NUCLEOSIDE TRANSPORTERS--STRUCTURE/FUNCTION AND REGULATI

核苷转运蛋白在将合成核苷药物转运到靶细胞中发挥着重要作用，这些药物在靶细胞中被代谢并产生细胞毒性。核苷类药物用于化疗(如：在白血病中的Ara C)和抗病毒药物(例如。AZT治疗艾滋病)。从药理学上讲，Na独立核苷转运系统可分为硝基苄基硫代肌苷（NBMPR）敏感系统（ES）和NBMPR不敏感系统（EI）。NBMPR是核苷转运的抑制剂。最近，我们实验室克隆了人类na不依赖的平衡核苷转运蛋白（ENT1（编码ES）和ENT2（编码EI）），并开发了核苷转运蛋白缺陷细胞系PK15NTD细胞。这将使我们能够研究核苷转运体的结构/功能关系以及作为核苷转运体底物的核苷和核苷药物的结构决定因素。通过在分子水平上对核苷转运的了解，一个长期目标是促进核苷类似物药物的设计，以治疗疾病。在这个应用中，我们将从功能和药理学上对克隆的在PK15NTD细胞中稳定表达的人类ENT1和ENT2进行表征（Aim 1）。从克隆蛋白中获得的结果将与人结肠癌细胞株T84的内源性ES和EI转运蛋白进行比较。利用动力学研究，我们将确定核苷通过ENT1和ENT2运输的结构决定因素。在Aim 2中，我们将研究ENT1和ENT2的结构/功能关系。我们将定义糖基化在ENT1和ENT2功能中的作用，因为糖基化对核苷运输很重要。核苷转运蛋白的功能是硫醇敏感的，在ENT2中硫醇敏感组是表面的，而在ENT1中是细胞质的。我们建议鉴定赋予ENT1和ENT2硫醇敏感性的半胱氨酸残基。ENT1和ENT2对NBMPR的敏感性相差3000倍，对鸟苷和胞苷转运的亲和力相差8-10倍，将构建ENT1/ENT2嵌合体来鉴定ENT1和ENT2的结合域和底物识别域。蛋白激酶C （PKC）在T84细胞中抑制克隆的ENT1和ENT2以及内源性ES和EI转运蛋白。第三个目的是通过实验研究PKC抑制ENT1和ENT2的分子机制。这些拟议的研究将提供对核苷转运的分子机制和激酶调控的见解，以及核苷转运体转运核苷/核苷药物的结构决定因素。

Nucleoside transporters are important in transporting synthetic nucleoside drugs into their target cells where these drugs are metabolized and become cytotoxic. Nucleoside drugs are used in chemotherapy (eg. Ara C in leukemia) and as antiviral agents (eg. AZT in the treatment of AIDS). Pharmacologically, the Na independent nucleoside transport systems can be separated into the nitrobenzylthioinosine (NBMPR) sensitive (ES) and the NBMPR insensitive (EI) systems. NBMPR is an inhibitor of nucleoside transport. Recently, our laboratory cloned the human Na-independent Equilibrative Nucleoside Transporters (ENT1(encoding ES) and ENT2 (encoding EI)) and developed a Nucleoside Transporter Deficient cell line, PK15NTD cells. This will allow us to study the structure/function relationship of the nucleoside transporters and the structural determinants of nucleosides and nucleoside drugs as substrates of the nucleoside transporters. Through the knowledge of nucleoside transport at the molecular level, a long term goal is to facilitate the design of nucleoside analogue drugs for the treatment of diseases. In this application, we will characterize functionally and pharmacologically the cloned human ENT1 and ENT2 stably expressed in PK15NTD cells (Aim 1). Results obtained from the cloned proteins will be compared with the endogenous ES and EI transporters in human colonic cancer cell line, T84. Using kinetic studies, we will identify structural determinants of nucleosides for the transport by ENT1 and ENT2. In Aim 2, we will study the structure/function relationship of ENT1 and ENT2. We will define the role of glycosylation in the function of ENT1 and ENT2 since glycosylation is important for nucleoside transport. Nucleoside transporter function is thiol-sensitive and the thiol sensitive group is exofacial in ENT2 but is cytoplasmic in ENT1. We propose to identify cysteine residues that confer thiol sensitivity of ENT1 and ENT2. ENT1 and ENT2 have a 3000 fold difference in sensitivity to NBMPR and an 8-10 fold difference in the affinity for guanosine and cytidine transport, ENT1/ENT2 chimeras will be constructed to identify the binding domain and substrate recognition domain in ENT1 and ENT2. Protein kinase C (PKC) inhibits the cloned ENT1 and ENT2 and endogenous ES and EI transporters in T84 cells. Experiments are proposed in the third aim to study the molecular mechanisms of how PKC inhibits ENT1 and ENT2. These proposed studies should provide insights into the molecular mechanisms and kinase regulation of nucleoside transport, and the structural determinants of nucleosides/nucleoside drugs for transport by the nucleoside transporters.