Signaling Mechanism in Salivary Gland Cells

唾液腺细胞的信号传导机制

• 批准号: 6516635
• 负责人: Shmuel Muallem
• 金额: $ 37.05万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-15 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6516635
• 关键词: biological signal transduction; calcium flux; cell membrane; endoplasmic reticulum; laboratory mouse; membrane channels; protein protein interaction; receptor expression; receptor sensitivity; salivary glands; tissue /cell culture

DESCRIPTION (provided by applicant): The polarized nature of salivary gland functions requires polarized organization and function of signaling complexes. Understanding assembly of signaling proteins into complexes within cellular microdomains is the central theme of this proposal. Based on our preliminary findings of polarized expression of Ca2+ signaling proteins in secretory cells, expression of multiple P2Rs in a membrane limited manner in salivary gland cells, regulation of GPCR signaling by RGS proteins and the regulation of Icrac channels by IP3R we will test the central hypothesis by 1. Studying the spatial features of Ca2+ signaling by P2Rs and GPCR. Imaging of Fura2, BTC and Mg-Fura2 will be used to capture Ca2+ waves and gradients in SMG cells and correlate them with expression patterns of P2Rs and Ca2 about signaling complexes. Function of individual P2Rs will be probed further by characterizing ionic current activated by ATP and by partial P2R agonist and the sensitivity of these currents to P2Rs inhibitory Abs. 2. Determine interaction of RGS proteins with GPCR. This will be achieved by measuring the potency of several RGS proteins to inhibit Ca2+ signaling evoked by GPCR in SMG cells. RGS proteins specific antibodies and dominant negatives will be used to identify the active RGS proteins in SMG cells and their physiological role. Known constitutively active mutants of the alfa-1BAR will be used in an attempt to identify the site of interaction of RGS proteins with GPCR. 3. Explore Ca2+ signaling complexes at the PM/ER junction: icrac-IP3R complexes. Determine if Imin is Icrac and characterize single channel properties of Icrac in excised patches from native SMG cells. Determine whether Icrac in SMG cells is gated by the newly discovered conformational coupling mode. Study regulation of Icrac by the Homer family of scaffolding proteins in the context of their role in assembly of 1crac-IP3R and signaling complexes. I believe that the tools available in my lab for the proposed projects, the expertise of the personnel in my lab and the collaborations we established will allow us to achieve our goals and provide insights of general relevance to cell signaling and to the function of salivary gland cells.

描述（由申请人提供）：唾液腺的两极分化性质 功能需要信号复合物的两极分化组织和功能。 了解信号蛋白将信号蛋白组装成细胞内的复合物 微域是该提案的中心主题。基于我们的初步 分泌细胞中Ca2+信号蛋白极化表达的发现， 在唾液腺中以膜有限的方式表达多个P2RS 细胞，RGS蛋白对GPCR信号的调节以及ICRAC的调节 IP3R的渠道我们将通过1。研究空间假设。 P2RS和GPCR的Ca2+信号传导的特征。 Fura2，BTC和MG-Fura2的成像 将用于捕获SMG细胞中的Ca2+波和梯度并相关 它们具有P2RS和CA2的表达模式，涉及信号复合物。 通过表征离子，将进一步探测单个P2R的功能 电流由ATP和部分P2R激动剂激活以及 这些流向P2RS抑制性ABS。 2。确定RGS蛋白的相互作用 与GPCR。这将通过测量几个RG的效力来实现 蛋白质抑制SMG细胞中GPCR引起的Ca2+信号传导。 RGS蛋白 特定的抗体和主要的负面因素将用于识别活动 SMG细胞中的RGS蛋白及其生理作用。构成众所周知 ALFA-1BAR的活动突变体将用于识别该站点 RGS蛋白与GPCR的相互作用。 3。探索CA2+信号传导复合物 在PM/ER连接处：ICRAC-IP3R复合物。确定Imin是否是ICRAC，​​并且 表征来自天然的切除贴片中ICRAC的单个通道特性 SMG细胞。确定SMG细胞中的ICRAC是否被新的 发现的构象耦合模式。荷马对ICRAC的研究调节 脚手架蛋白质家族在它们在组装中的作用的背景下 1CRAC-IP3R和信号传导复合物。我相信我的工具可用 拟议项目的实验室，我的实验室人员的专业知识和 我们建立的合作将使我们能够实现我们的目标并提供 与细胞信号传导和唾液功能的一般相关性的见解 腺细胞。