The structural basis of cis and trans protocadherin interactions

描述(由申请人提供)：拟议研究的总体目标是确定原钙粘附素(Pcdh)细胞表面蛋白的反式(细胞对细胞)和顺式(相同细胞表面)相互作用的性质。大多数哺乳动物的Pcdhs存在于三个大的基因簇中，这些簇的组织导致了巨大的单细胞多样性的产生。这种多样性被认为是单个神经元的分子条形码，它允许细胞区分自我和非自我。尤其是， 聚簇的Pcdhs已被证明是小鼠大脑和脊髓中树突自我回避和其他方面的神经回路组装所必需的。这一功能可能需要在相对的质膜表面不同的Pcdh亚型之间进行同亲相互作用。最初，细胞-细胞聚集试验将用于对PcdH异构体的亲和性进行全面的分析。用荧光标记的Pcdh Alpha、Beta和Guama亚型的选定子集将被克隆并在培养中导入哺乳动物细胞。转染相同或不同Pcdh亚型的细胞将混合和细胞 通过荧光显微镜下免疫荧光细胞聚集物的大小来量化聚集。通过构建和检测缺失特定胞外区的PcdH亚型来鉴定反式亲性相互作用域。一旦确定，相互作用的胞外结构域将接受生物物理分析，以确定多聚体状态和同亲结合亲和力。顺式Pcdh相互作用的表征将通过将多个Pcdh亚型导入细胞，然后进行细胞-细胞聚集分析来完成。将进行结构域删除研究，以绘制顺式相互作用区和这些接受生物物理分析的区域图。一旦确定，反式二聚体亲均结构域将在高水平表达，二聚体络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说将使用定点突变和细胞聚集分析作为关联结构和功能的一种手段进行检验。最后，将尝试生产和结晶全长Pcdh和围绕顺式界面的亚区及其由X射线结晶学确定的结构。如果成功，拟议的研究将提供深入的机械性见解，以了解聚集性Pcdh在调节自我回避和神经回路组装的其他方面所起的作用。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.