Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: Implications for nitric oxide signaling
Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: Implications for nitric oxide signaling
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DOI：
10.1073/pnas.93.13.6448
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发表时间：
1996-06-25
影响因子：
11.1
通讯作者：
Sessa, WC
中科院分区：
文献类型：
--
作者：
GarciaCardena, G;Oh, P;Sessa, WC
内皮型一氧化氮合酶(ENOS)的膜结合在血管内皮细胞一氧化氮(NO)的生物合成中起着重要作用。以前，我们已经证明，在培养的内皮细胞和完整的血管中，eNOS主要发现在细胞的核周区和质膜的离散区域，这表明蛋白质从高尔基体运输到特殊的质膜结构。在这里，我们发现eNOS存在于培养的牛主动脉内皮细胞制备的Triton X-100不溶性膜中，并与小窝的外壳蛋白小窝蛋白在培养的牛肺微血管内皮细胞中共定位。为了验证eNOS是否真的存在于小窝中，我们直接从完整的、灌流的大鼠肺中提纯了管腔内皮细胞质膜及其小窝。内皮型一氧化氮合酶存在于腔内质膜中，在纯化的小窝中明显富含eNOS。由于内皮型一氧化氮合酶的棕榈酰化并不显著影响其膜结合，我们接下来研究了这种修饰是否会影响内皮型一氧化氮合酶的靶向性，野生型内皮型一氧化氮合酶，而不是棕榈酰化突变型的内皮型一氧化氮合酶在NIH3T3细胞中与小凹蛋白共定位于细胞表面，证明了内皮型一氧化氮合酶的棕榈酰化是其靶向小凹的必要条件。这些数据表明，eNOS对小窝的亚细胞靶向可以将NO信号限制在细胞表面有限的微环境中的特定靶点，并可能影响小窝的信号转导。
The membrane association of endothelial nitric oxide synthase (eNOS) plays an important role in the biosynthesis of nitric oxide (NO) in vascular endothelium. Previously, we have shown that in cultured endothelial cells and in intact blood vessels, eNOS is found primarily in the perinuclear region of the cells and in discrete regions of the plasma membrane, suggesting trafficking of the protein from the Golgi to specialized plasma membrane structures. Here, we show that eNOS is found in Triton X-100-insoluble membranes prepared from cultured bovine aortic endothelial cells and colocalizes with caveolin, a coat protein of caveolae, in cultured bovine lung microvascular endothelial cells as determined by confocal microscopy. To examine if eNOS is indeed in caveolae, we purified luminal endothelial cell plasma membranes and their caveolae directly from intact, perfused rat lungs. eNOS is found in the luminal plasma membranes and is markedly enriched in the purified caveolae. Because palmitoylation of eNOS does not significantly influence its membrane association, we next examined whether this modification can affect eNOS targeting to caveolae, Wild-type eNOS, but not the palmitoylation mutant form of the enzyme, colocalizes with caveolin on the cell surface in transfected NIH 3T3 cells, demonstrating that palmitoylation of eNOS is necessary for its targeting into caveolae. These data suggest that the subcellular targeting of eNOS to caveolae can restrict NO signaling to specific targets within a limited microenvironment at the cell surface and may influence signal transduction through caveolae.