CELL BIOLOGY OF BIOACTIVE PEPTIDE SECRETION

生物活性肽的生产涉及前体、酶、在分泌颗粒中的储存、颗粒到释放位点的路径和调节释放的机制。肽甘氨酸酰胺化单加氧酶（PAM）是为数不多的跨越颗粒膜的肽加工酶之一。在之前的资助期间，我们发现PAM的表达影响at -20促皮质性肿瘤细胞中黑色素原皮质素（POMC）和激素原转化酶1 （PC1）的运输和细胞骨架的组织。Kalirin是GTP小结合蛋白的GDP/GTP交换因子Dbl家族的新成员，通过其与PAM细胞质结构域的路径决定因子相互作用而被鉴定出来。at -20细胞中Kalirin的表达改变了细胞骨架组织和POMC运输。通过探索PAM的管腔结构域与颗粒含量蛋白的相互作用，以及加里林介导的PAM与细胞质成分的相互作用，我们将了解管腔环境的状态是如何与相关的细胞质因子沟通的。目的1是评估管腔和细胞质相互作用在PAM和POMC运输中的相互作用，以及细胞骨架的改变。我们将使用可诱导表达PAM的单个结构域的at -20细胞来鉴定参与运输和细胞骨架组织的结构域。目的2是确定Kalirin与PAM等蛋白相互作用的功能意义。将寻找垂体中的加里林同源物。Kalirin与分泌途径和细胞骨架相互作用的机制将通过完整的和通透的at -20细胞和垂体原代培养来检查。目的3是确定PAM胞质结构域控制其与加里林相互作用的特征。不能与Kalirin相互作用的膜PAM蛋白将用于研究PAM影响调节分泌的机制。目的4是确定加里林及其同源物的功能域。Kalirin与PAM细胞质结构域相互作用所必需的光谱样区域将被描绘出来，GDP/GTP交换因子结构域的活性将被评估。将鉴定Kalirin的pleckstrin同源和src同源3结构域的结合相互作用。

Production of bioactive peptides involves precursors, enzymes, storage in secretory granules, routing of granules to release sites and the machinery for regulated release. Peptidyiglycine alpha-Amidating Monooxygenase (PAM) is one of the few peptide processing enzymes spanning the granule membrane. During the previous grant period we found that expression of PAM affects trafficking of proopiomelanocortin (POMC) and prohormone convertase 1 (PC1) and organization of the cytoskeleton in AtT-20 corticotrope tumor cells. Kalirin, a new member of the Dbl family of GDP/GTP exchange factors for small GTP binding proteins, was identified by its interaction with routing determinants in the cytosolic domain of PAM. Expression of Kalirin in AtT-20 cells alters cytoskeletal organization and POMC trafficking. By exploring interactions of the lumenal domains of PAM with granule content proteins, and the Kalirin-mediated interactions of PAM with cytosolic components, we will develop an understanding of how the status of the lumenal milieu is communicated to relevant cytosolic factors. Aim 1 is to evaluate the interplay of lumenal and cytosolic interactions in the trafficking of PAM and POMC, and in alterations in the cytoskeleton. We will use AtT-20 cells exhibiting inducible expression of individual domains of PAM to identify domains involved in trafficking and cytoskeletal organization. Aim 2 is to determine the functional significance of the interaction of Kalirin with PAM and other proteins. A Kalirin homologue in pituitary will be sought. The mechanisms through which Kalirin interacts with the secretory pathway and the cytoskeleton will be examined using intact and permeabilized AtT-20 cells and primary pituitary cultures. Aim 3 is to identify features of the PAM cytosolic domain governing its interactions with Kalirin. Membrane PAM proteins unable to interact with Kalirin will be used to study the mechanism through which PAM affects regulated secretion. Aim 4 is to define the functional domains of Kalirin and its homologues. The spectrin-like region essential for the interaction of Kalirin with the cytosolic domain of PAM will be delineated and the activity of the GDP/GTP exchange factor domain will be assessed. Binding interactions of the pleckstrin homology and src homology 3 domains of Kalirin will be identified.