Biosynthesis, Processing And Secretion Of Neuropeptides

在神经内分泌细胞中，前神经肽向调节分泌通路（RSP）的细胞内分选对活性激素的加工、储存和释放至关重要。研究了促肾上腺皮质激素（POMC）、促acth /内啡肽（acth /endorphin）和胰岛素原在调节分泌通路（RSP）上的分选。我们发现，当这些前蛋白从内质网的合成位点穿过细胞到达反式高尔基网络（TGN）时，作为一个浓缩步骤，它们经历了同型寡聚化，在那里它们被分类成受调节的分泌途径的致密核颗粒进行加工和分泌。定点诱变研究发现了一个一致的分选基序，由两个酸性残基组成，彼此相距12-15A，暴露在这些分子的表面，以及两个疏水残基，距离酸性残基5-7A，这是分选到RSP所必需的。我们鉴定出一种对POMC和前胰岛素分选信号具有特异性的RSP分选受体为膜羧基肽酶E （CPE）。激素原分选基序中的两个酸性残基特异性地与分选受体羧基肽酶E （CPE）的两个碱性残基Arg255和Lys260相互作用，对RSP进行分选。我们发现CPE是一种跨膜蛋白，通过其c端两亲结构域锚定在TGN中独特的富含胆固醇-鞘糖脂的微结构域，称为筏。用洛伐他汀处理细胞或截断CPE的c端导致胆固醇消耗，导致CPE和POMC对RSP缺乏分选。因此，膜结合对于激素原分选受体功能和CPE在TGN的分选是必不可少的。在神经2a细胞的CPE零克隆中转染Arg255和Lys260突变为丙氨酸的CPE突变体，并将CPE显性阴性突变体转染到at -20细胞中，导致POMC对组成通路的错误分选，这表明CPE分选域的碱性残基在体内与POMC分选信号中的酸性残基相互作用，从而对RSP进行分选。这些研究为在神经内分泌细胞中将前激素定向到受调节的分泌通路的分选信号/受体介导机制提供了证据。此外，我们的研究还表明，高胰岛素原血症患者血浆中高水平分泌的突变原胰岛素是由于突变原胰岛素在被调节的分泌途径上的分选缺陷，这是由于其结构的遗传改变导致它们无法与分选受体CPE结合。我们的研究还表明，CPE是通过一种与脂筏相关的机制进行分类的。此外，CPE在胞吐作用后通过ARF6依赖途径再循环到TGN并重复使用。

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.