Formation of the Regulated Secretory Pathway

描述（由申请方提供）：肽类激素、神经肽、生长因子和单胺的调节释放取决于它们在能够调节胞吐作用的大致密核心囊泡（LDCV）内的储存。然而，我们仍然非常了解蛋白质如何进入这种调节分泌途径（RSP），而不是赋予大多数新合成蛋白质立即释放的组成性分泌途径。在trans-Golgi网络（TGN）中，注定用于LDCV的蛋白聚集形成致密的核心，这表明内腔或可能的膜相互作用驱动LDCV生物发生，在随后的LDCV成熟过程中，注定用于其他细胞器的蛋白被去除。然而，我们以前已经确定了一个细胞质基序所需的囊泡单胺转运蛋白VMAT 2到LDCV的排序，这表明细胞溶质机械的作用。该基序中的突变增加了转运蛋白的细胞表面表达，显然是通过将其从调节途径转移到组成途径。 由于LDCV生物发生中的缺陷应表型复制VMAT 2中这些突变的影响，我们筛选了对RNAi高度敏感的果蝇S2细胞中转运蛋白的细胞表面表达增加。我们发现，S2细胞表达RSP和显着的，果蝇VMAT（dVMAT）包含相同的排序基序的哺乳动物转运蛋白，在这个基序的突变也增加了S2细胞的细胞表面表达。筛选7000果蝇序列保守的哺乳动物通过流式细胞术增加野生型dVMAT的表达，我们确定了一个小数目的基因，影响调节蛋白分泌。聚焦于异源四聚体衔接蛋白AP-3，因为其中两个亚基在筛选中得分为阳性，我们发现AP-3的缺失也会使哺乳动物细胞的分泌失调。虽然LDCV仍然形成在AP-3的情况下，我们发现，他们缺乏的蛋白质，如调节释放所需的突触结合蛋白。在前两个目标中，我们将确定AP-3如何有助于RSP的形成，通过检验AP-3的功能，以隔离货物注定为RSP的假设，在它的情况下，两个分泌途径混合。我们还发现AP-3相互作用蛋白VPS 41的敲低会使蛋白分泌失调，并且在第三个目的中，将测试VPS 41作为AP-3适配器的外壳蛋白的假设。我们还将分析扩展到缺乏AP-3和VPS 41的小鼠。这些结果将为未来研究LDCV形成的分子机制以及对生理、发育和疾病的影响提供基础。 公共卫生相关性：神经系统的信息处理以及整个生物体的许多生理过程都依赖于肽激素、神经肽和生长因子的调节释放，但我们对这些蛋白质如何靶向能够调节分泌的大而致密的核心囊泡知之甚少。在这个建议中，我们解决了神经内分泌细胞的这一基本特性的分子基础，对生理学，发育，行为和疾病具有重要意义。

DESCRIPTION (provided by applicant): The regulated release of peptide hormones, neural peptides, growth factors and monoamines depends on their storage inside large dense core vesicles (LDCVs) capable of regulated exocytosis. However, we still understand remarkably little about how proteins sort into this regulated secretory pathway (RSP) rather than the constitutive secretory pathway that confers the immediate release of most newly synthesized proteins. In the trans-Golgi network (TGN), proteins destined for LDCVs aggregate to form a dense core, suggesting that lumenal or possibly membrane interactions drive LDCV biogenesis, with proteins destined for other organelles removed during the subsequent process of LDCV maturation. However, we have previously identified a cytoplasmic motif required for the sorting of vesicular monoamine transporter VMAT2 into LDCVs, suggesting a role for cytosolic machinery. Mutations in this motif increase cell surface expression of the transporter, apparently by diverting it from the regulated to the constitutive pathway. Reasoning that a defect in LDCV biogenesis should phenocopy the effect of these mutations in VMAT2, we screened for increased cell surface expression of the transporter in Drosophila S2 cells, which are highly susceptible to RNAi. We find that S2 cells express an RSP and remarkably, Drosophila VMAT (dVMAT) contains the same sorting motif as the mammalian transporter, with mutations in this motif also increasing cell surface expression in S2 cells. Screening 7000 Drosophila sequences conserved to mammals by flow cytometry for increased expression of wild type dVMAT, we identified a small number of genes that affect regulated protein secretion. Focusing on the heterotetrameric adaptor protein AP-3 because two of the subunits scored positive in the screen, we found that loss of AP-3 also dysregulates secretion in mammalian cells. Although LDCVs still form in the absence of AP-3, we find that they lack the proteins such as synaptotagmin required for regulated release. In the first two aims, we will determine how AP-3 contributes to formation of the RSP by testing the hypothesis that AP-3 functions to segregate cargo destined for the RSP, and in its absence, the two secretory pathways mix. We have also found that knockdown of the AP-3-interacting protein VPS41 dysregulates protein secretion, and in the third aim, will test the hypothesis that VPS41 functions as a coat protein for the AP-3 adaptor. We will also extend the analysis to mice lacking AP-3 and VPS41. The results will provide a foundation for future work on the molecular mechanisms involved in LDCV formation and the consequences for physiology, development and disease. PUBLIC HEALTH RELEVANCE: Information processing by the nervous system and indeed many physiological processes throughout the organism depend on the regulated release of peptide hormones, neural peptides and growth factors, but we understand little about how these proteins target to the large dense core vesicles capable of regulated secretion. In this proposal, we address the molecular basis for this fundamental property of neuroendocrine cells with important implications for physiology, development, behavior and disease.