Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease.
Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease.
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从内皮丰富的来源分离的富含小窝素膜结构域的表征：对人类疾病的影响。
DOI：
10.1083/jcb.126.1.111
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发表时间：
1994-07
影响因子：
7.8
通讯作者：
Sargiacomo, M
中科院分区：
文献类型：
--
作者：
Lisanti, M P;Scherer, P E;Vidugiriene, J;Tang, Z;Hermanowski-Vosatka, A;Tu, Y H;Cook, R F;Sargiacomo, M
小窝是50-100 nm的膜微域，代表质膜的一个亚室。以前的形态学研究表明，小凹涉及：(A)大分子(包括低密度脂蛋白和修饰的低密度脂蛋白)穿过毛细血管内皮细胞；(B)通过涉及GPI连接的受体分子和未知的阴离子转运蛋白的称为细胞吞噬作用的过程摄取小分子；(C)与基于肌动蛋白的细胞骨架相互作用；以及(D)某些信号分子的区域化，包括G蛋白偶联受体。小窝蛋白是一种22kD的完整膜蛋白，是小窝的重要结构成分，在劳斯肉瘤病毒转化细胞中首次被确定为主要的v-Src底物。这一发现最初暗示了小窝蛋白、跨膜信号和细胞转化之间的关系。我们最近开发了一种从培养细胞中分离富含小窝蛋白的膜域的方法。为了便于生化操作，我们将这一程序应用于肺组织--一种富含内皮和小窝蛋白的来源--允许大规模制备这些复合体。这些膜结构域保留了大约85%的小窝蛋白和大约55%的GPI连接的标记蛋白，而它们排除了-gt；或=98%的完整质膜蛋白标记和=99.6%的其他细胞器特异的膜标记。通过微测序和免疫印迹对这些复合体的表征揭示了已知的修饰形式的低密度脂蛋白(清道夫受体：CD36和RAGE)、多个GPI连接蛋白、阴离子转运体(质膜孔蛋白)、细胞骨架元件和细胞质信号分子的受体-包括Src样激酶、异源三聚体G蛋白和Rap家族的三个成员(Rap 1-Ras肿瘤抑制蛋白、Rap 2和TC21)。这些复合体中至少有一部分肌动蛋白出现单体(G-肌动蛋白)，提示这些结构域可能是微丝在信号转导过程中成核/组装的膜结合部位。鉴于这些蛋白质中的大多数是已知分子，我们目前的研究为评估体内这些相互作用提供了系统的基础。
Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G- protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue--an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno- blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules--including Src-like kinases, hetero- trimeric G-proteins, and three members of the Rap family of small GTPases (Rap 1--the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G- actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.