MODULATION OF TE IP3/DAG 2ND MESSENGER PATHWAY

描述：（改编自申请人的摘要） 该提案的目标是确定RC 3（神经颗粒蛋白）和 相关的PKC底物调节第二信使途径耦合到7- 哺乳动物前脑中的跨膜受体。 RC 3是一个神经元 蛋白激酶C（PCK）的特异性底物， 主要神经元树突棘的兴奋性不对称突触连接 前脑神经元 该项目的中心假设是PKC- 磷酸化RC 3选择性调节IP 3/DAG第二信使 与突触后7-跨膜受体偶联的通路，特别是 代谢型谷氨酸受体 在特异性目的1中，野生型或突变型 RC 3的形式将与5 HT 1C受体在非洲爪蟾中共表达 卵母细胞，并测试它们在增强Ca 2 + - 由血清素诱发的激活的氯离子电流。 这些研究将确定 RC 3中的特定氨基酸残基介导其调节 IP 3/DAG第二信使途径。 在具体目标2中，RC 3的潜力 将研究与Go、PLC或IP 3受体的相互作用 在非洲爪蟾卵母细胞的功能试验中，通过测量 佛波酯诱发和马槟榔素诱发选择性药物氯电流 抑制剂的 潜在的RC 3调节GTP γ S结合和 Go蛋白的ADP核糖基化、磷脂酶C β活性或结合 还将用纯化的大鼠 与RC 3 -78合成肽孵育的脑部分。 在特定 目的3、RC 3在神经元内IP 3相关信号转导中的作用 在三种不同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.