Real-time imaging of alpha2 adrenergic receptor subtypes in living cells using green fluorescent protein

背景和目的：尽管对β 2肾上腺素能受体的认识不断增加，但激动剂诱导的α 2肾上腺素能受体细胞内转运的研究仅限于免疫细胞化学分析。因此，我们使用绿色荧光蛋白（GFP）和人α 2肾上腺素能受体亚型的融合蛋白来检测活细胞中α 2肾上腺素能受体的实时亚型特异性运输。材料和方法：利用重组DNA技术，将每个α 2a、B和C肾上腺素受体亚型的C端融合到GFP的N端（α 2 AR-GFP）。每个alpha2 AR-GFP的DNA序列通过对两条链进行双脱氧测序来确认。在放射性标记配体结合实验中，将克隆到Pcdna3载体上的重组构建物用DEAE dextram法瞬时转染COS-1细胞。用不同浓度的3 [H]-RX-821002孵育膜，测定饱和结合等温线，并测定非特异性结合。利用非线性最小二乘测量拟合技术（GraphPDA Software Inc., San Diego， CA）从饱和等温线确定平衡解离常数。根据Lowry等人的方法测定膜制剂的蛋白质含量。利用脂质体法将重组构建体瞬时转染HEK293细胞进行活细胞成像。我们利用附着在epifluorescence显微镜上的冷却CCD相机，研究了alpha2 AR-GFP在右美托咪定作用下的动态转运方式。结果和讨论：：在稳定状态下，α 2a和2B AR亚型定位于质膜，尽管α 2c AR亚型的很大一部分定位于细胞内池。我们观察到，alpha2B AR亚型经历了激动剂诱导的内化，并且似乎在15分钟内遵循与bet2 AR相同的内体途径。alpha2A和2C AR亚型激动剂治疗导致一些受体内化减少

Background and Goals: Despite the increasing knowledge of beta2 adrenergic receptor, the study of agonist-induced intracellular trafficking of alpha2 adrenoceptor has been limited to immunocytochemical analysis. Thus, we examine the real time subtype-specific trafficking of alpha2 adrenoceptors in living cells using a respective fusion protein of green fluorescent protein (GFP) and human alpha2 adrenoceptor subtypes.Material and Methods: Using recombinant DNA techniques, the C terminus of each alpha2A, B, and C adrenoceptor subtype was fused to the N terminus of GFP (alpha2 AR-GFP). The DNA sequence of each alpha2 AR-GFP was confirmed by dideoxy sequencing both strands. For the radiolabeled ligand binding assay, the recombinant constructs cloned into Pcdna3 vector were transfected transiently in COS-1 cells using DEAE dextram method. Satruration binding isotherms were performed by incubating membranes with varying cocentrations of 3 [H]-RX-821002, and nonspecific binding was determined … More by adding 10 Um rauwolscine. Equilibrium dissociation constants were determined from saturation isothems using a nonlinear least-square surve-fitting technique (GraphPDA Software Inc., San Diego, CA). Protein content for the membrane preparations were assayed according to Lowry et al. The recombinant constructs were transfected transiently in HEK293 cells using liposome method for the living cell imaging. We examined dynamic manners of trafficking of alpha2 AR-GFP in response to dexmedetomidne using cooled CCD camera attached to an epifluorescent microscope. The distributon into endosomal compartments were also observed using three-dimensional analysis of time-lapse imagesResults and Discussions: : At steady state, alpha2A and 2B AR subtypes are localized in the plasma membrane, though a significant portion of alpha2C AR subtype is localized in an intracellular pool. We observed that alpha2B AR subtype underwent agonist-induced internalization and seemed to follow the same endosomal pathway within 15 min used by beta2 AR. Agonist treatment of alpha2A and 2C AR subtypes results in some receptor internalization Less