Chen Maxine, Lucas Kenyatta G, Akum Barbara F, Balasingam Gaithri, Stawicki Tamara M, Provost Janine M, Riefler Gary M, Jörnsten Rebecka J, Firestein Bonnie L
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854-8082, USA.
Mol Biol Cell. 2005 Nov;16(11):5103-14. doi: 10.1091/mbc.e05-02-0165. Epub 2005 Aug 24.
Temporal and spatial assembly of signal transduction machinery determines dendrite branch patterning, a process crucial for proper synaptic transmission. Our laboratory previously cloned and characterized cypin, a protein that decreases PSD-95 family member localization and regulates dendrite number. Cypin contains zinc binding, collapsin response mediator protein (CRMP) homology, and PSD-95, Discs large, zona occludens-1 binding domains. Both the zinc binding and CRMP homology domains are needed for dendrite patterning. In addition, cypin binds tubulin via its CRMP homology domain to promote microtubule assembly. Using a yeast two-hybrid screen of a rat brain cDNA library with cypin lacking the carboxyl terminal eight amino acids as bait, we identified snapin as a cypin binding partner. Here, we show by affinity chromatography and coimmunoprecipitation that the carboxyl-terminal coiled-coil domain (H2) of snapin is required for cypin binding. In addition, snapin binds to cypin's CRMP homology domain, which is where tubulin binds. We also show that snapin competes with tubulin for binding to cypin, resulting in decreased microtubule assembly. Subsequently, overexpression of snapin in primary cultures of hippocampal neurons results in decreased primary dendrites present on these neurons and increased probability of branching. Together, our data suggest that snapin regulates dendrite number in developing neurons by modulating cypin-promoted microtubule assembly.
信号转导机制的时空组装决定树突分支模式，这一过程对于正常的突触传递至关重要。我们实验室之前克隆并鉴定了cypin，一种可降低PSD - 95家族成员定位并调节树突数量的蛋白质。Cypin包含锌结合、塌陷反应介导蛋白（CRMP）同源结构域以及PSD - 95、盘状大蛋白、紧密连接蛋白 - 1结合结构域。树突模式形成需要锌结合结构域和CRMP同源结构域。此外，cypin通过其CRMP同源结构域与微管蛋白结合以促进微管组装。利用酵母双杂交筛选大鼠脑cDNA文库，以缺失羧基末端八个氨基酸的cypin作为诱饵，我们鉴定出snapin为cypin的结合伴侣。在此，我们通过亲和层析和免疫共沉淀表明，snapin的羧基末端卷曲螺旋结构域（H2）是cypin结合所必需的。此外，snapin与cypin的CRMP同源结构域结合，而微管蛋白也结合于此。我们还表明，snapin与微管蛋白竞争结合cypin，导致微管组装减少。随后，在海马神经元原代培养物中过表达snapin会导致这些神经元上的初级树突减少，分支概率增加。总之，我们的数据表明，snapin通过调节cypin促进的微管组装来调控发育中神经元的树突数量。
