Signaling Mechanisms in Salivary Gland Cells

唾液腺细胞的信号传导机制

• 批准号: 7558563
• 负责人: Shmuel Muallem
• 金额: $ 37.65万
• 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 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7558563
• 关键词: Adrenergic Receptor; Agonist; Antibodies; Attenuated; Binding; Biological Assay; Biological Models; Biotinylation; Brain; Cell membrane; Cell physiology; Cells; Complex; Data; Development; Electrolytes; Endoplasmic Reticulum; Epidermal Growth Factor Receptor; Fluids and Secretions; Fluorescence; Functional disorder; G Protein-Coupled Receptor Genes; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Glycerol; Goals; Homologous Gene; Hormones; Knock-out; Lead; Liquid substance; Mediating; Membrane Potentials; Modeling; Mouth Diseases; Mus; Muscarinics; N-terminal; Neurabin; Neurotransmitters; Play; Proteins; Pump; RGS Proteins; RGS2 gene; Radiation; Regulation; Reporting; Research Personnel; Retrieval; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; STIM1 gene; Salivary Glands; Scaffolding Protein; Scheme; Signal Transduction; Signaling Protein; Sjogren' s Syndrome; Techniques; Testing; Western Blotting; Work; Xerostomia; basolateral membrane; in vivo; luminal membrane; novel; parotid cell; programs; receptor; scaffold; spinophilin; trafficking

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.

描述(申请人提供)：钙信号调节唾液腺(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中钙信号复合体的极化组织和功能。支架蛋白在钙信号蛋白在复合体中的组装和调节中起着关键作用。钙信号复合体的中心成分是钙内流，它是由TRPC通道介导的。在SG中，TRPC3和TRPC6是主要通道。亲刺素(SPL)、神经降压素(NRB)和Hmer如何调节GPCRs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对钙信号的调节，Hmer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钙信号转导中的作用。1.确定SPL/NRB在RGS蛋白调节SG细胞GPCRs钙信号通路中的作用。这将通过a)确定与RGS蛋白结合的NRB结构域以及SPL与RGS蛋白的NRB结合之间的关系，b)确定NRB在RGS2-/-和NRB-/-细胞中的钙信号转导中的作用，以及c)表征SPL-/-和NRB-/-小鼠SG细胞中的钙信号。2.通过研究TRPC3/6在SG细胞中的转位和恢复，以及贮备物耗竭和Hmer在这两种活动中的作用，探讨Hmer在TRPC通道转位和钙内流中的作用；b)通过生物素化和TIRF方法研究在HEK细胞中表达的TRPC3/6-YFP的转位，从而扩展了在天然细胞中的发现。3.研究SPL/NRB对TRPC3/6的调节：a)鉴定与TRPC3/6相互作用的两个SPL/NRB结构域；b)确定SPL/NRB在TRPC3/6转位和恢复中的作用；c)利用SPL-/-和NRB-/-细胞在体内表征SPL/NRB对TRPC3/6通道活性的调节。4.研究STIM1对TRPC3/6的调控：a)确定STIM1对天然和表达的TRP3/6通道的调控；b)探讨STIM1调控TRPC3/6的机制。本工作探索了钙信号中新的调控机制及其与脑脊液和电解质分泌的调节。