Graduate Program in Pharmaceutical Sciences and Pharmacogenomics, University of California San Francisco, San Francisco, California, United States of America ; Departments of Physiology and Neurology, University of California San Francisco, San Francisco, California, United States of America.
PLoS Genet. 2013;9(9):e1003812. doi: 10.1371/journal.pgen.1003812. Epub 2013 Sep 26.
The regulated secretion of peptide hormones, neural peptides and many growth factors depends on their sorting into large dense core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network, but the mechanisms that sort proteins to this regulated secretory pathway and the cytosolic machinery that produces LDCVs remain poorly understood. Recently, we used an RNAi screen to identify a role for heterotetrameric adaptor protein AP-3 in regulated secretion and in particular, LDCV formation. Indeed, mocha mice lacking AP-3 have a severe neurological and behavioral phenotype, but this has been attributed to a role for AP-3 in the endolysosomal rather than biosynthetic pathway. We therefore used mocha mice to determine whether loss of AP-3 also dysregulates peptide release in vivo. We find that adrenal chromaffin cells from mocha animals show increased constitutive exocytosis of both soluble cargo and LDCV membrane proteins, reducing the response to stimulation. We also observe increased basal release of both insulin and glucagon from pancreatic islet cells of mocha mice, suggesting a global disturbance in the release of peptide hormones. AP-3 exists as both ubiquitous and neuronal isoforms, but the analysis of mice lacking each of these isoforms individually and together shows that loss of both is required to reproduce the effect of the mocha mutation on the regulated pathway. In addition, we show that loss of the related adaptor protein AP-1 has a similar effect on regulated secretion but exacerbates the effect of AP-3 RNAi, suggesting distinct roles for the two adaptors in the regulated secretory pathway.
肽激素、神经肽和许多生长因子的调节分泌依赖于它们被分拣到能够进行调节性胞吐的大致密核心囊泡（LDCV）中。LDCV 在高尔基体内形成，但将蛋白质分拣到这种调节分泌途径的机制以及产生 LDCV 的细胞质机制仍知之甚少。最近，我们使用 RNAi 筛选鉴定了异源四聚体衔接蛋白 AP-3 在调节分泌中的作用，特别是在 LDCV 形成中的作用。事实上，缺乏 AP-3 的 mocha 小鼠具有严重的神经和行为表型，但这归因于 AP-3 在溶酶体而非生物合成途径中的作用。因此，我们使用 mocha 小鼠来确定 AP-3 的缺失是否也会在体内失调肽的释放。我们发现，mocha 动物的肾上腺嗜铬细胞瘤显示出可溶性货物和 LDCV 膜蛋白的组成型胞吐作用增加，从而降低了对刺激的反应。我们还观察到 mocha 小鼠胰岛细胞中胰岛素和胰高血糖素的基础释放增加，表明肽激素的释放存在普遍紊乱。AP-3 存在于普遍存在的和神经元亚型中，但对缺失每种亚型的小鼠以及同时缺失这两种亚型的小鼠的分析表明，缺失这两种亚型是重现 mocha 突变对调节途径的影响所必需的。此外，我们还表明，相关衔接蛋白 AP-1 的缺失对调节性分泌具有相似的影响，但会加剧 AP-3 RNAi 的影响，这表明这两种衔接蛋白在调节分泌途径中具有不同的作用。
