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由以及用于调节释放的机制。 肽基甘氨酸α-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。 在先前的资助期间，我们发现PAM的表达影响AtT-20促肾上腺皮质激素肿瘤细胞中阿黑皮素原（POMC）和激素原转化酶1（PC 1）的运输以及细胞骨架的组织。 Kalirin是Dbl家族中的一个新成员，它与PAM胞质结构域中的路由决定簇相互作用，从而确定了小GTP结合蛋白的GDP/GTP交换因子。 Kalirin在AtT-20细胞中的表达改变细胞骨架组织和POMC运输。 通过探索PAM与颗粒内容物蛋白质的内腔结构域的相互作用，以及Kalirin介导的PAM与胞质组分的相互作用，我们将了解内腔环境的状态如何与相关的胞质因子进行通信。 目的1是评估在PAM和POMC的运输中，以及在细胞骨架的改变中，内腔和胞质相互作用的相互作用。 我们将使用AtT-20细胞表现出诱导表达的个别领域的PAM，以确定参与运输和细胞骨架组织的领域。 目的2是确定Kalirin与PAM和其他蛋白质相互作用的功能意义。 将寻找垂体中的Kalirin同系物。 Kalirin与分泌途径和细胞骨架相互作用的机制将使用完整和透化的AtT-20细胞和原代垂体培养物进行检查。 目的3是确定PAM胞质结构域的特点，它与Kalirin的相互作用。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Kalirin及其同系物的功能结构域。 将描绘Kalirin与PAM胞质结构域相互作用所必需的血影蛋白样区域，并评估GDP/GTP交换因子结构域的活性。 将鉴定Kalirin的pleckstrin同源性和src同源性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.