The Role of Megalin in Craniofacial Development

描述（由申请人提供）：颅面畸形，包括一系列前脑缺陷，统称为前脑无裂畸形（HPE），是人类最常见的出生缺陷。定义HPE的特征的变化表明多个基因导致该综合征。Sonic Hedgehog（Shh）基因的突变是一种信号蛋白，以其在组织图案中的关键作用而闻名，包括面部，大脑，脊髓和眼睛的结构已被证明会导致HPE。HPE表型也已在巨蛋白（一种与LDL受体相关的内吞受体）表达缺陷的小鼠中描述。我们的研究揭示了megalin，Shh和Shh受体patched-1（Ptc-1）之间以前未知的关系。这些发现包括巨蛋白结合Shh并介导其内吞作用，以及巨蛋白还影响Ptc-I的亚细胞运输和蛋白水解加工。因此，W8假设巨蛋白活性是Shh控制颅面形态发生的机制的组成部分。为了验证这一假设，实验提出了调查巨蛋白介导的细胞内运输的Shh。我们的研究结果表明，巨蛋白内化的Shh绕过溶酶体。鉴于已知巨蛋白介导几种配体的胞吞转运的事实，主要重点是确定巨蛋白是否介导Shh的跨上皮转运（胞吞转运）。这种转胞吞作用将代表一种新的机制来解释发育过程中的长距离Shh信号传导。实验还将集中于建立megalin和Ptc-1之间的关系，包括表征megalin在调节Ptc-1靶向溶酶体和Ptc-1的蛋白水解加工中的作用。Ptc-1的蛋白水解加工是一个新的观察，我们发现Ptc-1的羧基末端片段易位到核仁，提示在基因表达调控中的作用，这使得Ptc-1的蛋白水解加工更有趣。另外的实验是针对建立的作用，巨蛋白调节Shh依赖的基因表达重要的眼睛发育和神经元的规范。拟议的实验预计将导致对Shh信号控制颅面发育的机制的新见解。

DESCRIPTION (provided by applicant): Craniofacial malformations, including a range of forebrain defects collectively known as holoprosencephaly (HPE), are the most common birth defects that occur in humans. The variation in the characteristics defining HPE suggests that multiple genes contribute to the syndrome. Mutation of the gene for sonic hedgehog (Shh), a signaling protein known for its crucial role in patterning tissues, including structures of the face, brain, spinal cord and eye has been shown to cause HPE. A HPE phenotype has also been described in mice that are deficient in the expression of megalin, an endocytic receptor related to the LDL receptor. Our studies have revealed previously unknown relationships between megalin, Shh and the Shh receptor, patched-1 (Ptc-1). These include the findings that megalin binds Shh and mediates its endocytosis and that megalin also influences subcellular trafficking and proteolytic processing of Ptc-l. W8 therefore hypothesize that megalin activity is an integral part of the mechanism by which Shh acts to control craniofacial morphogenesis. To test this hypothesis, experimentation is proposed to investigate megalin-mediated intracellular trafficking of Shh. Our results indicate that megalin-internalized Shh bypasses lysosomes. Given the fact that megalin is known to mediate transcytosis of several ligands; a major emphasis is to determine if megalin mediates transepithelial transport (transcytosis) of Shh. Such transcytosis would represent a novel mechanism to explain long range Shh signaling during development. Experimentation will also focus on establishing the relationship between megalin and Ptc-1, including characterization of the role of megalin in regulating targeting of Ptc-1 to lysosomes and proteolytic processing of Ptc-l. Proteolytic processing of Ptc-1 is a new observation that is made more interesting by our finding that a carboxy-terminal fragment of Ptc-1 is translocated to the nucleolus, suggestive of a role in regulation of gene expression. Additional experimentation is directed towards establishing the role of megalin in regulating Shh-dependent gene expression important for eye development and neuron specification. The proposed experimentation is expected to lead to new insights into the mechanism by which Shh signaling controls craniofacial development.