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

描述(申请人提供)：致密的核心颗粒是储存和释放生物活性多肽的囊泡，存在于大多数真核生物中。在过去的二十年里，已经开发了详细的模型来解释针对分泌颗粒的蛋白质靶标。这些模型最基本的方面是，这一途径中的蛋白质分类不依赖于规范的跨膜受体或受体适配器。然而，最近在哺乳动物神经元上的实验导致了令人惊讶的发现，骨源性神经营养因子(BDNF)是一种对神经元维持至关重要的多肽，其靶向是涉及经典溶酶体分选受体sortilin的途径中的分泌颗粒，该途径尚未被描述。此外，山梨素受体家族变异体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着直接作用。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，这表明AP-3适配器复合体参与了分泌颗粒的形成。目前还没有模型可以解释索氏菌素或AP-3的数据。分泌颗粒是嗜热四膜虫纤毛虫的显著特征。通过研究活跃颗粒合成期间的基因表达，最近发现在这些条件下，四膜虫索蒂林同系物与一组被认为是分泌颗粒形成基础的基因结合在一起高度上调。这些基因产物包括推定的山梨素配体，以及AP-3接头和其他可能参与山梨素运输的细胞质机制。初步数据验证了这种基于基因表达的方法在揭示颗粒发生机制方面的价值，并证明了对四膜目主要颗粒蛋白类别的分类需要山梨醇。因此，该提案旨在了解山梨素是如何促进蛋白质靶向颗粒的，以及涉及哪些其他细胞机制。为此，这项建议的目的包括分析山梨素配体和与山梨素细胞质结构域相互作用的适配蛋白。

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.