MODULATION OF TE IP3/DAG 2ND MESSENGER PATHWAY

描述：(改编自申请者摘要)长期 该提案的目标是确定RC3(神经颗粒素)和 相关的PKC底物调节偶联到7-蛋白的第二信使通路 哺乳动物前脑中的跨膜受体。RC3是一种神经元- 蛋白激酶C(PCK)的特定底物 大鼠树突棘的兴奋性非对称性突触连接 前脑神经元。该项目的中心假设是PKC- 磷酸化RC3选择性调节IP3/DAG第二信使 与突触后7-跨膜受体偶联的通路，特别是 代谢性谷氨酸受体。在特定目标1中，野生型或突变体 RC3的不同形式将与5HT1C受体在非洲爪哇共表达 并测试它们在增强钙离子方面的相对有效性。 5-羟色胺激活的氯离子电流。这些研究将确定 RC3中的特定氨基酸残基，介导其对 IP3/DAG第二信使通路。在特定目标2中，RC3的S潜力 将研究与GO、PLC或IP3受体的相互作用 在非洲爪哇卵母细胞的功能分析中通过测量 佛波酯和5-羟色胺诱导的选择性药物氯电流 抑制剂。GTPGammaS结合和GTPGammaS结合的潜在RC3调节 GO蛋白的ADP-核糖化、磷脂酶Cβ活性或结合 对IPC受体的作用也将用纯化的大鼠进行生化检测 脑组分与RC3-78合成肽孵育。具体而言 目的3、RC3在神经细胞IP3相关信号转导中的作用 在三种不同类型的组织培养实验中得到解决。

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.