CFTR and interacting proteins from shark rectal gland

描述（由申请人提供）： 囊性纤维化跨膜传导调节因子（CFTR）糖蛋白 囊性纤维化患者中基因突变的产物是 已知最大的一类膜蛋白，ABC（腺嘌呤核苷酸结合 盒式磁带）超家族。然而，CFTR是一种新的ABC蛋白， 已知是离子通道。虽然有很多争论，但 现在普遍认为，ABC蛋白质的结构组织， 在进化上如此成功，也适应了后生动物， 磷酸化和核苷酸调控的上皮氯离子通道 在特征性ABC核苷酸结合结构域（NBD）处的相互作用。一 这是更快地阐明 CFTR蛋白质的结构功能关系一直缺乏 用于纯化的蛋白质的丰富天然或重组来源。的 在异种哺乳动物细胞表达中可达到的表达水平 系统比其他ABC系统低一个数量级以上 蛋白质如P-糖蛋白，其低分辨率3维 通过二维电子衍射获得了结构 晶体我们之前已经纯化并重建了从 昆虫细胞，但聚集的强烈倾向阻碍了获得 晶体阵列我们最近发现，天然CFTR可以更 很容易从鲨鱼的直肠腺中提纯出来， 可以获得显著量的蛋白质。因此第一 本项目的具体目标是进一步探讨 纯化足够量的功能性CFTR以能够结晶 试验和结构确定。第二个相关的具体目标是 确定另一种电导率较大的氯化物的分子身份 通道与CFTR一起存在于直肠腺管的顶膜中 细胞这一目标将根据两个不同的假设来实现： 或者该较大的传导通道可以是 CIC通道家族，或者由于它被环AMP激活，它可以 直接与CFTR互动。这两种假设都可能 稍等然而，使用这种方法的可行性证明 用于这些目的高度特化的非哺乳动物模型组织 应该有助于促进对CFTR运作机制的理解 并控制氯化物分泌。

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein product of the gene mutated in patients with cystic fibrosis is a member of the largest known class of membrane proteins, the ABC (adenine nucleotide binding cassette) superfamily. However, CFTR is a novel ABC protein as the only one known to be an ion channel. Although there has been considerable debate, it is now generally agreed that the ABC protein structural organization which has been so successful evolutionarily was also adapted in metazoa to form an epithelial chloride channel regulated by phosphorylation and by nucleotide interactions at the characteristic ABC nucleotide binding domains (NBDs). A major impediment to more rapid advances in elucidating the structure‑function relationships of the CFTR protein has been the lack of a rich natural or recombinant source of the protein for purification. The levels of expression achievable in heterolgous mammalian cell expression systems is more than an order of magnitude lower than that of other ABC proteins such as P-glycoprotein for which a low resolution 3-dimensional structure has been obtained by electron diffraction of two dimensional crystals. We have previously purified and reconstituted CFTR expressed from insect cells but a strong tendency to aggregate has hindered attempts to obtain crystalline arrays. We have more recently found that native CFTR can be more readily purified from shark rectal gland, the only accessible solid tissue from which significant amounts of the protein can be obtained. Therefore the first specific aim of the present project is to further explore the feasibility of purifying sufficient quantities of functional CFTR to enable crystallization trials and structure determination. The second related specific aim is to determine the molecular identity of the other larger conductance chloride channel present with CFTR in the apical membranes of the rectal gland tubular cells. This objective will be pursued on the basis of two distinct hypotheses: either that this larger conductance channel may be an isoform of a member of the CIC channel family or that, since it is activated by cyclic AMP, it may interact directly with CFTR. It is possible that both of these hypotheses may hold. However, demonstration of the feasibility of employing this highly-specialized non-mammalian model tissue for either of these purposes should serve to advance understanding of the mechanism whereby CFTR functions and controls chloride secretion.