CELL BIOLOGY OF CARBOXYPEPTIDASE D

许多神经内分泌多肽和蛋白质经过翻译后的蛋白水解处理以产生生物活性形式。例如，胰岛素和胰岛素受体都需要蛋白水解处理。直到最近，只有羧基肽酶E （CPE）与神经内分泌细胞中的肽/蛋白加工有关。我们发现，肥胖小鼠由于点突变而缺乏CPE活性，但仍然能够有限的肽/蛋白质加工，这表明另一种羧肽酶在体内参与了这一加工步骤。在神经内分泌细胞分泌途径中寻找其他cpe样酶的过程中，我们发现了羧基肽酶（CPD），这是一种完整的膜蛋白，含有58个残基细胞质c端尾部。考虑酶的生理作用的一个关键问题是细胞内的位置。我们发现CPD主要存在于反式高尔基网络（TGN）中，循环到细胞表面并返回TGN。CPD磷酸化对贩运的作用将是第一个具体目标的重点。CPD的细胞质尾部包含几个公认的磷酸化位点。这些和其他元件在CPD细胞质尾部的功能将在Aim 2中通过分析缺失和点突变来研究。由于对TGN蛋白的详细研究很少，因此对CPD的研究对于了解TGN蛋白转运的特定元件将具有重要意义。在目标3中，我们将研究与CPD的C端区域相互作用的蛋白质。最近，已经确定了几种与CPD细胞质区相互作用的蛋白质，其总体目标是确定这些相互作用的生理意义。此外，我们还将使用亲和层析和酵母双杂交方法筛选其他cpd结合蛋白。这将提供一个更好的理解蛋白质，无论是已知的还是新的，与CPD相互作用。这些关于CPD的研究将促进我们对神经内分泌细胞中蛋白质运输的认识。

Many neuroendocrine peptides and proteins undergo post- translational proteolytic processing to generate the bioactive forms. For example, both insulin and the insulin receptor require proteolytic processing. Until recently, only carboxypeptidase E (CPE) was associated with peptide/protein processing in neuroendocrine cells. Our finding that fat/fat mice lack CPE activity due to a point mutation but are still capable of limited peptide/protein processing suggests that another carboxypeptidase contributes to this processing step in vivo. A search for additional CPE-like enzymes in the secretory pathway of neuroendocrine cells led to our discovery of carboxypeptidase (CPD), an integral membrane protein that contains a 58 residue cytoplasmic C-terminal tail. A key issue in considering the physiological role of an enzyme is the intracellular location. We have found that CPD is primarily found in the trans Golgi network (TGN), cycles to the cell surface and returns to the TGN. The role of phosphorylation of CPD on trafficking will be the focus of the first specific aim. The cytoplasmic tail of CPD contains several consensus sites for phosphorylation. The function of these and other elements within the cytoplasmic tail of CPD will be studied in Aim 2 by analysis of deletion and point mutations. Since few TGN proteins have been studied in detail, the studies on CPD will be important for understanding the specific elements involved in trafficking of TGN proteins. In Aim 3, we will investigate proteins that interact with the C- terminal region of CPD. Recently, several proteins that interact with the cytosolic region of CPD have been identified, an he overall goal is to determine the physiological significance of these interactions. In addition, we will also screen for other CPD-binding proteins using affinity chromatography and the yeast two-hybrid method. This will provide a better understanding of the proteins, either known or novel, that interact with CPD. These studies on CPD will advance our knowledge of protein trafficking in neuroendocrine cells.