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

项目概要/摘要 在大脑发育过程中，神经元连接的产生受到突触形成的调节 依赖于树突的形态，树突是从神经元延伸出来的树枝状结构。 因此，树突形成的中断可以导致非典型的突触连接， 神经发育障碍，如自闭症、精神分裂症和Cri-du-chat综合征。我们的建议是 以更好地理解树突形态下的分子过程。这将通过 我们最近发现了两种新的蛋白质复合物，每种都涉及δ-连环蛋白。δ-连环蛋白 属于连环蛋白的p120-亚家族，其成员以其在 与钙粘蛋白细胞粘附蛋白和细胞间隙相关的伙伴，包括小， GTP酶。δ-连环蛋白定位于神经元树突和突触，在那里它在发育中起作用， 体内平衡δ-连环蛋白具有大多数连环蛋白中存在的中央犰狳重复结构域，但它 在其末端C-末端还含有PDZ结合配体。这种PDZ配体结合到许多 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显示 δ-连环蛋白的PDZ-配体和PDZ-结构域蛋白Magi 1和Pdlim 5之间的新相互作用。Magi1 是膜相关鸟苷酸激酶（MAGUK）家族的成员，其沿沿着表达 在早期阶段在尖端内富集，可能与枝晶延伸的作用有关。Pdlim 5是一个 PDZ-LIM蛋白也存在于树突中，但被认为是负调节轴突生长锥， 树突变长了我们对大鼠海马神经元的初步研究表明，Magi 1 表达促进树突的延长，而Pdlim 5似乎反而增强分支。这 向我们提出了一个有趣的想法，即两种看似相反的蛋白质可以分别结合δ- 连环蛋白。对这些相互作用的分析揭示了δ-连环蛋白PDZ配体中的一个关键磷酸化位点 这似乎决定了两种相互作用中的哪一种发生，可能有助于空间和 枝晶形态的时间控制。使用原代大鼠海马神经元以及HEK 293细胞， 本申请中提出的实验检查磷酸依赖性（Aim 1）和细胞和 δ-连环蛋白：Magi 1和δ-连环蛋白：Pdlim 5复合物的发育意义（目的2）。 实验方法包括（除其他外）选择性破坏这两个δ-连环蛋白复合物 使用击倒/加回策略。鉴于所研究的三种蛋白质中的每一种的功能障碍 涉及神经系统疾病，解决它们的相互作用和树突发育的贡献 可以为神经发育障碍的进展提供见解。

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.