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性加工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质分解过程。直到最近，只有羧基肽酶E(CPE)与神经内分泌细胞中的肽/蛋白质加工有关。我们的发现，脂肪/肥胖小鼠由于点突变而缺乏CPE活性，但仍然能够进行有限的肽/蛋白质处理，这表明另一种羧基肽酶在体内参与了这一处理步骤。在神经内分泌细胞的分泌途径中寻找其他类似CPE的酶导致我们发现了羧基肽酶(CPD)，它是一种完整的膜蛋白，含有58个残基的细胞质C末端尾巴。在考虑酶的生理作用时，一个关键问题是细胞内的位置。我们已经发现，CPD主要存在于跨高尔基体网络(TGN)，循环到细胞表面，然后返回TGN。CPD的磷酸化对贩运的作用将是第一个具体目标的重点。CPD的细胞质尾部含有几个共同的磷酸化位点。在目标2中，将通过缺失和点突变分析来研究CPD胞质尾部内的这些元件和其他元件的功能。由于对TGN蛋白的详细研究很少，因此对CPD的研究对于了解参与TGN蛋白运输的特定元件具有重要意义。在目标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，一些与CPD胞浆区域相互作用的蛋白质已被鉴定，一个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。这将使我们更好地了解与CPD相互作用的蛋白质，无论是已知的还是新颖的。这些关于CPD的研究将促进我们对蛋白质在神经内分泌细胞中运输的了解。

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.