Cell Biology of Bioactive Peptide Secretion

性状（由申请人提供）：分泌颗粒在不同成熟阶段需要非常不同的颗粒成分和膜成分。目前的数据表明，大量的信号发生在分泌颗粒膜上，而不是将这些高度专门化的细胞器与细胞的其他部分隔离和保护起来。肽酰胺化单加氧酶（PAM），一种颗粒膜蛋白的表达，引起细胞骨架组织的变化。已知的PAM胞质相互作用蛋白包括Trio和Kalirin，能够影响细胞骨架组织和基因表达的多功能Rho GDP/GTP交换因子（GEF）。提出的实验集中在PAM及其与Trio/Kalirin在垂体内分泌细胞中的相互作用。目的1通过检查降低Trio或Kalirin水平的垂体细胞中调节的分泌和细胞骨架组织来检验Trio和Kalirin发挥不同作用的假设。将Trio/Kalirin的单个Rho GEF结构域的能力与完整分子的能力进行比较。目的2探索Trio和Kalirin的非催化结构域的结构和功能作用。将探索Trio/Kalirin启动的途径，涉及Pak（一种已知调节细胞骨架组织的Rac活化激酶）和Cdk 5（一种已知影响β细胞和垂体前叶内分泌细胞的神经突生长和激素分泌的Pro定向激酶）。初步的数据表明，卡林和邻近的Pro丰富的序列的第一个SH 3结构域的分子内相互作用的调节作用，将追求使用结构域缺失和细胞渗透肽抑制剂。目的3将定义PAM胞质结构域中的分泌颗粒进入信号，并分离和表征PAM与其他蛋白质的复合物。将寻找与分泌颗粒膜结合依赖于内腔pH值的细胞溶质蛋白，并评价Trio/Kalirin在组织信号传导途径中的支架作用。随着对分泌颗粒与周围胞质溶胶沟通的一些方式的机械理解，可以寻求控制激素分泌的治疗手段。

DESCRIPTION (provided by applicant): Secretory granules require, at different stages of maturity, very different granule constituents and membrane components. Instead of regarding these highly specialized organelles as sequestered and protected from the rest of the cell, current data suggest that substantial signaling occurs across secretory granule membranes. Expression of the peptide amidating monooxygenase (PAM), a granule membrane protein, causes changes in cytoskeletal organization. Known PAM cytosolic interactor proteins include Trio and Kalirin, multifunctional Rho GDP/GTP exchange factors (GEFs) capable of affecting cytoskeletal organization and gene expression. The experiments proposed focus on PAM and its interactions with Trio/Kalirin in pituitary endocrine cells. Aim 1 tests the hypothesis that Trio and Kalirin play different roles by examining regulated secretion and cytoskeletal organization in pituitary cells with reduced levels of either Trio or Kalirin. The capabilities of the individual Rho GEF domains of Trio/Kalirin will be compared to those of the intact molecules. Aim 2 explores the structure and functional roles of the non-catalytic domains of Trio and Kalirin. Trio/Kalirin-initiated pathways involving Pak, a Rac-activated kinase known to modulate cytoskeletal organization, and Cdk5, a Pro-directed kinase known to affect neurite outgrowth and hormone secretion from beta-cells and anterior pituitary endocrine cells, will be explored. Preliminary data demonstrating a regulatory role for intramolecular interactions of the first SH3 domain of Kalirin and neighboring Pro-rich sequences will be pursued using domain deletions and cell permeant peptide inhibitors. The secretory granule entry signal in the cytosolic domain of PAM will be defined in Aim 3 and complexes of PAM with other proteins will be isolated and characterized. Cytosolic proteins whose binding to secretory granule membranes is dependent on lumenal pH will be sought and a scaffolding role for Trio/Kalirin in organizing signaling pathways will be evaluated. With a mechanistic understanding of some of the ways in which secretory granules communicate to the surrounding cytosol, therapeutic means of controlling hormone secretion can be sought.