Elucidation of molecular signaling mechanism of axonal guidance

溃缩蛋白反应介导蛋白（CRMPs）已被牵连在轴突导向的信号传导，包括信号蛋白。我以前已经确定了一种新的CRMP相关蛋白，命名为CRMP相关分子的CRAM，属于unc-33基因家族。推导的氨基酸序列表明，CRAM基因编码563个氨基酸的蛋白质，并显示与二氢嘧啶酶（DHdR）的57%的同源性和与CRMPs的50- 51%的同源性。系统发育树分析表明，CRAM序列与DHE 2的相似性最高，与4种CRMP的差异最大，表明CRAM与DHE 2的亲缘关系较近。CRAM在脑中的表达是显著的，并且在胎儿和新生儿中尤其高，但在成人脑中降低到非常低的水平。因此，CRAM是该基因家族的一个独特的新成员，并且可能在轴突引导信号传导中发挥不同于其他四种CRMP的作用。此外，我最近报道了CRAM与几种细胞蛋白，包括酪氨酸激酶Fes/Fps和线粒体Septin相关。免疫组织化学分析显示CRAMin在生长锥的丝状伪足中积累。使用细胞松弛素D的实验表明，CRAM的丝状伪足定位是独立的丝状肌动蛋白。CRAM在神经元细胞中的过表达显著促进丝状伪足的生长，并导致额外生长锥的形成，从而获得对semaphorin-3A刺激的抗性。最后，敲低CRAM通过使用RNA干扰阻断丝状伪足的形成，并揭示了异常形态的生长锥。我建议，CRAM调节丝状伪足的动态和生长锥的发展，从而限制生长锥的反应，排斥指导线索。

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. I have previously identified a novel CRMP-associated protein, designated CRAM for CRMP-Associated Molecule that belongs to the unc-33 gene family. The deduced amino acid sequence reveals that the CRAM gene encodes a protein of 563 amino acids and shows 57 % identity with dihydropyrimidinase (DHPase) and 50-51 % identity with CRMPs. A phylogenetic tree analysis indicates that CRAM sequence shows the greatest similarity with DHPase and divergence from the four CRMPs, indicating that CRAM is more closely related to DHPase than to the four CRMPs. The expression of CRAM is remarkable for brain, and especially high in fetal and neonatal, but decreases to very low levels in adult brain. Thus, CRAM is a unique new member of this gene family and may play a role in axonal guidance signaling distinct from other four CRMPs. Furthermore I have recently reported that CRAM was associated with several cellular proteins including tyrosine kinase Fes/Fps and mitochondrial septin.In this study, I have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAMin the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. I propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.