Signaling Mechanisms in Salivary Gland Cells

描述（由申请人提供）：Ca 2+信号调节唾液腺（SG）细胞的液体和电解质分泌。SG的这种极化功能决定了细胞微区中Ca 2+信号复合物的极化组织和功能。支架蛋白在复合物内Ca 2+信号蛋白的组装和调节中起着关键作用。Ca 2+信号复合物的中心组分是由TRPC通道介导的Ca 2+内流。TRPC 3和TRPC 6是SG的主要通道。Spinophilin（SPL）、Neurabin（NRB）和Homer 1支架如何调节GPCR和TRPC 3/6通道的作用是本提案的主题。为了实现我们的目标，我们发现了SPL/NRB对对Ca 2+信号传导的调节，Homer 1在TRPC通道运输中的作用，SPL和新发现的STIM 1对TRPC 6活性的调节。这些发现导致了以下特定目标的发展，以探索支架在SG Ca 2+信号传导中的作用。1.确定SPL/NRB在SG细胞中通过RGS蛋白调节GPCR Ca 2+信号传导中的作用。这将通过以下方式实现：a）鉴定结合RGS蛋白的NRB结构域以及RGS蛋白的SPL和NRB结合之间的关系，B）确定NRB在RGS 2-/-和NRB-/-细胞中的Ca 2+信号传导中的作用，以及c）表征来自SPL-/-和NRB-/-小鼠的SG细胞中的Ca 2+信号传导。2.通过以下方式探索Homer 1在TRPC通道易位和Ca 2+内流中的作用：a）研究SG细胞中TRPC 3/6的易位和回收以及储存耗尽和Homer 1在这两种活性中的作用; B）通过生物素化和TIRF测定研究HEK细胞中表达的TRPC 3/6-YFP的易位来扩展天然细胞中的发现。3.通过以下方式研究SPL/NRB对TRPC 3/6的调节：a）鉴定与TRPC 3/6相互作用的两个SPL/NRB结构域; B）确定SPL/NRB在TRPC 3/6易位和回收中的作用; c）使用SPL-/-和NRB-/-细胞在体内表征SPL/NRB对TRPC 3/6通道活性的调节。4.研究STIM 1对TRPC 3/6的调节：a）确定STIM 1对NATIVE和表达的TRP 3/6通道的调节，B）探索STIM 1调节TRPC 3/6的机制。拟议的工作探讨新的监管机制，Ca 2+信号及其相关的SG液和电解质分泌的调节。

DESCRIPTION (provided by applicant): Ca2+ signaling regulate fluid and electrolyte secretion by salivary gland (SG) cells. This polarized function of SG dictates polarized organization and functioning of Ca2+ signaling complexes in cellular microdomains. Scaffolding proteins plays critical roles in the assembly AND regulation of Ca2+ signaling proteins within the complexes. A central component of the Ca2+ signaling complexes is Ca2+ influx, which is mediated by TRPC channels. TRPC3 and TRPC6 are the dominant channels in SG. How the scaffolds Spinophilin (SPL), Neurabin (NRB) and Homerl regulates the action of GPCRs and TRPC3/6 channels is the theme of this proposal. Towards achieving our goals we found the regulation of Ca2+ signaling by the SPL/NRB pair, the role of Homerl in trafficking of TRPC channels, regulation of TRPC6 activity by SPL and by the newly discovered STIM1. These findings led to development of the following specific aims to probe the role of scaffolds in SG Ca2+ signaling. 1. Determine the role of SPL/NRB in regulating GPCRs Ca2+ signaling by RGS proteins in SG cells. This will be achieved by a) Identifying the NRB domain that binds RGS proteins and the relationship between SPL and NRB binding of RGS proteins, b) Determining the role of NRB in Ca2+ signaling in RGS2-/- and NRB-/- cells and c) Characterizing Ca2+ signaling in SG cells from SPL-/- and NRB-/- mice. 2. Explore the role of Homerl in TRPC channels translocation and Ca2+ influx by: a) studying translocation and retrieval of TRPC3/6 in SG cells and the role of store depletion and Homerl in both activities; b) Extend the findings in native cells by studying translocation of TRPC3/6-YFP expressed in HEK cells by biotinylation and TIRF assays. 3. Study regulation of TRPC3/6 by SPL/NRB by: a) Identifying the two SPL/NRB domains that interact with TRPC3/6; b) determine the effect of SPL/NRB in TRPC3/6 translocation and retrieval; c) characterize the regulation of TRPC3/6 channel activity by SPL/NRB in vivo using SPL-/- and NRB-/- cells. 4. Study regulation of TRPC3/6 by STIM1 by: a) determine the regulation of NATIVE and expressed TRP3/6 channels by STIM1, b) explore the mechanism by which STIM1 regulates TRPC3/6. The proposed work explores novel regulatory mechanisms in Ca2+ signaling and their relevance to regulation of SG fluid and electrolyte secretion.