Real-time optical monitoring of the cell surface sorting and the agonist-promoted internalization of α1b-adrenoceptor with green fluorescent protein.

由于缺乏有效的生物试剂，研究G蛋白偶联受体（GPCR）在活细胞中的信号转导和行为在技术上是困难的。我们发现一个全功能的带有绿色荧光蛋白标记的α 1b -肾上腺素能受体（α1B-AR）可以用来确定活细胞内化的分子机制。利用这种方法，我们研究了α1B-AR细胞运输的分子机制，包括合成受体蛋白到细胞表面的分选过程，以及激动剂诱导的内化。分别用α1B-AR诱导的DDTIMF-2细胞进行分选，用稳定表达α1B-AR的CHO细胞进行激动剂促进内化。我们研究了细胞松弛素D和mycalolide B（肌动蛋白解聚剂）、去乙酰胆碱（微管破坏剂）、brefeldin a（囊泡运输和高尔基功能抑制剂）、巴霉素A1（液泡质子泵的特异性抑制剂）或高渗蔗糖处理（可能抑制网格蛋白介导的内吞作用）对这些过程的影响。我们发现激动剂促进的内化被细胞松弛素D和mycalolide B阻断，而细胞表面分选过程被brefeldin A特异性阻断，表明这两个过程涉及细胞内吞机制的不同组成部分。此外，我们发现激动剂促进α1B-AR内化与PLC激活密切相关，并且依赖于PKC激活，但不依赖于[Ca2+]i的增加。本研究表明，实时光学监测α1B-AR（以及其他GPCR）在活细胞中的亚细胞定位是可行的，这可能为进一步研究受体定位的生化机制提供有价值的系统。

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.