Signaling Mechanism in Salivary Gland Cells

唾液腺细胞的信号传导机制

• 批准号: 6634697
• 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/6634697
• 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）：唾液腺的极化性质 功能需要信号复合体的极化组织和功能。 了解信号蛋白在细胞内组装成复合物 微域是该提案的中心主题。根据我们的初步 分泌细胞中 Ca2+ 信号蛋白极化表达的发现， 唾液腺中多个 P2R 以膜限制方式表达 细胞、RGS 蛋白对 GPCR 信号传导的调节以及 Icrac 的调节 通过 IP3R 的通道，我们将通过 1. 研究空间来检验中心假设 P2R 和 GPCR 的 Ca2+ 信号传导特征。 Fura2、BTC 和 Mg-Fura2 的成像 将用于捕获 SMG 细胞中的 Ca2+ 波和梯度并进行关联 它们与 P2R 和 Ca2 有关信号复合物的表达模式有关。 将通过表征离子来进一步探讨单个 P2R 的功能 由 ATP 和部分 P2R 激动剂激活的电流以及 这些电流流向 P2Rs 抑制性抗体。 2. 确定 RGS 蛋白的相互作用 与GPCR。这将通过测量多个 RGS 的效力来实现 抑制 SMG 细胞中 GPCR 诱发的 Ca2+ 信号传导的蛋白质。 RGS蛋白 特异性抗体和显性失活将用于识别活性 SMG 细胞中的 RGS 蛋白及其生理作用。本构已知 alfa-1BAR 的活性突变体将用于尝试识别该位点 RGS 蛋白与 GPCR 的相互作用。 3. 探索 Ca2+ 信号复合物 在 PM/ER 连接处：icrac-IP3R 复合物。确定 Imin 是否为 Icrac 并且 表征从天然体中切除的斑块中 Icrac 的单通道特性 SMG 细胞。确定 SMG 细胞中的 Icrac 是否由新门控 发现了构象耦合模式。研究荷马对 Icrac 的规定 支架蛋白家族在其组装中的作用 1crac-IP3R 和信号复合物。我相信我的可用工具 拟议项目的实验室、我实验室人员的专业知识以及 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普遍相关的见解 腺细胞。