Cell Biology of Bioactive Peptide Secretion

描述(由申请人提供)：分泌颗粒在不同的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，而不是认为这些高度专门化的细胞器与细胞的其余部分隔离和保护。氨化单加氧酶(PAM)是一种颗粒膜蛋白，其表达导致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，以及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)。这些实验提出了重点研究PAM及其与Trio/Kalirin在垂体内分泌细胞中的相互作用。目的1通过检测TRIO或Kalirin水平降低的垂体细胞的调节分泌和细胞骨架组织，验证Trio和Kalirin发挥不同作用的假说。将Trio/Kalirin的单个Rho全环基金结构域的能力与完整分子的能力进行比较。目的研究Trio和Kalirin的非催化结构域的结构和功能。Trio/Kalirin启动的通路涉及Pak和CDK5，Pak是一种RAC激活的激酶，已知可以调节细胞骨架组织，而CDK5是一种Pro导向的激酶，已知会影响轴突生长和β细胞和垂体前叶内分泌细胞的激素分泌。初步数据表明Kalirin的第一个SH3结构域与邻近的Pro-Rich序列的分子内相互作用的调节作用将通过结构域缺失和细胞意思多肽抑制剂来进行。目标3中将定义PAM胞浆结构域中的分泌颗粒进入信号，并将分离和表征PAM与其他蛋白质的复合体。将寻找与分泌颗粒膜结合依赖于管腔pH的胞浆蛋白，并将评估Trio/Kalirin在组织信号通路中的支架作用。通过对分泌颗粒与周围胞浆沟通的一些机制的了解，可以寻找控制激素分泌的治疗方法。

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.