Wang L H, Strittmatter S M
Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06250, USA.
J Neurochem. 1997 Dec;69(6):2261-9. doi: 10.1046/j.1471-4159.1997.69062261.x.
The cytoplasmic collapsin response mediator protein CRMP62 is involved in the signaling cascade initiated by collapsin-1, which collapses neuronal growth cones. To investigate the mechanism of CRMP action, we screened mouse and human fetal cDNA libraries by the yeast two-hybrid method with CRMP as bait. Clones encoding CRMP1 and CRMP4 were isolated, suggesting that the CRMPs form multimers. This finding was confirmed by expressing various rat CRMP cDNAs in the yeast two-hybrid system. Rat CRMP isoforms show differential association with one another. Heterooligomerization is preferred in both two-hybrid and in vitro binding assays. Purified bovine brain CRMP migrates as a tetramer during size exclusion chromatography. Examination of binding with truncated forms of CRMPs indicates that the avid association of CRMPs requires nearly intact proteins. Through the analysis of CRMP chimeras, CRMP amino acids 8-134 and 281-435 are found to be essential for CRMP oligomerization. The tetrameric structure of CRMP resembles that of liver dihydropyrimidinase (DHPase), a protein that shares sequence similarity with the CRMPs. Although purified brain CRMP does not hydrolyze several DHPase substrates, it is likely that a related activity accounts for CRMP participation in neuronal growth cone signaling.
细胞质塌陷反应介导蛋白CRMP62参与由塌陷蛋白-1启动的信号级联反应，塌陷蛋白-1可使神经元生长锥塌陷。为了研究CRMP的作用机制，我们以CRMP为诱饵，通过酵母双杂交法筛选小鼠和人类胎儿cDNA文库。分离出了编码CRMP1和CRMP4的克隆，这表明CRMPs形成多聚体。在酵母双杂交系统中表达各种大鼠CRMP cDNA证实了这一发现。大鼠CRMP异构体彼此之间的结合存在差异。在双杂交和体外结合试验中，异源寡聚化更为常见。纯化的牛脑CRMP在尺寸排阻色谱中以四聚体形式迁移。对与截短形式的CRMPs结合的研究表明，CRMPs的紧密结合需要近乎完整的蛋白质。通过对CRMP嵌合体的分析，发现CRMP的8-134和281-435位氨基酸对于CRMP寡聚化至关重要。CRMP的四聚体结构类似于肝脏二氢嘧啶酶（DHPase），一种与CRMPs具有序列相似性的蛋白质。尽管纯化的脑CRMP不能水解几种DHPase底物，但很可能一种相关活性解释了CRMP参与神经元生长锥信号传导的现象。
