The Role of Afadin in Coordinating Cell Adhesion Molecules during Synaptogenesis

描述（由申请人提供）：轴突和树突之间的突触连接形成是由细胞间粘附分子发起的，这些分子通过结合的细胞内效应物，形成突触前神经递质释放和突触后神经递质接受所需的蛋白质的相互募集。类似粘附体连接的连接在突触发生过程中形成。钙粘蛋白是上皮细胞粘附连接形成的细胞间粘附分子。对钙粘蛋白在神经元中的功能的研究表明，钙粘蛋白及其相关的连环蛋白在调节突触数量和突触前膜和突触后膜的成熟中发挥作用。表达研究发现，钙粘蛋白异构体不是普遍表达的，而是定位于不同的神经回路，这表明钙粘蛋白可能在突触特异性中发挥作用。根据对上皮细胞的研究，粘附连接被认为是由连接素在反式中的相互作用引发的，随后是钙粘蛋白的募集。连接蛋白依赖于它们与腺嘌呤的PDZ结构域的相互作用来招募和激活钙粘蛋白。通过相同的PDZ结构域，黄嘌呤可以结合其他细胞表面受体，这些受体在突触发生中具有确定的作用。我建议通过检查失去黄嘌呤表达的影响来检查黄嘌呤介导的细胞表面受体与钙粘蛋白在突触发生中的作用。这种分子连接的缺失可能会阻断钙粘蛋白与几个细胞表面受体家族的结合。由于在培养的上皮细胞中失去afadin已被证明会导致连接素和钙粘蛋白粘附的丧失，我们预计突触形成和/或稳定性会严重减少。由于先前的分析没有令人满意地证明afadin如何与α -连环蛋白和p120-连环蛋白结合，进一步的分析将针对检查结合这些连环蛋白的afadin结构域。我将通过确定这些突变体在体外挽救黄嘌呤零表型的能力，来研究黄嘌呤与这些连环蛋白的关联作用。最后，我们将确定失去黄嘌呤对这些其他细胞表面受体启动的信号传导的影响。

DESCRIPTION (provided by applicant): Synaptic junction formation is initiated between axons and dendrites by intercellular adhesion molecules, which through bound intracellular effectors, nucleate the reciprocal recruitment of proteins required for presynaptic neurotransmitter release and postsynaptic neurotransmitter reception. Junctions analogous to adherens junctions are formed during synaptogenesis. Cadherins are intercellular adhesion molecules central to the formation of adherens junctions in epithelial cells. Studies on cadherin function in neurons have demonstrated a role for cadherins and their associated catenins in regulating synapse number and maturation of both pre- and post-synaptic membranes. Expression studies have found that cadherin isoforms are not expressed ubiquitously, but are localized to distinct neural circuits, suggesting that cadherins may play a role in synaptic specificity. Based upon studies in epithelial cells, adherens junctions are thought to be initiated by nectin interaction in trans followed by cadherin recruitment. Nectins rely upon their interaction with the PDZ domain of afadin to recruit and activate cadherins. Through this same PDZ domain, afadin can bind other cell surface receptors, which have an established role in synaptogenesis. I am proposing to examine the role of afadin-mediated cell surface receptor's association with cadherins on synaptogenesis, by examining the affect of loss of afadin expression. Loss of this molecular link will likely block cadherin association with several cell surface receptor families. As loss of afadin in cultured epithelial cells has been shown to lead to a loss of nectin and cadherin adhesion, we expect a severe reduction in synapse formation and/or stabilization. As previous analysis has not satisfactorily demonstrated how afadin associates with alpha-catenin and p120-catenin, additional analysis will be directed at examining the domains of afadin that binds these catenins. I will examine the role of afadin association with these catenins, by determining the ability of these mutants to rescue the afadin null phenotype in vitro. Finally, the effect of loss of afadin on signaling initiated by these other cell surface receptors will be determined. Proper nervous system functioning requires the ordered synaptic connection of disparate brain regions between seemingly unrelated cells. While much research has documented the molecules required for guiding axons to the right brain regions, we are interested in determining how axons then connect with the proper neuron and on the proper part of the neuron. This proposal is studying the associations between different classes of adhesion proteins and the role of this association in creating and modifying synapses, which is likely to play a role in generating synaptic specificity.