CONTROL OF SECRETION BY SMALL GTP-BINDING PROTEINS

描述：（改编自申请人的摘要和具体目标。） 受刺激的胞吐作用是细胞调控的关键过程 信号和效应分子释放到环境中， 并控制其表面膜的组成。 它有 最近变得明显，小Ras相关的GTP酶的拉布 家族在细胞膜上起着普遍而关键的作用 包括胞吐作用。 像其他家庭成员一样， 已经发现Rab蛋白具有扩展的GT3超家族， 可预测的生物化学活性，如受调控的核苷酸 交易所和受监管的GTTRUNK活动。 这些生化活动 提供了一个强有力的理由，以确定相关的 proteins. 本申请的中心假设是， 大鼠嗜碱性白血病（RBL）细胞中推定的胞吐性Rab，Rab 3“e”， 可以作为生物化学和分子遗传学的工具， RBL细胞中受调节的胞吐机器的其他组件。 的 具体目的是：1）从RBL细胞文库中克隆Rab 3“e”cDNA; 2)建立Rab 3“e”在肺泡T2中的细胞特异性定位 原位杂交和北方印迹法检测RBL细胞和RBL细胞的DNA 3）建立Rab 3“e”的亚细胞定位， 通过表位标记的构建体的表达从T2细胞和从RBL细胞 在RBL细胞中，以及在T2细胞和RBL细胞中通过免疫细胞化学 使用抗肽抗血清和Rab 3“e”特异性抗全蛋白的细胞 血清; 4）从T2细胞建立Rab 3“e”的分子功能， 从RBL细胞中通过丧失胞吐功能调节胞吐 通过显性抑制突变体的表达和使用 RBL细胞中的反义寡核苷酸; 5）分析结构/功能 RBL细胞中Rab 3“e”的关系，使用瞬时和稳定 突变体和嵌合Rab 3“e”的转染，以及通过使用 来自“效应”区和其他结构域的肽; 6）使用RBL 细胞作为发现Rab 3“e”-相关组分的系统， 使用基于蛋白质生物化学测定的受调节的胞吐机器 核苷酸交换和GTP酶活性; 7）鉴定其它蛋白质， 其与Rab 3“e”的相互作用无法通过生物化学预测， 物理和遗传学方法; 8）肺泡T2细胞鉴定 通过使用探针同源克隆RBL胞吐机器的同源物 来源于RBL细胞。 的分子组分的解析 受调节的胞吐机器可能是理解和 在试图阻止影响分泌功能的过程中， 肺泡T2细胞和组织肥大细胞。

DESCRIPTION: (Adapted from the applicant's abstract and Specific Aims.) Stimulated exocytosis is a key regulatory process by which cells control the release of signalling and effector molecules into their environment, and also control the composition of their surface membranes. It has recently become apparent that small Ras-related GTPases of the Rab family play a ubiquitous and critical role in intracellular membrane transfers, including exocytosis. Like other family members of the extended GTPase superfamily, Rab proteins have been found to possess predictable biochemical activities, such as regulated nucleotide exchange and regulated GTPase activities. These biochemical activities provide a powerful rationale for the identification of associated proteins. It is the central hypothesis of this application that the putative exocytic Rab in rat basophilic leukemia (RBL) cells, Rab3"e", can be used as a biochemical and molecular genetic tool to discover other components of the regulated exocytic machine in RBL cells. The Specific Aims are to: 1) clone Rab3"e" cDNA from an RBL cell library; 2) establish the cell-specific localization of Rab3"e" from alveolar T2 cells and from RBL cells by in situ hybridization and Northern blot hybridization; 3) establish the subcellular localization of Rab3"e" from T2 cells and from RBL cells by expression of epitope-tagged constructs of both in RBL cells, and by immunocytochemistry in T2 cells and RBL cells using anti-peptide antisera and Rab3"e"-specific anti-holoprotein sera; 4) establish the molecular function of Rab3"e" from T2 cells and from RBL cells in regulated exocytosis by loss of exocytic function through the expression of dominant suppressor mutants and the use of antisense oligonucleotides in RBL cells; 5) analyze structure/function relationships for Rab3"e" in RBL cells using transient and stable transfections of mutant and chimeric Rab3"e", and through the use of peptides from the "effector" region and other domains; 6) employ the RBL cell as a system for discovery of Rab3"e"-associated components of the regulated exocytic machine using protein biochemical assays based on nucleotide exchange and GTPase activities; 7) identify other proteins, whose interactions with Rab3"e" cannot be biochemically predicted, by physical and genetic methods; and 8) identify alveolar T2 cell homologues of the RBL exocytic machine by homology cloning using probes derived from RBL cells. Elucidation of the molecular components of the regulated exocytic machine may be an important step in understanding and in attempting to pharmacologically influence secretory function in alveolar T2 cells and tissue mast cells.