FUNCTIONAL ANALYSIS OF A PDGF-INDUCIBLE CYTOKINE

单核细胞趋化蛋白-1(MCP-1)是近年来发现的 已定义的趋化因子家族称为“趋化因子”，它们是 值得注意的是，它们有能力吸引特定的白细胞亚群。MCP-1， 最初被克隆为PDGF诱导的je基因，吸引单核细胞和 记忆T淋巴细胞，以排除其他白细胞，如 中性粒细胞。因为它的吸引性和它的图案 表达，MCP-1被认为在急性髓系白血病中起致病作用。 具有单核细胞炎症成分的各种疾病，例如 动脉硬化。然而，到目前为止，还没有直接的 单核细胞趋化蛋白-1 S因果参与疾病的演示。 在这笔赠款的赞助下，确定了结构/活动分析 证明了MCP-1的趋化活性所需的区域 MCP-1作为二聚体，产生了一个显性抑制突变体。 此外，过量表达MCP-1的转基因小鼠提供了证据 MCP-1参与了对L. 单核细胞增多症和结核分枝杆菌。这项拨款续期旨在：(1)至 将结构/活性分析扩展到MCP-1及其 相互作用部位的受体；以及(2)建立MCP-1缺陷小鼠 验证单核细胞趋化蛋白-1‘S参与疾病的假说。以下是 具体目标旨在实现以下目标： 特定目标1：先前定义为关键的MCP-1区域 将通过扫描突变来分析活性，以确定特定的 参与受体相互作用的氨基酸。以前未经审查的区域 还将对MCP-1进行分析。MCP-1受体B的区域也将是 通过替换IL-8受体1的胞外结构域和通过 定点突变。基于这些发现的多肽将被 测试它们的抑制或激活特性。 特定目的2：我的实验室最近培育出MCP-1缺陷小鼠 通过定向基因破坏。这些老鼠的特征将是他们的 基线表型及其对免疫学和感染性的反应 挑战。单核细胞趋化蛋白-1‘S在动脉粥样硬化中的作用将由 让这些小鼠接受致动脉粥样硬化的饮食，并通过繁殖被破坏的 等位基因进入易患动脉粥样硬化的遗传背景。 特定目的3：许多趋化因子具有重叠的性质，提示 这种冗余可能会防止出现异常的表型。 MCP-1基因缺陷小鼠。由于趋化因子基因聚集，多个 同时进行的“击倒”在技术上是难以实现的 繁衍后代。相反，表达显性抑制基因的转基因小鼠 趋化因子的变异体将被构建。它们的交配会产生小鼠 多个趋化因子同时中断。

Monocyte chemoattractant protein-1 (MCP-1) is a member of a recently defined family of chemotactic factors called "chemokines," which are notable for their ability to attract specific leukocyte subsets. MCP-1, originally cloned as the PDGF-inducible JE gene, attracts monocytes and memory T lymphocytes to the exclusion of other leukocytes, such as neutrophils. Because of its attractant properties and its patterns of expression, MCP-1 has been hypothesized to play a pathogenetic role in a variety of diseases having a monocyte inflammatory component, such as atherosclerosis. So far, however, there have been no direct demonstrations of MCP-1's causal participation in disease. Under the auspices of this grant, structure/activity analyses defined regions of MCP-1 required for its chemoattractant activity, demonstrated that MCP-1 acts as a dimer, and generated a dominant suppressor mutant. In addition, transgenic mice overexpressing MCP-1 provided evidence that MCP-1 is involved in resistance to intracellular pathogens such as L. monocytogenes and M. tuberculosis. This grant renewal seeks: (1) to extend the structure/activity analyses to test regions of MCP-1 and its receptor for sites of interaction; and (2) to create MCP-1-deficient mice to test hypotheses about MCP-1's involvement in disease. The following specific aims are designed to accomplish these goals: SPECIFIC AIM 1: Regions of MCP-1 previously defined as critical for activity will be analyzed by scanning mutagenesis to identify specific amino acids involved in receptor interaction. Previously unexamined areas of MCP-1 will also be analyzed. Regions of MCP-1 receptor B will also be tested by substituting extracellular domains of IL-8 receptor 1 and by site-directed mutagenesis. Peptides based on these findings will be tested for their inhibitory or activating properties. SPECIFIC AIM 2: My laboratory has recently developed MCP-1-deficient mice by targeted gene disruption. These mice will be characterized for their baseline phenotype and their responses to immunologic and infectious challenges. MCP-1's role in atherosclerosis will be investigated by subjecting these mice to atherogenic diets and by breeding the disrupted alleles into an atherosclerosis-prone genetic background. SPECIFIC AIM 3: Many chemokines have overlapping properties, suggesting that redundancy might prevent the appearance of an abnormal phenotype in MCP-1-deficient mice. Since chemokine genes cluster, multiple simultaneous "knockouts" would be technically difficult to construct by breeding. Instead, transgenic mice expressing dominant suppressor chemokine variants will be constructed. Their mating will produce mice multiple simultaneous disruption of several chemokines.