Signaling Mechanisms in Salivary Gland Cells

唾液腺细胞的信号传导机制

• 批准号: 7761190
• 负责人: JAMES T STULL
• 金额: $ 37.28万
• 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/7761190
• 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（由申请人提供）：CA2+信号传导通过唾液腺（SG）细胞调节流体和电解质分泌。 SG的极化功能决定了细胞微区中Ca2+信号复合物的两极化组织和功能。脚手架蛋白在复合物中Ca2+信号蛋白的组装和调节中起关键作用。 Ca2+信号传导复合物的一个中心分量是Ca2+流入，它是由TRPC通道介导的。 TRPC3和TRPC6是SG中的主要通道。该提案的主题是如何调节GPCR和TRPC3/6通道的作用的脚手架（SPL），神经蛋白（NRB）和HOMERL是该提案的主题。为了实现我们的目标，我们发现了SPL/NRB对对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的的发展，以探测支架在SG Ca2+信号传导中的作用。 1。确定SPL/NRB在SG细胞中RGS蛋白调节GPCRS Ca2+信号传导中的作用。这将通过a）识别结合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RGS2 - / - / - 和NRB - / - / - 和C中的Ca2+信号传导中的作用。 2。探索HOMERL在TRPC通道中的作用和Ca2+流入的作用： b）通过研究通过生物素化和TIRF分析在HEK细胞中表达的TRPC3/6-YFP的转运来扩展本机细胞中的发现。 3。通过SPL/NRB对TRPC3/6的研究调节：a）识别与TRPC3/6相互作用的两个SPL/NRB域； b）确定SPL/NRB在TRPC3/6易位和检索中的影响； c）使用SPL - / - 和NRB - / - 细胞来表征通过体内SPL/NRB对TRPC3/6通道活性的调节。 4。通过Stim1对TRPC3/6的研究调节：a）通过Stim1确定天然和表达的TRP3/6通道的调控，b）探索STIM1调节TRPC3/6的机制。拟议的工作探讨了Ca2+信号传导中的新调节机制及其与SG流体和电解质分泌的调节相关性。