MCP--MV/C3B/C4B BINDING SITES AND ISOFORM VARIATION

描述（改编自申请人的摘要）：超过 二十年来，申请人已经在一个 努力了解它在健康和疾病中的作用。 早些时候 研究集中在豚鼠、小鼠和人类C4的生物学上。 以来 20世纪80年代，注意力集中在补体受体和调节因子上。 proteins. 一种这样的调节剂，膜辅因子蛋白（MCP; CD 46）， 通过作为辅因子与因子I一起沿着起作用来保护宿主组织， 在自体细胞上存款的C3 b和C4 b。 以来 申请人在1984年鉴定MCP为C3结合蛋白，其作用 逐渐扩展超出仅补体抑制剂的范围。 MCP 最近被鉴定为麻疹病毒（MV）受体， 用作相关病毒受体（牛瘟和犬瘟）的探针 犬瘟热）。 此外，MCP在生殖生物学方面也很有意义， 在精子和母胎界面的表达。 作为 作为补体激活的抑制剂，MCP被设计成 异种移植物并作为可溶性治疗剂产生。 因为 这些和其他显着的相互作用的MCP，三个具体的目标是 提出了 首先，为了鉴定MV、C3 b和 将表达和评价C4 b缺失/置换构建体 用于配体相互作用。 第二，MCP的细胞保护特性 将通过使用稳定转染的克隆比较同种型， 配体功能分析中同种型的等效拷贝数 结合、辅因子活性和细胞保护。 第三，功能性 将分析MCP的两个胞质尾的意义，因为它们 涉及高甘露糖前体（pro-MCP）的加工， 磷酸化 尾介导的四重 将通过使用MCP来确定pro-MCP处理的差异 表达每种尾、尾嵌合体和尾突变体的转染子， 确定是否存在内质网（ER）转运加速因子， ER或其他过程中的寡聚化解释了这一点 差 MCP的胞质尾区具有几个假定的 磷酸化位点。 为了确定MCP是否被磷酸化， 将评估尾部、MCP转染子、人类细胞和细胞系 在通过补体激活（或MCP上调）攻击后， 战略 完成本提案的具体目标将 不仅为补体系统提供相关信息， 也适用于其他领域，如生殖免疫学、异种移植 和传染病。

DESCRIPTION (Adapted from the applicant's abstract): For more than twenty years, the applicant has examined the complement system in an effort to understand its workings in health and disease. Earlier studies focused on the biology of guinea pig, mouse and human C4. Since the 1980's, attention has centered on complement receptors and regulatory proteins. One such regulator, Membrane Cofactor Protein (MCP; CD46), protects host tissue by acting as a cofactor, along with factor I, to inactivate C3b and C4b that deposit on autologous cells. Since the applicant identified MCP as a C3-binding protein in 1984, its role has progressively expanded beyond that of only a complement inhibitor. MCP was recently identified as the measles virus (MV) receptor and is being used as a probe for related virus receptors (rinderpest and canine distemper). Additionally, MCP is of interest in reproductive biology due to its expression on sperm and at the maternal-fetal interface. As an inhibitor of complement activation, MCP is being engineered into xenografts and produced as a soluble therapeutic agent. Because of these and other remarkable interactions of MCP, three specific aims are proposed. First, to identify the binding sites on MCP of MV, C3b and C4b, deletion/substitution constructs will be expressed and evaluated for ligand interaction. Second, the cytoprotective properties of MCP isoforms will be compared by using stably transfected clones bearing equivalent copy numbers of isoforms in functional analyses of ligand binding, cofactor activity and cytoprotection. Third, the functional significance of the two cytoplasmic tails of MCP will be analyzed as they relate to processing of high mannose precursors (pro-MCP) and to phosphorylation. The mechanism governing the tail-mediated fourfold difference in processing of pro-MCP will be determined by using MCP transfectants expressing each tail, tail chimeras and tail mutants to determine if an endoplasmic reticulum (ER) transport accelerating factor, oligomerization in the ER or another process accounts for this difference. The cytoplasmic tails of MCP possess several putative phosphorylation sites. To determine if MCP is phosphorylated on the tail, MCP transfectants, human cells and cell lines will be evaluated following challenge by a complement activating (or MCP upregulating) strategy. Completion of the specific goals of this proposal will provide relevant information not only for the complement system, but also for other areas such as reproductive immunology, xenotransplantation and infectious diseases.