Signaling Mechanism in Salivary Gland Cells

描述(申请人提供)：唾液腺的极化性质 功能需要两极化的信号复合体的组织和功能。 了解细胞内信号蛋白组装成复合体的过程 微域是这项提议的中心主题。根据我们的初步调查 分泌细胞中钙信号蛋白极化表达的研究 多个P2Rs在唾液腺中的膜限制性表达 细胞、RGS蛋白对GPCR信号的调节和Icrc的调节 通过IP3R的通道，我们将通过1检验中心假设。研究空间 用P2Rs和GPCR研究钙信号转导的特点。FURA2、BTC和镁-FURA2的成像 将被用来捕捉SMG细胞中的钙波和梯度并关联 它们具有关于信号复合体的P2Rs和Ca2+的表达模式。 将通过表征离子来进一步探讨单独的P2Rs的功能 ATP和部分P2R激动剂激活的电流及其敏感性 这些电流作用于P2Rs抑制抗体。2.确定RGS蛋白之间的相互作用 与GPCR.这将通过测量几个RG的效力来实现 抑制GPCR在SMG细胞中诱导的钙信号的蛋白质。RGS蛋白 将使用特定抗体和显性阴性来识别活跃的 SMG细胞中的RGS蛋白及其生理作用。宪法上已知的 将使用ALFA-1BAR的活性突变体来尝试确定该位置 RGS蛋白与GPCR的相互作用。3.探索钙信号复合体 在PM/ER交界处：ICRAC-IP3R复合体。确定伊明是不是ICRAC和 在从天然斑块切除的斑块中表征ILAC的单通道特性 SMG细胞。确定SMG细胞中的iCRAC是否由新的 发现了构象偶联模式。论荷马对ICRAC的调控 支架蛋白家族在组装过程中的作用 1CRAC-IP3R和信号复合体。我相信我的工具中的可用工具 提议的项目的实验室，我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 对细胞信号和唾液功能的普遍相关性的见解 腺体细胞。

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.