Biosynthesis, Processing And Secretion Of Neuropeptides

在神经内分泌细胞处理、储存和释放活性激素的过程中，神经肽前体在调节分泌途径(RSP)中的分选是必不可少的。研究了前阿片黑素皮质素(POMC，前ACTH/内啡肽)和胰岛素原对调节分泌途径(RSP)的分选作用。我们发现，作为一个浓缩步骤，这些原蛋白经历了同型齐聚，因为它们从内质网中的合成部位穿过细胞到反高尔基网络(TGN)，在TGN中它们被分类为调节分泌途径的致密核心颗粒进行处理和分泌。定点突变研究发现，这些分子表面暴露的两个相距12-15A的酸性残基和两个疏水残基距离RSP所需的酸性残基5-7A，构成了一个整合的排序基序。一种专用于POMC和胰岛素原分选信号的RSP分选受体被鉴定为膜羧肽酶E(CPE)。前激素分选基序中的两个酸性残基与分选受体羧肽酶E(CPE)的两个碱性残基Arg255和Lys260特异地相互作用，影响对RSP的分选。我们发现CPE是一种跨膜蛋白，它通过其C末端的两亲性结构域锚定到TGN中独特的富含胆固醇-鞘糖脂的微结构域，即RAFT。用洛伐他汀处理细胞或截断CPE的C末端导致胆固醇耗竭，导致CPE和POMC对RSP缺乏分选。因此，膜结合对于前激素分选受体的功能和CPE在TGN的分选是必不可少的。将突变的CPE与Arg255和Lys260突变为丙氨酸的CPE分别导入Neuro2a细胞的CPE阴性克隆中，再将显性阴性的CPE突变株导入AtT-20细胞中，导致POMC向本构途径的错误分选，表明CPE分选结构域中的碱性残基与体内POMC分选信号中的酸性残基相互作用而影响对RSP的分选。这些研究为神经内分泌细胞中前激素靶向调节分泌途径的分选信号/受体调节机制提供了证据。此外，我们的研究还表明，高胰岛素血症患者血浆中分泌的高水平突变的胰岛素是由于突变的胰岛素不能与分类受体CPE结合而导致的缺陷，这是由于它们的结构发生了遗传变化。我们的研究还表明，CPE是通过与脂筏相关的机制分类的。此外，CPE在胞吐时通过ARF6依赖的途径循环回到TGN并重复使用。 在另一个项目中，我们发现了一个主要的开/关开关，嗜铬粒素A(CGA)，它控制着神经内分泌细胞中TGN大致密核颗粒(LDCG)的形成。在缺乏CGA、LDCGs和激素分泌调控的突变型内分泌细胞系6T3中，CGA基因的转染使其恢复了野生型表型。我们利用基因芯片比较了缺乏LDCGs的6T3细胞和稳定转染CGA的6T3细胞的基因表达。在表达CGA的6T3细胞中，水通道蛋白1(AQ1)和颗粒素基因高度上调，表明CGA在转录水平上发挥了新的作用。这些蛋白质似乎存在于分泌颗粒中，可能在调节内分泌和神经内分泌细胞的胞吐和激素分泌方面发挥重要作用。

The intracellular sorting of pro-neuropeptides to the regulated secretory pathway (RSP) is essential for processing, storage and release of active hormones in the neuroendocrine cell. The sorting of pro-opiomelanocortin (POMC, pro-ACTH/endorphin) and proinsulin to the regulated secretory pathway (RSP) was investigated. We show that these pro-proteins undergo homotypic oligomerization, as a concentration step, as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network(TGN)where they are sorted into dense-core granules of the regulated secretory pathway for processing and secretion. Site-directed mutagenesis studies identified a concensus sorting motif consisting of two acidic residues, 12-15A apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7A away from the acidic residues which are necessary for sorting to the RSP. A RSP sorting receptor that was specific for the sorting signal of POMC and pro-insulin was identified as membrane carboxypeptidase E (CPE). The two acidic residues in the prohormone sorting motif specifically interact with two basic residues, Arg255 and Lys260, of the sorting receptor, carboxypeptidase E (CPE), to effect sorting to the RSP . We showed that CPE is a transmembrane protein which is anchored via its C-terminal amphipathic domain to unique cholesterol-glycosphingolipid rich microdomains known as rafts, in the TGN. Cholesterol depletion by treatment of cells with lovastatin or truncation of C-terminus of CPE resulted in lack of sorting of CPE and POMC to the RSP. Thus membrane association is essential for the prohormone sorting receptor function and the sorting of CPE at the TGN. Transfection of a mutant CPE with Arg255 and Lys260 mutated to alanine in a CPE null clone of Neuro2a cells, and transfection of a dominant negative CPE mutant into AtT-20 cells caused missorting of POMC to the constitutive pathway, indicating that the basic residues in the sorting domain of CPE interacts with the acidic residues in the POMC sorting signal in vivo to effect sorting to the RSP. These studies provide evidence for a sorting signal/receptor mediated mechanism for targeting prohormones to the regulated secretory pathway in neuro-endocrine cells. In addition, our studies also showed that high levels of secreted mutant proinsulins in the plasma of patients with hyperproinsulinemia are due to defects in sorting of the mutant proinsulins to the regulated secretory pathway, stemming from their inability to bind to the sorting receptor, CPE as a result of genetic alterations to their structure. Our studies also show that CPE is sorted by a mechanism involving association with lipid rafts. Furthermore, CPE, upon exocytosis is recycled back to the TGN by an ARF6 dependent-pathway and reused. In another project, we uncovered a master on/off switch , chromogranin A (CgA), that controls the formation of large dense-core granules(LDCG) at the TGN in neuroendocrine cells. In a mutant endocrine cell line, 6T3, lacking CgA, LDCGs and regulated hormone secretion, transfection of CgA restored the wild type phenotype in these cells. We compared gene expression in 6T3 cells lacking LDCGs and 6T3cells stably transfected with CgA using microarrays. Aquaporin 1(AQ1, a water channel) and the granuphilin genes were highly up-regulated in 6T3 cells expressing CgA, indicating a new role of CgA at the transcriptional level. These proteins appear to be in secretory granules and may play an important role in regulating exocytosis and hormone secretion in endocrine and neuroendocrine cells.