A phospho-switch in delta-catenin: relationship to PDZ-domain proteins and neuron development

项目摘要/摘要 神经元连通性的产生在大脑发育过程中受到调节，伴随着突触的形成 依赖于树突的形态，树枝状结构从神经元延伸出来。 因此，树突形成的中断可导致非典型的突触连接，与 神经发育障碍，如自闭症、精神分裂症和Cri-Du-chat综合征。我们的建议旨在 为了更好地了解树枝晶形态背后的分子过程。这将通过以下方式实现 对我们最近发现的两个新的蛋白质复合体进行了检查，每个复合体都涉及增量-连环蛋白。增量连锁反应 属于连环蛋白的p120亚家族，其成员最为人所知的是 与钙粘附素、细胞黏附蛋白和细胞骨架相关伙伴的关系 GTP酶。Delta-catenin定位于神经元树突和突触，在那里它在发育和 动态平衡。Delta-catenin具有一个存在于大多数连环蛋白中的中央螳螂重复结构域，但它 此外，在其末端还含有PDZ结合配体。这种PDZ-配体与许多 PDZ结构域蛋白对突触和树突功能至关重要。我们的初步调查结果揭示了两个 DeltaCatenin的PDZ配体与PDZ结构域蛋白Magi1和Pdlim5的新相互作用。MAGI1 是膜相关鸟苷酸激酶(MAGUK)家族中的一员，该家族沿着树突表达 在早期阶段，在顶端富集物，这可能与树枝晶伸展有关。Pdlim5是一个 PDZ-LIM蛋白也在树突中发现，但被认为可以负向调节轴突生长锥体停止生长 树枝状物在变长。我们使用大鼠海马神经元的初步工作表明，Magi1 Pdlim5的表达促进了树突的延长，而Pdlim5的出现反而增强了分支。这 向我们展示了一个耐人寻味的想法，两种看似相反的蛋白质都可以与Delta结合- 连锁素。对这些相互作用的分析揭示了DeltaCatenin的PDZ配体中的一个关键的磷酸化位点 这似乎决定了两种相互作用中的哪一种发生，潜在地促进了空间和 树枝状形态的时间控制。使用原代大鼠海马神经元以及HEK293细胞， 本申请中提出的实验检查了磷酸依赖(目标1)和细胞和 Delta-catenin：Magi1和Delta-catenin：Pdlim5复合体的发育意义(目标2)。 实验方法包括(除其他外)选择性地破坏这两个三角洲-连环蛋白复合体 使用击倒/加回策略。考虑到正在研究的三种蛋白质中每一种的功能障碍 与神经系统疾病有关，解决它们之间的相互作用和对树突发育的贡献 可以为神经发育障碍的进展提供洞察。

PROJECT SUMMARY/ABSTRACT Generation of neuronal connectivity is regulated during brain development, with the formation of synapses being dependent on the morphology of dendrites, branch-like structures extending out from neurons. Disruptions in dendrite formation can thus lead to atypical synaptic connectivity, associated with neurodevelopmental disorders such as autism, schizophrenia and Cri-du-chat syndrome. Our proposal seeks to better understand molecular processes underlying the morphology of dendrites. This will occur through examination of two novel protein complexes we recently revealed, each involving delta-catenin. delta-Catenin belongs the p120-subfamily of catenin proteins, whose members have are best known for their functions in association with cadherin cell-adhesion proteins and cytoskeletal-associated partners including small- GTPases. delta-Catenin localizes to neuronal dendrites and synapses where it functions in development and homeostasis. Delta-catenin possesses a central armadillo-repeat domain present in most catenins, but it additionally contains a PDZ-binding ligand at its extreme C-terminus. This PDZ-ligand binds to a number of PDZ-domain proteins crucial for synaptic and dendritic functions. Our preliminary findings have revealed two novel interactions between delta-catenin's PDZ-ligand and the PDZ-domain proteins Magi1 and Pdlim5. Magi1 is a member of the membrane-associated guanylate kinase (MAGUK) family that is expressed along dendrites with enrichment within the tips at early stages, potentially relevant to a role in dendrite extension. Pdlim5 is a PDZ-LIM protein also found in dendrites, but is believed to negatively regulate the neurite growth cone to halt the dendrite's lengthening. Our preliminary work using rat hippocampal neurons suggests that Magi1 expression promotes the lengthening of dendrites, while Pdlim5 appears instead to enhance branching. This presents us with the intriguing thought that two proteins with seemingly opposing roles can each bind delta- catenin. Analysis of these interactions has revealed a critical phosphorylation site in delta-catenin's PDZ-ligand that appears to determine which of the two interactions occurs, potentially contributing to the spatial and temporal control of dendritic morphologies. Using primary rat hippocampal neurons as well as HEK293 cells, the experiments proposed in this application examine the phospho-dependency (Aim 1) and the cellular and developmental significance of the delta-catenin:Magi1 and delta-catenin:Pdlim5 complexes (Aim 2). Experimental approaches include (among others) the selective disruption of these two delta-catenin complexes using knock-down/ add-back strategies. Given that the dysfunction of each of the three proteins under study are implicated in neurologic diseases, addressing their interactions and contributions to dendrite development could provide insight to the progression of neurodevelopmental disorders.