CELL BIOLOGY OF CARBOXYPEPTIDASE D

许多神经内分泌肽和蛋白质经过翻译后蛋白水解加工而产生生物活性形式。 例如，胰岛素和胰岛素受体都需要蛋白水解加工。 直到最近，只有羧肽酶E（CPE）与神经内分泌细胞中的肽/蛋白质加工有关。 我们的发现，脂肪/脂肪小鼠缺乏CPE活性，由于点突变，但仍然能够有限的肽/蛋白质加工表明，另一种羧肽酶有助于这一处理步骤在体内。 在神经内分泌细胞的分泌途径中寻找额外的CPE样酶导致我们发现羧肽酶（CPD），一种包含58个残基的细胞质C-末端尾的完整膜蛋白。 考虑酶的生理作用的一个关键问题是细胞内的位置。我们已经发现CPD主要存在于反式高尔基体网络（TGN）中，循环到细胞表面并返回到TGN。第一个具体目标的重点是使持续专业发展磷酸化对贩运的作用。 CPD的胞质尾区含有几个磷酸化的共有位点。 在目的2中，将通过分析缺失和点突变来研究CPD胞质尾区内这些和其他元件的功能。 由于很少有TGN蛋白的详细研究，CPD的研究将是重要的了解参与运输的TGN蛋白的特定元件。 在目的3中，我们将研究与CPD的C-末端区域相互作用的蛋白质。最近，已经鉴定了几种与CPD的胞质区域相互作用的蛋白质，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了解蛋白质，无论是已知的或新的，与CPD相互作用。CPD的这些研究将推进我们的知识在神经内分泌细胞中的蛋白质贩运。

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.