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

使用绿色荧光蛋白对活细胞中的 α2 肾上腺素受体亚型进行实时成像

• 批准号: 13671605
• 负责人: MIZOBE Toshiki
• 金额: $ 2.56万
• 依托单位: Kyoto Prefectural University of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671605/
• 关键词: adrenergic receptor; green fluorescent protein (GFP)

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

背景和目标：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肾上腺素能受体胞内转运的研究仅限于免疫细胞化学分析。因此，我们研究了真实的时间亚型特异性贩运的α 2肾上腺素受体在活细胞中使用各自的融合蛋白的绿色荧光蛋白（GFP）和人α 2肾上腺素受体subtypes.Material和方法：使用重组DNA技术，每个α 2 A，B，和C肾上腺素受体亚型的C末端融合到N末端的GFP（α 2 AR-GFP）。通过双脱氧测序两条链来确认每个α 2 AR-GFP的DNA序列。对于放射性标记的配体结合试验，使用DEAE-dextram方法将克隆到Pcdna 3载体中的重组构建体瞬时转染COS-1细胞中。通过用不同浓度的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 ...更多信息 加入10 μ m萝芙木碱使用非线性最小二乘测量拟合技术（GraphPDA Software Inc.，San Diego，CA）。根据Lowry等人测定膜制备物的蛋白质含量。使用脂质体方法将重组构建体瞬时转染到HEK 293细胞中用于活细胞成像。我们使用连接到落射荧光显微镜的冷却CCD相机检查了响应于右美托咪啶的α 2 AR-GFP的运输的动态方式。结果和讨论：在稳态时，α 2A和2B AR亚型定位于质膜，尽管α 2C AR亚型的相当一部分定位于细胞内池。我们观察到α 2 B AR亚型经历激动剂诱导的内化，并且似乎在15分钟内遵循与β 2 AR相同的内体途径。α 2A和2C AR亚型的激动剂治疗导致一些受体内化

项目成果

MIZOBE T: "β adrenergic receptor knockout mouse and cardiovascular control"The Autonomic Nervous System. 37. 204-209 (2000)

MIZOBE T：“β肾上腺素能受体敲除小鼠和心血管控制”自主神经系统。 37. 204-209 (2000)

溝部俊樹: "βアドレナリン受容体ノックアウトマウスと血圧調整"自律神経. 37(2). 204-209 (2000)

Toshiki Mizobe：“β-肾上腺素受体敲除小鼠和血压调节”自主神经系统37(2)。

溝部俊樹: "アドレナリン受容体とノックアウトマウス(2)"麻酔. 50(1). 12-19 (2001)

Toshiki Mizobe：“肾上腺素受体和基因敲除小鼠（2）”麻醉50（1）。

Mizobe T. et al.: "Searching for structural similarity of beta 2 adrenoceptor to bacterior hodopsin or opioid M-receptor by making chimeric receptors"The management of acute and chronic pain. 65-70 (2000)

Mizobe T. 等人：“通过制造嵌合受体来寻找 β2 肾上腺素受体与细菌视紫红质或阿片类 M 受体的结构相似性”急性和慢性疼痛的治疗。

溝部俊樹: "βアドレナリン受容体ノックアウトマウスと血圧調節"自律神経. 37(2). 204-209 (2000)

Toshiki Mizobe：“β-肾上腺素受体敲除小鼠和血压调节”自主神经系统37(2)。