the sorting process and CHO cells stably expressing α1B-AR for the agonist-promoted internalization. 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 process. We found that the agonist-promoted internalization is blocked by cytochalasin D and mycalolide B, while the cell surface sorting process is specifically blocked by brefeldin A, indicating that the two processes involve different components of the cellular endocytic machinery. Furthermore, agonist-promoted internalization of α1B-AR was found to be closely linked to PLC activation, and was dependent on PKC activation, but was independent of [Ca2+]i increase. This study demonstrated that real-time optical monitoring of the subcellular localization of α1B-AR (as well as other GPCR) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of receptor localization.