MODULATION OF TE IP3/DAG 2ND MESSENGER PATHWAY

TE IP3/DAG 第二信使通路的调节

• 批准号: 2270777
• 负责人: Joseph B. Watson
• 金额: $ 16.23万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-04 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2270777
• 关键词: ADP ribosylation; G protein; Xenopus oocyte; chemical binding; chloride channels; diacylglycerols; enzyme activity; gene mutation; glutamate receptor; guanosine triphosphate; inositol phosphates; laboratory rat; neurotransmitter receptor; phorbols; phospholipase C; prosencephalon; protein kinase C; serotonin; serotonin receptor; synthetic peptide; tissue /cell culture

DESCRIPTION: (adapted from Applicant's Abstract) The long-term objectives of the proposal are to identify how RC3 (neurogranin) and related PKC substrates modulate second messenger pathways coupled to 7- transmembrane receptors in the mammalian forebrain. RC3 is a neuron- specific substrate of protein kinase C (PCK) that accumulates near excitatory asymmetric synaptic juctions in dendritic spines of primarily forebrain neurons. The central hypothesis for the project is that PKC- phosphorylated RC3 selectively modulates IP3/DAG second messenger pathways coupled to postsynaptic 7-transmembrane receptors, particularly metabotropic glutamate receptors. In Specific Aim 1, wildtype or mutant forms of RC3 will be co-expressed with the 5HT1C receptor in Xenopus oocytes and tested for their relative effectiveness in enhancing Ca2+ - activated Cl- currents evoked by serotonin. These studies will identify specific amino acid residues in RC3 that mediate its modulation of the IP3/DAG second messenger pathway. In Specific Aim 2, RC3's potential interaction with either Go, PLC or the IP3 receptor will be investigated in functional assays in Xenopus oocytes by measuring the sensitivity of phorbol ester-evoked and serotonin-evoked Cl- currents to selective drug inhibitors. Potential RC3 modulation of either GTPgammaS binding and ADP-ribosylation of Go protein, phospholipase Cbeta activity, or binding to the IPc receptor will also be assayed biochemically with purified rat brain fractions incubated with a RC3-78 synthetic peptide. In Specific Aim 3, RC3 functions in IP3-related signal transduction in neurons will be addressed in three different kinds of tissue culture experiments.

描述：（改编自申请人的摘要）长期 该提案的目标是确定RC3（Neurogranin）和 相关的PKC底物调节耦合到7-的第二信使途径 哺乳动物前脑中的跨膜受体。 RC3是神经元 - 蛋白激酶C（PCK）的特异性底物积聚在附近 树突状刺激的兴奋性不对称突触夹具主要是 前脑神经元。 该项目的中心假设是PKC- 磷酸化的RC3有选择地调节IP3/DAG第二使者 与突触后7跨膜受体相连的途径，特别是 代谢性谷氨酸受体。 在特定的目标1中，野生型或突变体 RC3的形式将与Xenopus中的5HT1C受体共表达 卵母细胞并测试了它们在增强Ca2+ - 的相对有效性 血清素引起的活化cl液。 这些研究将确定 RC3中的特定氨基酸残基介导了其调节 IP3/DAG第二通路通路。 在特定的目标2中，RC3的潜力 将研究与GO，PLC或IP3受体的相互作用 通过测量爪蟾卵母细胞的功能测定 凤凰酯诱发的和5-羟色胺诱发的cl曲子至选择性药物 抑制剂。 GTPGAMMAS结合的潜在RC3调制和 GO蛋白，磷脂酶CBETA活性或结合的ADP-核糖基化 向IPC受体也将用纯化的大鼠进行生化测定 与RC3-78合成肽一起孵育的脑部分子。 具体 AIM 3，RC3在IP3中与神经元中与IP3相关信号转导的功能将 可以在三种不同类型的组织培养实验中解决。