The structural basis of cis and trans protocadherin interactions

描述（由申请方提供）：拟定研究的总体目标是确定原钙粘蛋白（Pcdh）细胞表面蛋白的反式（细胞-细胞）和顺式（相同细胞表面）相互作用的性质。大多数哺乳动物Pcdhs存在于三个大的基因簇中，这些簇的组织导致产生巨大的单细胞多样性。这种多样性被认为是单个神经元的分子“条形码”，使细胞能够区分自我和非自我。特别是 已经显示成簇的Pcdh是小鼠脑和脊髓中树突状自我回避和神经回路组装的其它方面所需的。这种功能可能需要不同的Pcdh异构体之间的嗜同性相互作用在相对的质膜表面。最初，将使用细胞-细胞聚集试验对Pcdh同种型嗜同性特异性进行全面分析。将用荧光标记物标记的Pcdh α、β和guama同种型的选定子集克隆并转染到培养的哺乳动物细胞中。将用相同或不同的Pcdh同种型转染的细胞混合，并将细胞培养。 通过荧光显微术，通过免疫荧光细胞聚集体的大小来定量聚集。将通过构建和测试缺失特异性胞外结构域的Pcdh同种型来鉴定反式嗜同性相互作用结构域。一旦鉴定，将对相互作用的胞外域进行生物物理学分析以确定多聚体状态和嗜同性结合亲和力。顺式Pcdh相互作用的表征将通过用多种Pcdh同种型转染细胞，然后进行细胞-细胞聚集测定来完成。将进行结构域缺失研究以绘制顺式相互作用区域，并对这些区域进行生物物理分析。一旦鉴定，反式二聚体亲同结构域将以高水平表达，并且二聚体复合物结晶并进行三维原子结构分析。基于三维结构的结构假设将使用定点诱变和细胞聚集试验作为相关结构和功能的方法进行检测。最后，将尝试生产和结晶全长Pcdhs和子区域，包括顺式界面和它们的结构由X-射线晶体学确定。如果成功的话，这些研究将为集群Pcdhs在介导自我回避和神经回路组装的其他方面的作用提供深入的机制见解。此外，这些研究可能揭示Pcdh介导的分子识别密码的存在和性质。

DESCRIPTION (provided by applicant): The overall objective of the proposed research is to determine the nature of trans (cell-to cell) and cis (same cell surface) interactions of protocadherin (Pcdh) cell surface proteins. The majority of mammalian Pcdhs are present in three large gene clusters, and the organization of these clusters leads to the generation of enormous single cell diversity. This diversity is thought to function as a molecular "barcode" for individual neurons, which allows cells to distinguish between self and non-self. In particular, the clustered Pcdhs have been shown to be required for dendritic self-avoidance and other aspects of neural circuit assembly in the mouse brain and spinal cord. This function is likely to require homophilic interactions between distinct Pcdh isoforms at the surface of opposing plasma membranes. Initially, a cell-cell aggregation assay will be used to carry out a comprehensive analysis of Pcdh isoform homophilic specificity. A selected subset of Pcdh Alpha, Beta and guama isoforms tagged with fluorescent labels will be cloned and transfected into mammalian cells in culture. Cells transfected with the same or different Pcdh isoforms will be mixed and cell aggregation quantitated by the size of the immunofluorescent cell aggregates by fluorescent microscopy. The trans-homophilic interaction domains will be identified by constructing and testing Pcdh isoforms missing specific extracellular domains. Once identified the interacting ectodomains will be subjected to biophysical analyses to determine the multimeric state and homophilic binding affinities. Characterization of cis Pcdh interactions will be accomplished through the transfection of cells with multiple Pcdh isoforms, followed by cell-cell aggregation assays. Domain deletion studies will be carried out to map the cis- interacting regions and these regions subjected to biophysical analyses. Once identified, trans dimeric homophilic domains will be expressed at high levels, and the dimeric complexes crystallized and subjected to three-dimensional atomic structure analyses. Structural hypothesis based on the three dimensional structure will be tested using site-directed mutagenesis and cell aggregation assays as a means a correlating structure and function. Finally, attempts will be made to produce and crystallize full-length Pcdhs and sub regions that encompass the cis interface and their structures determined by x-ray crystallography. If successful, the proposed studies will provide deep mechanistic insights into the role of the clustered Pcdhs in mediating self-avoidance and other aspects of neural circuit assembly. In addition, the studies may reveal the existence and nature of a Pcdh-mediated molecular recognition code.