CFTR and interacting proteins from shark rectal gland

鲨鱼直肠腺的 CFTR 和相互作用蛋白

• 批准号: 6440235
• 负责人: JOHN R RIORDAN
• 金额: $ 15.7万
• 依托单位: MAYO CLINIC ARIZONA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6440235
• 关键词: chloride channels; crystallization; mass spectrometry; protein isoforms; protein protein interaction; protein purification; protein structure; proteomics; salt gland; sharks; western blottings

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.

描述（由申请人提供）： 囊性纤维化跨膜电导调节剂（CFTR）糖蛋白 囊性纤维化患者中突变的基因的产物是该基因的成员 最大的已知膜蛋白类别ABC（腺嘌呤核苷酸结合） 盒式）超家族。但是，CFTR是一种新型的ABC蛋白，是唯一的ABC蛋白 已知是离子通道。尽管有很多辩论，但 现在普遍同意，ABC蛋白结构组织具有 在进化上如此成功的进化也被改编在后生动物中以形成 由磷酸化和核苷酸调节的上皮氯化物通道 特征ABC核苷酸结合结构域（NBD）的相互作用。一个 阐明更快进步的主要障碍 CFTR蛋白的结构功能关系缺乏 蛋白质的丰富自然或重组来源。这 在杂哺乳动物细胞表达中可以达到的表达水平 系统比其他ABC低的数量级要高 蛋白质（例如P-糖蛋白）为低分辨率3维的蛋白质 通过二维的电子衍射获得了结构 晶体。我们以前已经纯化和重构了CFTR 昆虫细胞但强烈汇总的趋势阻碍了获得的尝试 结晶阵列。我们最近发现，本机CFTR可能更 很容易从鲨鱼直肠腺上纯化，这是唯一可以从 可以获得大量蛋白质。因此第一个 本项目的具体目的是进一步探索 纯化足够数量的功能CFTR以实现结晶 试验和结构确定。第二个相关的特定目的是 确定其他较大的氯化物的分子身份 直肠腺管的顶膜中的CFTR存在的通道 细胞。这一目标将根据两个不同的假设实现： 该较大的电导通道可能是成员的同工型 CIC渠道家族或该家族，由于它被循环放大器激活，因此可能 直接与CFTR相互作用。这两个假设都可能 抓住。但是，证明了采用此事的可行性 这些目的之一 应该有助于提高对CFTR功能的机制的理解 并控制氯化物分泌。