DISSECTION OF CAPS FUNCTION IN CHROMAFFIN CELL SECRETION

描述（改编自申请人的描述）：激素的分泌和 神经递质是一个多步骤的过程， 与它们的目标细胞进行交流。两种类型的分泌囊泡，小 清晰的突触囊泡（SSV）和大的致密核心囊泡（DCV）， 参与分泌过程。虽然它们有几个共同的特点， 在这两种囊泡类型的胞吐作用之间存在许多差异。 PI实验室的重点是分泌周期， 含儿茶酚胺的DCV。因此，我们有兴趣澄清 区分DCV和SSV胞吐作用的分子机制。最近 CAPS（Ca2+依赖性分泌激活蛋白） 作为一种145 kDa的蛋白质，似乎对DCV分泌途径具有特异性。 我们的初步数据表明，CAPS拮抗作用对细胞增殖产生了深刻的抑制作用。 对小牛肾上腺嗜铬细胞分泌儿茶酚胺的影响。其他 在透化的哺乳动物细胞和突触体中的研究表明，CAPS起着重要的作用， 在DCV而不是SSV的分泌中起作用。在本申请中，我们提出 详细分析CAPS在DCV分泌中的作用。有四个具体的 目的：（1）确定CAPS调控的胞吐途径的阶段 在生理条件下。我们将使用不同的协议来隔离 分泌的最后一步，并确定CAPS拮抗作用对此的影响 步骤，然后确定CAPS是否参与分泌的其他阶段， 级联。(2)确定CAPS是否是DCV胞吐的Ca2+传感器， 研究CAPS与其他候选钙传感器的关系， 突触结合蛋白(3)研究CAPS的PH结构域在其 功能(4)评估CAPS功能是否受到PKC依赖性 磷酸化，并确定这是否在PKC增强 DCV分泌。为了实现这些目标，我们将使用多学科 包括生物化学、分子和单细胞的方法 嗜铬细胞的电生理方法。技术如 电容和安培记录，这是建立在PI的 实验室，将用于解剖分泌动力学与毫秒 解析并精确定位CAPS在胞吐过程中的作用位点。 这个项目将加强我们对CAPS在胞吐中的功能的理解， 阐明了DCV和SSV分泌之间的关键差异之一， 途径。

DESCRIPTION (Adapted from Applicant's Description): Secretion of hormones and neurotransmitters is a multistep process by which endocrine cells and neurons communicate with their target cells. Two types of secretory vesicles, small clear synaptic vesicles (SSVs) and large dense-core vesicles (DCVs), are involved in the secretory process. While they share several common features, a number of differences exist between the exocytosis of these two vesicle types. The focus of the PI's laboratory is the secretory cycle of catecholamine-containing DCVs. As such, we are interested in the elucidation of the molecular machinery that distinguishes DCV and SSV exocytosis. Recent studies have identified CAPS (Ca2+-dependent Activator Protein for Secretion) as a 145 kDa protein that appears to be specific for the DCV secretory pathway. Our preliminary data show that CAPS antagonism exerts a profound inhibitory effect on catecholamine secretion in calf adrenal chromaffin cells. Other studies in permeabilized mammalian cells and synaptosomes show that CAPS plays a role in secretion by DCVs but not SSVs. In this application we propose to analyze in detail the role of CAPS in DCV secretion. There are four specific aims: (1) Determine the stage(s) of the exocytotic pathway regulated by CAPS under physiological conditions. We will use different protocols to isolate the final step of secretion and determine the effect of CAPS antagonism on this step, then determine if CAPS is involved at other stages of the secretory cascade. (2) Determine if CAPS is a Ca2+-sensor for DCV exocytosis and investigate the relationship of CAPS to other candidate Ca sensors like synaptotagmin. (3) Investigate the role of the PH domain of CAPS in its function. (4) Assess whether CAPS function is affected by PKC-dependent phosphorylation and determine if this plays a role in the PKC augmentation of DCV secretion. To accomplish these aims we will use a multidisciplinary approach that includes biochemical, molecular and single-cell electrophysiological methods in chromaffin cells. Techniques such as capacitance and amperometric recordings, that are well-established in the PI's laboratory, will be used to dissect secretory kinetics with millisecond resolution and pinpoint the site of action of CAPS in the exocytotic process. This project will enhance our understanding of CAPS function in exocytosis and illuminate one of the critical differences between the DCV and SSV secretory pathways.