原钙粘蛋白“恒定区”胞内域与钠钾ATP酶β1亚基的相互作用及其对神经突触形成的影响

Protocadherins (Pcdh) are a subfamily of cell adhesion molecules that have been proposed to play an important role in the formation of synapse，but little is known about molecular mechanisms of the Pcdh involving in the synapse formation currently. In a preliminary study aiming to identify interacting proteins for the constant intracelluar domain (constICD) of Pcdh by yeast two-hybrid screening, we identified Na,K-ATPaseβ1 subunit as a potential interacting protein for the Pcdh constICD. In this proposal, we would like to ① further investigate whether Pcdh constICD could interact with Na,K-ATPaseβ1 specifically; ② elucidate whether Pcdh constICD interacts with Na,K-ATPaseβ1 directly or through an intermediate molecule; ③ map the binding domain in both Pcdh constICD and Na,K-ATPaseβ1; ④ determine whether the Pcdh and Na,K-ATPaseβ1 could be associated specifically at the physiological condition in brain; ⑤ explore whether molecular interaction between Pcdh constICD and Na,K-ATPaseβ1 could play a role in regulating the Pcdh cellular trafficking, cell membrane localization and synapse formation by utilizing co-immunoprecipitation assay, HPLC-tandem mass spectrometry, GST pull-down assay, truncated protein test, PCR-mediated site-specific mutagenesis, siRNA knock-down experiment, live cell imaging system and immunostaining ect. Thus, our proposal aiming to decipher the Pcdh cell trafficking and cellular localization regulation mediated by the Pcdh constICD would likely provide a critical insight into the understanding of the molecular mechanisms involving in the synapse formation

原钙粘蛋白（Pcdh）是一类细胞粘附分子，对神经突触的形成至关重要，但是具体分子机制还知之甚少。前期的研究结果提示：钠钾ATP酶β1亚基（Na,K-ATPaseβ1）能够与Pcdh的"恒定区"胞内域（constICD）相互作用。本研究拟采用免疫共沉淀实验和液相色谱-质谱分析，进一步确认Pcdh constICD与Na,K-ATPaseβ1存在特异性相互作用、明确其直接或间接性；利用截短蛋白测试、基因定点突变和GST 融合蛋白沉降技术，分析二者相互作用的位点；通过免疫共沉淀实验，确定Pcdh与Na,K-ATPaseβ1在脑组织内存在特异性相互作用；采用基因沉默、细胞工作站技术等方法，探讨该相互作用的改变对Pcdh胞内运输、胞膜定位和突触形成的影响。首次从蛋白相互作用的角度入手，探讨constICD在Pcdh胞内运输、胞膜定位过程中的作用，为阐明Pcdh在神经突触形成过程中的分子机制提供线索

原钙粘蛋白（Pcdh）是一类细胞粘附分子，对神经突触的形成至关重要。钠钾ATP酶β1亚基（Na,K-ATPaseβ1）作为分子伴侣，在钠钾ATP酶α亚基的构象稳定、胞内运输以及胞膜定位的过程中发挥重要作用。前期的酵母双杂交的实验结果提示： Na,K-ATPaseβ1能够与Pcdh的“恒定区”胞内域（constICD）相互作用。本研究采用免疫共沉淀实验、超高效液相色谱-质谱分析以及免疫组织化学染色，对二者之间的相互作用进行研究，并对二者在脑组织内的表达和分布进行观察。结果表明，Pcdh constICD与Na,K-ATPaseβ1之间存在特异性的直接相互作用，而且二者具有相同的神经细胞内分布特征。本研究为了解Pcdh的胞内运输、胞膜定位的机制，进而为阐明Pcdh在神经突触形成过程中的分子机制提供线索