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首页> 外文期刊>Life sciences >Subtype-specific differences in subcellular localization and chlorethylclonidine inactivation of alpha1-adrenoceptors.
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Subtype-specific differences in subcellular localization and chlorethylclonidine inactivation of alpha1-adrenoceptors.

机译：亚型特异性差异在α1-肾上腺素受体的亚细胞定位和氯乙基可乐定灭活中。

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摘要

Chlorethylclonidine (CEC) inactivation has been used as one criterion to subclassify the alpha1-adrenoceptors (AR); however, the extent of CEC inactivation can vary depending on the CEC treatment. By constructing the FLAG-tagged (N-terminus) and green fluorescent protein (GFP)-fused (C-terminus) alpha1-ARs, we have determined the relationship between CEC sensitivity and the cellular localization of alpha1-AR subtypes using COS-7 cells. In GFP-expressing cells, flow cytometry analysis with anti-FLAG N-terminus antibody detected strong fluorescent signals in most of alpha1B-AR-expressing cells, but low signals in alpha1A-AR-expressing cells. Further examination with confocal microscopy showed that fluorescent signals densely localized intra-cellularly in alpha1A-AR-expressing cells, while most of alpha1B-AR localized on the cell surface. Furthermore, radioligand binding studies with [125I]HEAT showed that CEC (10 microM) treatment of intact cells inactivated approximately 30-40% of alpha1A-AR and >90% of alpha1B-AR, while the CEC treatment of membrane preparations resulted in >80% decrease in the alpha1A-AR density and >90% of alpha1B-AR density, respectively. The results showed that the hydrophilic alkylating agent CEC inactivated only alpha1-AR on the cell surface irrespective of its subtype, and that the subtype-specific sorting is a major determinant for CEC inactivation of alpha1-AR. Subtype-specific cellular localization suggests a new class of functional properties that may explain the signal and functional diversity of homologous alpha1-AR (as well as other G protein-coupled receptors) subtypes.
机译：氯乙基可乐定（CEC）失活已被用作对α1-肾上腺素能受体（AR）进行亚分类的标准。但是，CEC灭活的程度可能会因CEC治疗而异。通过构建标记FLAG（N末端）和绿色荧光蛋白（GFP）融合（C末端）的alpha1-ARs，我们已经确定了COS-7使用COS-7的CEC敏感性与alpha1-AR亚型细胞定位之间的关系。细胞。在表达GFP的细胞中，用抗FLAG N末端抗体进行的流式细胞仪分析在大多数表达alpha1B-AR的细胞中检测到强荧光信号，但是在表达alpha1A-AR的细胞中检测到低信号。共聚焦显微镜的进一步检查表明，荧光信号密集地定位在表达α1A-AR的细胞内，而大多数α1B-AR则定位在细胞表面。此外，用[125I] HEAT进行的放射性配体结合研究表明，完整细胞的CEC（10 microM）处理可灭活约30-40％的alpha1A-AR和> 90％的alpha1B-AR，而膜制剂的CEC处理可导致> alpha1A-AR密度分别降低了80％，而alpha1B-AR密度则降低了90％以上。结果表明，亲水性烷基化剂CEC不管其亚型都仅使细胞表面的alpha1-AR失活，并且亚型特异性分选是α1-AR的CEC失活的主要决定因素。亚型特异性细胞定位表明一类新的功能特性，可以解释同源的α1-AR（以及其他G蛋白偶联受体）亚型的信号和功能多样性。

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