Functional and Structural Studies of CD74 Activation

描述（由申请人提供）：CD 74是一种细胞表面受体，但更为人所知的是人类组织相容性白细胞抗原（HLA）II类缔合不变链，其将II类从内质网转运至与核内体融合的囊泡。CD 74在细胞表面上的存在的生理作用尚不清楚。一些实验表明，这是必要的快速摄取的HLA-DR特异性单克隆抗体进入早期内体和肽加载在细胞表面上，但细胞表面CD 74并不总是与细胞表面表达的HLA II类分子。最近，CD 74已被鉴定为细胞因子巨噬细胞迁移抑制因子（MIF）的细胞表面受体。MIF是一种促炎蛋白，在暴露于微生物或其产物时由细胞分泌。这种蛋白质的水平增加已被证明介导急性炎症（内毒素血症）和脓毒性休克（盲肠结扎和穿孔）小鼠模型中的致死性。MIF的抑制在关节炎、急性结肠炎、急性感染和多种形式的癌症的动物模型中显示出治疗益处。在人类中，高水平的MIF与这些疾病有关。该建议的假设是，CD 74介导的MIF的生物学效应。本提案旨在（1）检查负责MIF生物学的各种信号通路中对CD 74的需求（MAP激酶细胞质PLA 2活化、考克斯-2的表达和活化、Toll样受体4的表达、糖皮质激素抗炎作用的反调节、p53的抑制），（2）表征MIF催化位点和CD 74结合之间的任何潜在关系，（3）表征MIF和CD 74胞外域（sCD 74）之间的复合物的三维结构，和（4）使用定点和基于结构的诱变来确定MIF和CD 74上对于结合和/或信号传导重要的残基。

DESCRIPTION (provided by applicant): CD74 is a cell surface receptor, but is better known as the human histocompatibility leukocyte antigen (HLA) class ll-assoociated invariant chain that transports class II from the endoplasmic reticulum to vesicles which fuse with endosomes. The physiological role for the presence of CD74 on the cell surface is not known. Some experiments show that it is necessary for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes and for peptide loading on the cell surface, but cell-surface CD74 does not always correlate with cell-surface expression of HLA class II molecules. More recently, CD74 has been identified as a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF). MIF is a pro-inflammatory protein that is secreted by cells upon exposure to microbes or their products. Increased levels of this protein have been shown to mediate lethality in mice models of acute inflammation (endotoxemia) and septic shock (cecal ligation and puncture). Inhibition of MIF shows therapeutic benefits in animal models of arthritis, acute colitis, acute infections, and multiple forms of cancer. In humans, high levels of MIF are associated with these diseases. The hypothesis of this proposal is that CD74 mediates the biological effects of MIF. This proposal aims to (1) examine the requirement for CD74 in various signaling pathways responsible for MIF biology (MAP kinase cytoplasmic PLA2 activation, expression and activation of COX-2, expression of Toll-like receptor 4, counter-regulation of glucocorticoid anti-inflammatory effects, inhibition of p53), (2) characterize any potential relationship between the MIF catalytic site and CD74 binding, (3) characterize the three dimensional structure of the complex between MIF and the ectodomain of CD74 (sCD74), and (4) use site-directed and structure-based mutagenesis to determine residues on MIF and CD74 that are important for binding and/or signaling.