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（由申请人提供）：Ca2+信号传导调节唾液腺（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 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节中发挥着关键作用。 Ca2+ 信号复合物的核心组成部分是 Ca2+ 内流，由 TRPC 通道介导。 TRPC3 和 TRPC6 是 SG 中的主导通道。本提案的主题是支架材料 Spinophilin (SPL)、Neurabin (NRB) 和 Homerl 如何调节 GPCR 和 TRPC3/6 通道的作用。为了实现我们的目标，我们发现了 SPL/NRB 对对 Ca2+ 信号传导的调节、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具体目标的发展，以探讨支架在 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。这将通过以下方式实现：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-/- 细胞中 Ca2+ 信号传导中的作用，以及 c) ) 表征 SPL-/- 和 NRB-/- 小鼠 SG 细胞中的 Ca2+ 信号传导。 2. 通过以下方式探索 Homerl 在 TRPC 通道易位和 Ca2+ 流入中的作用： a) 研究 SG 细胞中 TRPC3/6 的易位和恢复以及存储耗尽和 Homerl 在这两种活动中的作用； b) 通过生物素化和 TIRF 测定研究 HEK 细胞中表达的 TRPC3/6-YFP 的易位，从而扩展了在天然细胞中的发现。 3. 通过以下方式研究 SPL/NRB 对 TRPC3/6 的调节： a) 识别与 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) 使用 SPL-/- 和 NRB-/- 细胞表征体内 SPL/NRB 对 TRPC3/6 通道活性的调节。 4. 研究STIM1对TRPC3/6的调节：a)确定STIM1对NATIVE和表达的TRP3/6通道的调节，b)探索STIM1调节TRPC3/6的机制。这项工作探索了 Ca2+ 信号传导的新调节机制及其与 SG 液体和电解质分泌调节的相关性。