Lopez-Ilasaca Marco, Liu Xiushi, Tamura Koichi, Dzau Victor J
Cardiovascular Research Laboratories, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Biol Cell. 2003 Dec;14(12):5038-50. doi: 10.1091/mbc.e03-06-0383. Epub 2003 Sep 5.
Our group identified angiotensin II type 1 (AT1) receptor-associated protein (ATRAP) in a yeast two-hybrid screen for proteins that bind to the carboxyl-terminal cytoplasmic domain of the AT1. In this work, we characterize ATRAP as a transmembrane protein localized in intracellular trafficking vesicles and plasma membrane that functions as a modulator of angiotensin II-induced signal transduction. ATRAP contains three hydrophobic domains at the amino-terminal end of the protein, encompassing the amino acid residues 14-36, 55-77, and 88-108 and a hydrophilic cytoplasmic carboxyl-terminal tail from residues 109-161. Endogenous and transfected ATRAP cDNA shows a particulate distribution; electron microscopy reveals the presence of ATRAP in prominent perinuclear vesicular membranes; and colocalization analysis by immunofluorescence shows that ATRAP colocalizes in an intracellular vesicular compartment corresponding to endoplasmic reticulum, Golgi, and endocytic vesicles. Real-time tracking of ATRAP vesicles shows constitutive translocation toward the plasma membrane. Using epitope-tagged forms of ATRAP at either the amino or carboxyl end of the molecule, we determined the orientation of the amino end as being outside the cell. Mutant forms of ATRAP lacking the carboxyl end are unable to bind to the AT1 receptor, leading to the formation of prominent perinuclear vesicle clusters. Functional analysis of the effects of ATRAP on angiotensin II-induced AT1 receptor signaling reveals a moderate decrease in the generation of inositol lipids, a marked decrease in the angiotensin II-stimulated transcriptional activity of the c-fos promoter luciferase reporter gene, and a decrease in cell proliferation.
我们的研究小组在酵母双杂交筛选中，发现了与血管紧张素II 1型（AT1）受体羧基末端胞质结构域结合的蛋白，即AT1受体相关蛋白（ATRAP）。在本研究中，我们将ATRAP鉴定为一种跨膜蛋白，定位于细胞内运输囊泡和质膜中，作为血管紧张素II诱导信号转导的调节剂发挥作用。ATRAP在蛋白的氨基末端包含三个疏水结构域，涵盖氨基酸残基14 - 36、55 - 77和88 - 108，以及一个从残基109 - 161开始的亲水性胞质羧基末端尾巴。内源性和转染的ATRAP cDNA呈现颗粒状分布；电子显微镜显示ATRAP存在于明显的核周囊泡膜中；免疫荧光共定位分析表明，ATRAP与对应于内质网、高尔基体和内吞囊泡的细胞内囊泡区室共定位。对ATRAP囊泡的实时追踪显示其向质膜的组成型转运。通过在分子的氨基或羧基末端使用表位标记形式的ATRAP，我们确定氨基末端位于细胞外。缺乏羧基末端的ATRAP突变体形式无法与AT1受体结合，导致形成明显的核周囊泡簇。对ATRAP对血管紧张素II诱导的AT1受体信号转导影响的功能分析表明，肌醇脂质生成适度减少，血管紧张素II刺激的c-fos启动子荧光素酶报告基因的转录活性显著降低，细胞增殖减少。
