Biology of a New DAP12 Associated Receptor Family

描述(申请人提供)：通过快速克隆，我们已经识别出 一个与信号附件相关的新的小鼠受体家族 分子，DAP12。它们有一个类似免疫球蛋白的结构域，并且它们的表达 似乎仅限于单核/巨噬细胞系的细胞。因此，我们有 将它们命名为MDI受体(髓系细胞DAP12相关Ig样细胞 受体)。我们已经确定了3个包含MDI基因的重叠BAC，并已 将这些基因定位在染色体17D上。BAC的摘要显示，MDI 受体家族很小，可能仅限于MDI-1和MDI-1两个同源基因 MDI-2。MDI-1在MT2巨噬细胞中表达时与内源性 DAP12，并刺激一氧化氮的产生。只有3个其他受体是 已知在单核/巨噬细胞中与DAP12有关。缺铁小鼠 DAP12的免疫变化似乎存在于抗原提呈细胞中， 缺乏DAP12的人类患有神经和骨骼疾病，可能 分别反映小胶质细胞和破骨细胞的缺陷。因此，我们 希望确定MDI受体及其配体的功能。我们建议 五个具体目标： 具体目标1.确定MDI-1和MDI-2的配体。我们将创造出可溶的 MDI-1和MDI-2受体用于识别其他细胞上的配体，或者如果有指示， 从血清或其他来源，我们将寻求确定配体为 已知蛋白质，如果未知，则克隆配体(S)的cDNA. 明确目标2.确定MDI受体家族的范围。我们将进一步 探索单核/巨噬细胞cDNA文库以寻找MDI的其他成员 一家人。其次，我们将绘制MDI基因的图谱并对其进行测序 三个与MDI记录杂交的BAC。 明确MDI受体的表达范围。我们会 产生针对个别MDI受体的单抗，我们将使用 以评估MDI在不同细胞类型上的表面表达和在 细胞激活的不同阶段。 具体目标4.确定MDI结扎后的细胞反应。我们会研究 无论是受体转染型细胞还是新鲜制备的细胞。我们的研究将是 根据我们自己的结果和DAP12缺陷小鼠的表型和 人类。其中包括与杰森·赛斯特博士在趋化因子方面的合作 和趋化因子受体。 明确目的5.明确MDI-/-小鼠的表型。与 Nigel Killeen，我们将创造同时缺乏MDI-1和MDI-2的小鼠。再说一遍， 这些研究将以我们自己的结果以及表型为指导 DAP12基因缺陷的小鼠和人类。

DESCRIPTION (provided by the applicant): By express cloning, we have identified a new family of mouse receptors that associate with the signaling accessory molecule, DAP12. They have a single Ig-like domain, and their expression appears to be restricted to cells of monocyte/macrophage lineage. Thus, we have named them "MDI receptors" (for myeloid cell DAP12-associated Ig-like receptors). We have identified 3 overlapping BACs containing MDI genes and have mapped the genes to chromosome 17d. Digests of the BACs indicate that the MDI receptor family is small, probably limited to two homologous genes, MDI-1 and MDI-2. When expressed in MT2 macrophages, MDI-1 associates with endogenous DAP12 and stimulates the production of nitric oxide. Only 3 other receptors are known to associate with DAP12 in monocyte/macrophage cells. Mice deficient in Dap12 have immune alterations that appear to lie in antigen presenting cells, and humans deficient in DAP12 have neurologic and bone disorders that may reflect defects in microglial cells and osteoclasts, respectively. We therefore wish to define the functions of the MDI receptors and their ligands. We propose five specific aims: Specific Aim 1. Define the ligands for MDI-1 and MDI-2. We will create soluble MDI-1 and MDI-2 receptors to identify ligands on other cells or, if indicated, from serum or other sources, We will seek to identify the ligands either as known proteins or, if they are unknown, to clone the cDNA(s) for the ligand(s). Specific Aim 2. Define the extent of the MDI receptor family. We will further probe monocyte/macrophage cDNA libraries to seek additional members of the MDI family. Second we will map and sequence the MDI genes that are encoded in the three BACs that hybridize to MDI transcripts. Specific Aim 3. Define the range of expression of MDI receptors. We will produce monoclonal antibodies against individual MDI receptors, and we will use these to assess the surface expression of MDI on different cell types and at different stages of cell activation. Specific Aim 4. Define the cellular responses to ligation of MDI. We will study both receptor-transfected cells and freshly prepared cells. Our studies will be guided by our own results and by the phenotype of DAP12-deficient mice and humans. They include a collaboration with Dr. Jason Cyster regarding chemokines and chemokine receptors. Specific Aim 5. Define the phenotype of MDI-/- mice. In collaboration with Nigel Killeen, we will create mice deficient in both MDI-1 and MDI-2. Again, these studies will be guided by our own results as well as the phenotype of DAP12-deficient mice and humans.