Sortilin-dependent traffic to dense core secretory granules - Resubmission 01

描述（由申请人提供）：致密核颗粒是储存和释放生物活性肽的囊泡，存在于大多数真核生物中。在过去的二十年里，已经建立了详细的模型来解释蛋白质靶向分泌颗粒。这些模型最基本的方面是该途径中的蛋白质分选不依赖于典型的跨膜受体或受体接头。然而，最近在哺乳动物神经元中进行的实验令人惊讶地发现，骨源性神经营养因子（BDNF），一种对神经元维持很重要的肽，在一个尚未描述的途径中被靶向分泌颗粒，该途径涉及经典的溶酶体分选受体sortilin。此外，sortinin家族受体变异已被确定为糖尿病的危险因素，最近的研究表明它们在含胰岛素颗粒的形成中起直接作用。另一个挑战来自最近对果蝇和哺乳动物神经内分泌细胞的研究，这些研究表明AP-3接头复合物与分泌颗粒的形成有关。目前没有任何模型可以解释sortilin或AP-3的数据。分泌颗粒是纤毛虫嗜热四膜虫的一个显著特征。通过研究活性颗粒合成期间的基因表达，最近发现在这些条件下，四膜虫sortilin同源物与一组被认为是分泌颗粒形成基础的基因一起高度上调。这些基因产物包括假定的sortilin配体以及AP-3接头和其他可能参与sortilin贩运的细胞质机制。初步数据验证了这种基于基因表达的方法在揭示颗粒形成机制方面的价值，并证明了四膜虫中主要颗粒蛋白类别的分选需要排序蛋白。因此，该建议旨在了解sortinlins如何促进蛋白质靶向颗粒，以及涉及哪些其他细胞机制。为此，本提案的目的包括分析sortilin配体和与sortilin细胞质结构域相互作用的适配器蛋白。

DESCRIPTION (provided by applicant): Dense core granules are vesicles that store and release bioactive peptides, and are found throughout most eukaryotes. Over the past two decades, detailed models have been developed to explain protein targeting to secretory granules. The most fundamental aspect of these models is that protein sorting in this pathway does not rely on canonical transmembrane receptors or receptor adaptors. However, recent experiments in mammalian neurons led to the surprising finding that bone-derived neurotropic factor (BDNF), a peptide important for neuronal maintenance, is targeted to secretory granules in a pathway, as yet undescribed, that involves a classical lysosomal sorting receptor, sortilin. Moreover, sortilin-family receptor variants have been identified as risk factors for diabetes, and recent work suggests they play a direct role in formation of insulin-containing granules. Another challenge came from recent studies in Drosophila, and in mammalian neuroendocrine cells, which implicated the AP-3 adaptor complex in secretory granule formation. No current model explains the sortilin or AP-3 data. Secretory granules are a prominent feature in the ciliate Tetrahymena thermophila. By studying gene expression during periods of active granule synthesis, it was recently discovered that the Tetrahymena sortilin homologs are highly upregulated under these conditions, in combination with a set of genes that are proposed to underlie secretory granule formation. Those gene products include putative sortilin ligands as well as AP-3 adaptors and other cytoplasmic machinery that is likely to be involved in sortilin trafficking. Preliminary data validate the value of this gene expression-based approach to uncovering mechanisms involved in granulogenesis, and demonstrate that sorting of major classes of granule proteins in Tetrahymena requires sortilins. The proposal is therefore aimed at understanding how sortilins facilitate protein targeting to granules, and what other cellular machinery is involved. To that end, the aims of this proposal include the analysis of sortilin ligands and of adapter proteins that interact with sortilin cytoplasmic domains.