CFTR and interacting proteins from shark rectal gland

描述(由申请人提供)： 囊性纤维化跨膜电导调节因子糖蛋白 囊性纤维化患者基因突变的产物是 已知的最大的一类膜蛋白，ABC(腺核苷酸结合 卡带)超级家庭。然而，cftr是一个新的ABC蛋白，因为它是唯一的 已知是离子通道。尽管有相当多的争论，但它是 现在普遍认为ABC蛋白质结构组织中有 在进化上如此成功也被后生动物改编成了 磷酸化和核苷酸调节的上皮氯通道 特征的ABC核苷酸结合域(NBD)上的相互作用。一个 对更快地澄清这些问题的主要障碍 CFTR蛋白的结构和功能关系一直缺乏 用于纯化的丰富的天然或重组蛋白质来源。这个 异种哺乳动物细胞表达的可实现水平 系统比其他ABC系统低一个数量级以上 如P-糖蛋白等蛋白质的低分辨率三维 用二维电子衍射法得到了它的结构 水晶。我们之前已经纯化和重组了CFTR，从 昆虫细胞但强烈的聚集倾向阻碍了获得 水晶阵列。我们最近发现，本地CFTR可以比 容易从鲨鱼直肠腺中提纯，唯一可以接触到的固体组织来自 可以获得相当数量的蛋白质。因此第一个 本项目的具体目标是进一步探讨 提纯足够量的功能性CFTR以实现结晶 试验和结构确定。第二个相关的具体目标是 确定其他较大电导氯化物的分子同一性 直肠腺管顶膜中存在CFTR的通道 细胞。这一目标将以两个不同的假设为基础： 或者该较大的电导通道可以是 CIC通道家族，或者，由于它被循环AMP激活，它可以 与CFTR直接互动。这两个假设都有可能 等一下。然而，论证了采用这一技术的可行性 用于上述任一目的的高度专业化的非哺乳动物模型组织 应有助于增进对CFTR发挥作用的机制的理解 并控制氯化物的分泌。

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.