CONTROL OF SECRETION BY SMALL GTP-BINDING PROTEINS

描述：(根据申请者的抽象和具体目标改编。) 受刺激的胞吐作用是细胞控制的一个关键调控过程 将信号和效应器分子释放到环境中， 并控制其表面膜的组成。它有 最近发现，RAB中与RAS相关的小GTP酶 家族在细胞膜中起着无处不在的关键作用 转移，包括胞吐作用。像其他家庭成员一样 扩展的GTP酶超家族，Rab蛋白已被发现具有 可预测的生化活动，如受调控的核苷酸 交换和受调控的GTPase活性。这些生化活动 为确定相关联的 蛋白质。这个应用程序的中心假设是 大鼠嗜碱性粒细胞白血病(RBL)细胞中假定的胞外Rab，Rab3“e”， 可以作为一种生化和分子遗传学工具来发现 RBL细胞中受调节的胞外机的其他组件。这个 目的：1)从RBL细胞文库中克隆Rab3“e”基因； 2)建立肺泡T2中Rab3“e”的细胞特异性定位 细胞和RBL细胞的原位杂交和Northern印迹分析 3)建立了Rab3“e”的亚细胞定位。 T2细胞和RBL细胞的表位标记构建物的表达 在RBL细胞中表达，在T2细胞和RBL中表达 使用抗肽抗血清和Rab3“e”特异性抗完整蛋白的细胞 4)从T2细胞中建立Rab3“e”的分子功能； 来自RBL细胞通过丧失胞外功能调节胞吐 通过显性抑制突变体的表达和利用 RBL细胞中的反义寡核苷酸；5)结构/功能分析 用瞬变和稳定方法研究RBL细胞中Rab3“e”的相互关系 突变体和嵌合体Rab3“e”的转化，并通过使用 来自“效应区”和其他结构域的多肽；6)利用RBL 细胞作为发现Rb_3“e”相关组分的系统 基于蛋白质生化分析的调节胞外机 核苷酸交换和GTPase活性；7)鉴定其他蛋白质， 它与Rab3“e”的相互作用不能用生化方法预测， 物理和遗传学方法；8)鉴定肺泡T2细胞 利用探针进行同源克隆的RBL胞外机同源基因 来源于RBL细胞。黄曲霉毒素的分子组成研究进展 调控的排胞机可能是理解和理解 在试图从药物上影响小鼠的分泌功能时 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.