The Role of Afadin in Coordinating Cell Adhesion Molecules during Synaptogenesis

描述(申请人提供)：突触连接的形成是由细胞间黏附分子在轴突和树突之间启动的，通过结合的细胞内效应器，核化突触前神经递质释放和突触后神经递质接收所需的蛋白质的相互招募。在突触发生过程中形成了类似于贴壁连接的连接。钙粘附素是细胞间黏附分子，在上皮细胞中黏附连接的形成中起中心作用。对神经元中钙粘附素功能的研究表明，钙粘附素及其相关的连接素在调节突触数量和突触前膜和突触后膜的成熟方面发挥了作用。表达研究发现，钙粘附素亚型并不是普遍表达，而是定位于不同的神经回路，提示钙粘附素可能在突触特异性中发挥作用。根据对上皮细胞的研究，黏附连接被认为是由反式凝集素相互作用启动的，随后是钙粘附素募集。Nectins依赖它们与afadin的PDZ结构域的相互作用来招募和激活钙粘附素。通过这个相同的PDZ结构域，afadin可以与其他细胞表面受体结合，这些受体在突触发生中具有既定的作用。我建议通过检测afadin表达缺失的影响，来研究afadin介导的细胞表面受体与钙粘附素的关联在突触发生中的作用。这种分子连接的缺失可能会阻止钙粘附素与几个细胞表面受体家族的联系。由于培养上皮细胞中afadin的丢失已被证明导致粘附素和钙粘附素的丧失，我们预计突触的形成和/或稳定性将严重减少。由于以前的分析没有令人满意地证明afadin如何与α-连环蛋白和p120-连环蛋白相关联，将进行额外的分析，以检查与这些连环蛋白结合的afadin的结构域。我将通过确定这些突变体在体外拯救afadin缺失表型的能力，来研究afadin与这些连环蛋白相关的作用。最后，将确定失去afadin对这些其他细胞表面受体启动的信号转导的影响。 神经系统的正常运作需要看似不相关的细胞之间不同的大脑区域之间有序的突触连接。虽然许多研究已经记录了将轴突引导到右侧大脑区域所需的分子，但我们感兴趣的是确定轴突如何与适当的神经元以及神经元的适当部分连接。这项提议正在研究不同类别的黏附蛋白之间的联系，以及这种联系在创建和修改突触方面的作用，这可能在产生突触特异性方面发挥作用。

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.