Functional and Structural Studies of CD74 Activation

描述(申请人提供)：CD74是一种细胞表面受体，但更为人所知的是人类组织相容白细胞抗原(HLA11)类关联的不变链，它将II类从内质网运输到与内体融合的小泡。CD74在细胞表面存在的生理作用尚不清楚。一些实验表明，人类白细胞抗原DR特异性单抗的快速摄取和细胞表面的多肽负载是必需的，但细胞表面的CD74并不总是与细胞表面的人类白细胞抗原II类分子的表达相关。最近，CD74被鉴定为细胞因子巨噬细胞移动抑制因子(MIF)的细胞表面受体。MIF是一种促炎蛋白，细胞暴露在微生物或其产物中时会分泌这种蛋白。在急性炎症(内毒素血症)和败血症休克(盲肠结扎和穿孔)模型中，这种蛋白水平的增加被证明是介导致死性的因素。抑制MIF在关节炎、急性结肠炎、急性感染和多种形式癌症的动物模型中显示出治疗效果。在人类中，高水平的MIF与这些疾病有关。该提议的假设是CD74介导了MIF的生物学效应。本研究旨在(1)研究与MIF生物学有关的各种信号通路(MAP激酶胞浆PLA2激活、COX-2的表达和激活、Toll样受体4的表达、糖皮质激素抗炎效应的反向调节、P53的抑制)对CD74的需求，(2)鉴定MIF催化位点与CD74结合之间的潜在关系，(3)表征MIF与CD74胞外区(SCD74)的复合体的三维结构，以及(4)使用定点和基于结构的突变来确定MIF和CD74上对结合和/或信号转导重要的残基。

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.