MOLECULAR REGULATION OF FOLATE AND ANTIFOLATE TRANSPORT

该项目旨在表征“经典”还原性叶酸载体（RFC）的结构、功能和调控，RFC介导还原性叶酸辅助因子进入人体细胞的摄取。充足的叶酸供应对所有组织的正常生长、功能和发育至关重要，越来越多的证据表明叶酸缺乏会导致染色体不稳定和恶性转化。RFC对甲氨蝶呤（methotrexate， MTX）及相关抗叶酸化疗药物的抗肿瘤效果也至关重要，RFC表达和功能的缺陷是MTX耐药发生的关键因素。然而，直到最近，RFC的分子表征是有限的，这在很大程度上是由于缺乏在人类细胞中研究该系统的关键试剂（抗体，cdna）。本申请中概述的研究将扩展我们实验室最近取得的进展，包括从异质转录本中分离和表征多种人类RFC cdna，确定人类RFC基因的组织和结构，以及双启动子的演示和假定的顺式调控元件的鉴定。制备并验证了人RFC的敏感抗体，并开始研究转运介导的MTX抗性的分子改变，以及RFC中关键氨基酸（Asn58, Gly44, Ser127）的功能重要性。为了这个项目的继续，我们将（Aim 1）通过表位插入诱变验证RFC膜拓扑的计算机模型，并通过亲和标记和位点定向和随机诱变建立RFC蛋白的结构和功能元件。我们将（Aim 2）在一个诱导启动子的控制下，在运输受损的人类细胞中表达表位标记的RFC，以确定转运功能随载体表达的变化，以及蛋白质与RFC相互作用在调节转运功能中的作用。进一步的研究将集中在RFC磷酸化在调节叶酸和反叶酸转运中的作用。我们将（目标3）进一步表征双RFC启动子中的顺式调控元件，并研究启动子利用或涉及上游非编码外显子的RNA剪接的变化如何调节正常组织和肿瘤中的RFC水平和功能。从这些实验中获得的知识将用于表征p53介导的RFC基因表达下调的机制，以及转录和转录后水平的改变如何导致耐药肿瘤中RFC表达降低和MTX转运受损。总的来说，我们的研究应该阐明RFC表达的结构和调控决定因素，叶酸和抗叶酸底物结合和膜易位的蛋白质决定因素，以及MTX耐药肿瘤细胞中RFC功能受损的分子基础。

This project is designed to characterize the structure, function, and regulation of the "classical" reduced folate carrier (RFC) which mediates uptake of reduced folate cofactors into human cells. Adequate supplies of folates are essential for normal growth, function, and development of all tissues and there is growing evidence that folate deficiency contributes to chromosomal instability and malignant transformation. RFC is also critical to the antitumor effectiveness of methotrexate (MTX) and related antifolate chemotherapeutic drugs, and defects in the expression and function of RFC are key factors in the development of MTX resistance. However, until recently, the molecular characterization of RFC was limited, in large part due to the lack of critical reagents (antibodies, cDNAs) for studying this system in human cells. The investigation outlined in this application will expand on recent advances made by our laboratory including the isolation and characterization of multiple human RFC cDNAs arising from heterogeneous transcripts, the determination of the organization and structure of the human RFC gene, and the demonstration of dual promoters and identification of putative cis regulatory elements. Sensitive antibodies to human RFC were prepared and verified, and studies were begun to characterize the molecular alterations responsible for transport- mediated MTX resistance, and the functional importance of key amino acids (Asn58, Gly44, Ser127) in RFC by site-directed mutagenesis. For the continuation of this project, we will (Aim 1) verify computer models of RFC membrane topology by epitope insertion mutagenesis, and establish the structural and functional elements of the RFC protein by affinity labeling and site-directed and random mutagenesis. We will (Aim 2) express epitope-tagged RFC in transport-impaired human cells under control of an inducible promoter, in order to identify changes in transport function with carrier expression, and the roles of protein interactions with RFC in regulating transport function. Additional studies will focus on the role of RFC phosphorylation in regulating folate and antifolate transport. We will (Aim 3) further characterize the cis regulatory elements in the dual RFC promoters and investigate how changes in promoter utilization or RNA splicing involving upstream non-coding exons regulate RFC levels and function in normal tissues and tumors. The knowledge gained from these experiments will be used to characterize the mechanisms of p53- mediated downregulation of RFC gene expression, and how alterations at both the transcriptional and posttranscriptional levels result in decreased RFC expression and impaired MTX transport in drug resistant tumors. Collectively, our studies should clarify the structural and regulatory determinants of RFC expression, the protein determinants of folate and antifolate substrate binding and membrane translocation, and the molecular bases of impaired RFC function in MTX resistant tumor cells.