Signaling Mechanisms in Salivary Gland Cells

描述（由申请人提供）：Ca2+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域Ca2+信号复合物的极化组织和功能。支架蛋白在复合物内Ca2+信号蛋白的组装和调控中起着关键作用。Ca2+信号复合物的一个中心组成部分是Ca2+内流，这是由TRPC通道介导的。TRPC3和TRPC6是SG的主要通道。Spinophilin (SPL), Neurabin （NRB）和Homerl支架如何调控gpcr和TRPC3/6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Ca2+信号的调节，Homerl在TRPC通道运输中的作用，SPL和新发现的STIM1对TRPC6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 Ca2+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 Ca2+信号传导中的作用。这将通过a)确定结合RGS蛋白的NRB结构域以及SPL和NRB结合RGS蛋白之间的关系，b)确定NRB在RGS2-/-和NRB-/-细胞中Ca2+信号传导的作用，以及c)表征SPL-/-和NRB-/-小鼠SG细胞中的Ca2+信号传导。2. 探讨Homerl在TRPC通道易位和Ca2+内流中的作用：a)研究TRPC3/6在SG细胞中的易位和恢复以及储存耗尽和Homerl在这两个活动中的作用；b)通过生物素化和TIRF实验研究HEK细胞中表达的TRPC3/6-YFP的易位，将研究结果扩展到天然细胞中。3. 通过以下方法研究SPL/NRB对TRPC3/6的调控：a)鉴定与TRPC3/6相互作用的两个SPL/NRB结构域；b)确定SPL/NRB对TRPC3/6易位和恢复的影响；c)利用SPL-/-和NRB-/-细胞表征SPL/NRB对体内TRPC3/6通道活性的调节。4. 通过以下途径研究STIM1对TRPC3/6的调控：a)确定STIM1对NATIVE及表达的TRP3/6通道的调控；b)探索STIM1调控TRPC3/6的机制。提出的工作探索Ca2+信号的新调控机制及其与SG流体和电解质分泌调节的相关性。

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.