Nectin-1: Synaptic processing and functions

描述（由申请人提供）：Nectin-1是一种定位于突触中点状粘附连接的细胞粘附分子。Nectin-1与其他蛋白质结合，共同参与神经元突触的形成。我们假设，nectin-1-经历了一个调节的多步骤的内切蛋白水解裂解事件的几个sheddases在活性依赖性的方式，这些事件调节突触发生，并有助于突触可塑性。我们的初步数据表明，nectin-1通过至少两个脱落酶进行胞外结构域脱落，导致产生两个C-末端片段（CTF）。这些CTF被γ-分泌酶进一步膜内切割并从质膜释放NE-ICD。释放的NEICD易位到细胞核中，我们假设它诱导基因表达。在特异性目标1中，我们将通过免疫亲和纯化，然后通过Edman降解测序来确定nectin-1的分泌酶切割位点。我们还将通过CodeLink Bioarrays研究海马神经元中NE-ICD调控的基因。然后，使用定量RT-PCR、ICC和Western印迹，我们将确认神经元中差异表达的基因。与NE-ICD相互作用的分子将通过酵母双杂交筛选来鉴定。一旦确定了相互作用物，将通过细胞和分子方法测定其生物学功能。在具体目标2中，我们将研究BACE 1在nectin-1加工中的生物学作用。最初的实验表明，BACE 1与nectin-1的脱落相关并参与脱落。我们将研究如何中断nectin-1脱落，通过丧失BACE 1功能，影响突触形成和突触可塑性的ICC，蛋白质印迹和囊泡回收试验。我们的初步数据表明，两个nectin-1点突变，T310 A和Y311 A，对BACE 1切割是难治的，并且可以/γ-显性干扰内源性nectin-1的加工。我们将研究这些点突变体如何影响突触的形成和突触功能的转导海马神经元和在体内成年海马与重组腺相关病毒载体。我们将通过ICC、活细胞成像和突触活动定量测量突触标记物、突触形态和大小的变化。随后，我们研究了反式显性nectin-1突变体的表达是否会影响海马依赖性学习

DESCRIPTION (provided by applicant): Nectin-1 is a cell adhesion molecule localized the puncta adherentia junctions in synapses. Nectin-1 associates with other proteins, which collectively participate in the formation of neuronal synapses. We hypothesize that nectin-1-undergoes a regulated multi-step set of endoproteolytic cleavage events by several sheddases in an activity-dependent manner and that these events regulate synaptogenesis and contribute to synaptic plasticity. Our preliminary data indicate that nectin-1 undergoes ectodomain shedding by at least two sheddases that result in the production of two C-terminal fragments (CTFs). These CTFs are further cleaved intramembraneously by y-secretase and liberate the NE-ICD from the plasma membrane. The released NEICD translocates into the nucleus and where we postulate it induces gene expression. In Specific Aim 1, we will determine the secretase cleavage sites of nectin-1 by immunoaffinity purification, followed by Edman degradation sequencing. We will also investigate which genes are regulated by NE-ICD in hippocampal neurons by CodeLink Bioarrays. Then, using quantitative RT-PCR, ICC, and Western blotting we will confirm differentially expressed genes in neurons. The molecules that interact with NE-ICD will be identified by a yeast two-hybrid screen. Once interactors are identified, their biological function will be assayed by cellular and molecular approaches. In Specific Aim 2, we will investigate the biological role of BACE1 in nectin-1 processing. Initial experiments indicate that BACE1 associates with and participates in the shedding of nectin-1. We will investigate how disruption of nectin-1 shedding, through loss of BACE1 function, affects synapse formation and synaptic plasticity by ICC, Western blotting and-vesicle recycling assays. Our preliminary data indicate that two nectin-1 point mutations, T310A and Y311A, are refractory to BACE1 cleavage and can /ra".y-dominantly interfere with processing of endogenous nectin-1. We will examine how these point mutants affect the synapse formation and synaptic function by transduction of hippocampal neurons and in vivo adult hippocampus with recombinant adeno-associated viral vectors. We will quantitatively measure the changes in synaptic markers, synapse morphology, and size by ICC, live cell imaging and synaptic activity. Subsequently, we examine whether expression of trans-dominant nectin-1 mutants will affect hippocampal dependent learning