Biology of a New DAP12 Associated Receptor Family

新 DAP12 相关受体家族的生物学

• 批准号: 6514711
• 负责人: William E Seaman
• 金额: $ 27.83万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514711
• 关键词: antireceptor antibody; cell migration; complementary DNA; cytokine; gene targeting; genetically modified animals; laboratory mouse; ligands; macrophage; monoclonal antibody; nitric oxide; polymerase chain reaction; protein structure function; receptor; receptor binding; receptor expression

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.

描述（由申请人提供）：通过快速克隆，我们已经鉴定出 与信号附件相关的一个新的小鼠受体家族 分子，DAP12。它们有一个类似 Ig 的结构域，并且它们的表达 似乎仅限于单核细胞/巨噬细胞谱系的细胞。因此，我们有 将它们命名为“MDI 受体”（骨髓细胞 DAP12 相关 Ig 样受体） 受体）。我们已经鉴定出 3 个包含 MDI 基因的重叠 BAC，并已 将基因定位到 17d 染色体。 BAC 的摘要表明 MDI 受体家族很小，可能仅限于两个同源基因，MDI-1 和 MDI-2。当在 MT2 巨噬细胞中表达时，MDI-1 与内源性结合 DAP12 并刺激一氧化氮的产生。只有 3 个其他受体 已知与单核细胞/巨噬细胞中的 DAP12 相关。小鼠缺乏 Dap12 的免疫改变似乎存在于抗原呈递细胞中， 缺乏 DAP12 的人类可能患有神经系统和骨骼疾病 分别反映小胶质细胞和破骨细胞的缺陷。我们因此 希望定义 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. 定义 MDI-1 和 MDI-2 的配体。我们将创造可溶 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，我们将寻求将配体鉴定为 已知蛋白质，或者如果未知，则克隆配体的 cDNA。 具体目标 2. 定义 MDI 受体家族的范围。我们将进一步 探测单核细胞/巨噬细胞 cDNA 文库以寻找 MDI 的其他成员 家庭。其次，我们将对编码在 MDI 基因中的 MDI 基因进行定位和测序。 三个与 MDI 转录本杂交的 BAC。 具体目标 3. 定义 MDI 受体的表达范围。我们将 生产针对单个 MDI 受体的单克隆抗体，我们将使用 这些用于评估 MDI 在不同细胞类型上的表面表达 细胞活化的不同阶段。 具体目标 4. 定义细胞对 MDI 连接的反应。我们将学习 受体转染细胞和新鲜制备的细胞。我们的学习将是 以我们自己的结果和 DAP12 缺陷小鼠的表型为指导 人类。其中包括与 Jason Cyster 博士在趋化因子方面的合作 和趋化因子受体。 具体目标 5. 定义 MDI-/- 小鼠的表型。与合作 Nigel Killeen，我们将创造出同时缺乏 MDI-1 和 MDI-2 的小鼠。再次， 这些研究将以我们自己的结果以及表型为指导 DAP12 缺陷小鼠和人类。