The Role of Megalin in Craniofacial Development

描述(申请人提供)：颅面畸形，包括一系列统称为无前脑(HPE)的前脑缺陷，是人类最常见的出生缺陷。定义HPE的特征的差异表明，多个基因与该综合征有关。Sonic hedgehog(Shh)是一种信号蛋白，以其在面部、大脑、脊髓和眼睛等组织的图案形成中的关键作用而闻名，已被证明是导致HPE的基因突变。巨蛋白是一种与低密度脂蛋白受体相关的内源性受体，在缺乏表达的小鼠中也被描述为HPE的表型。我们的研究揭示了先前未知的megalin、Shh和Shh受体Patched-1(PTC-1)之间的关系。这些发现包括：巨蛋白与Shh结合并介导其内吞作用，以及巨蛋白还影响PTC-L的亚细胞运输和蛋白分解过程。因此，W8假设巨蛋白活性是Shh控制头面部形态发生的机制的一个组成部分。为了验证这一假说，建议通过实验来研究巨蛋白介导的Shh的细胞内运输。我们的结果表明，巨蛋白内化的Shh绕过了溶酶体。鉴于已知的事实是，巨蛋白可以介导几种配体的跨细胞转运；一个主要的重点是确定巨蛋白是否介导Shh的跨上皮转运(跨细胞转运)。这种跨细胞作用将代表一种新的机制来解释发育过程中的长程Shh信号。实验还将侧重于建立巨蛋白和PTC-1之间的关系，包括表征巨蛋白在调节PTC-1靶向溶酶体和PTC-L的蛋白降解过程中的作用。PTC-1的蛋白降解过程是一个新的观察结果，我们的发现使其变得更加有趣，因为我们的发现将PTC-1的一个羧基末端片段移位到核仁，这暗示了它在基因表达调控中的作用。另外的实验旨在确定megalin在调节Shh依赖的基因表达中的作用，该基因对眼睛发育和神经元指定非常重要。这项拟议的实验有望为Shh信号控制头面部发育的机制带来新的见解。

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.