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，统称为全前脑畸形（HPE），是人类最常见的出生缺陷。定义HPE的特征的变化表明多种基因导致了该综合征。sonic hedgehog基因（Shh）是一种信号蛋白，在包括面部、大脑、脊髓和眼睛在内的组织结构中起着至关重要的作用，其基因突变已被证明会导致HPE。在与低密度脂蛋白受体相关的内吞受体meggalin表达缺乏的小鼠中也描述了HPE表型。我们的研究揭示了先前未知的meggalin、Shh和Shh受体补丁-1 （Ptc-1）之间的关系。这些发现包括meggalin结合Shh并介导其内吞作用，meggalin还影响亚细胞运输和ptc - 1的蛋白水解加工。因此，W8假设meggalin活性是Shh控制颅面形态发生机制的一个组成部分。为了验证这一假设，我们提出了研究巨噬细胞蛋白介导的Shh细胞内运输的实验。我们的研究结果表明，巨噬蛋白内化的Shh绕过溶酶体。已知meggalin介导几种配体的胞吞作用；一个主要的重点是确定meggalin是否介导Shh的经上皮转运（胞吞作用）。这种胞吞作用可能代表了一种解释发育过程中长距离Shh信号传导的新机制。实验还将重点建立巨噬细胞苷与Ptc-1之间的关系，包括表征巨噬细胞苷在调节Ptc-1靶向溶酶体和Ptc-1蛋白水解加工中的作用。Ptc-1的蛋白水解过程是一个新的观察结果，我们发现Ptc-1的一个羧基末端片段被转移到核仁上，这表明Ptc-1在基因表达的调节中起作用，这使得Ptc-1的蛋白水解过程变得更加有趣。另外的实验旨在确定meggalin在调节shh依赖性基因表达中的作用，这对眼睛发育和神经元规范很重要。这项拟议中的实验有望为Shh信号控制颅面发育的机制提供新的见解。

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.