MOLECULAR REGULATION OF MACROPHAGE CYTOCIDAL ACTIVITY

巨噬细胞杀细胞活性的分子调控

• 批准号: 2091072
• 负责人: ROBERT DAVID SCHREIBER
• 金额: $ 30.35万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-02-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2091072
• 关键词: biological signal transduction; cytokine receptors; enzyme activity; hamsters; human tissue; interferon gamma; laboratory mouse; laboratory rabbit; leukocyte activation /transformation; macrophage; monoclonal antibody; phosphorylation; protein sequence; protein structure function; protein tyrosine kinase; receptor binding; tissue /cell culture; transcription factor

Interferon-gamma (IFN-gamma) plays a pivotal role in promoting host defense to a variety of intracellular and extracellular microbial pathogens and neoplastic cells. For the past 12 years my laboratory has focused its efforts on elucidating IFNgamma's molecular mechanism of action and physiologic role in vivo. During the current funding period we cloned and characterized the ligand binding chain of the human and murine IFNgamma receptors (now termed the alpha chain), demonstrated the requirement for a second species matched polypeptide (now termed the beta chain), demonstrated the ligand dependent tyrosine phosphorylation of the IFNgamma receptor alpha chain and defined the mechanism by which IFNgamma receptor alpha chain tyrosine phosphorylation couples the receptor to its signal transduction system. On the basis of this work and work from other laboratories we have formulated a model of IFNgamma signal transduction that involves the following steps: (l) ligand induced assembly of an activated receptor complex (2) activation of receptor associated tyrosine kinases (3) phosphorylation of a specific tyrosine residues in the intracellular domain of the receptor a chain, (4) binding of a latent cytosolic/membrane associated transcription factor known as p9l to the phosphorylated receptor a chain, (5) phosphorylation/activation of p9l by the receptor associated active tyrosine kinases and (6) assembly of an activated p9l containing transcription factor complex that then translocates to the nucleus where it transcriptionally activates IFNgamma inducible genes. The focus of the research proposed in this renewal application is to critically test the model described above and to elucidate at the molecular level the interactions that occur between the IFNgamma receptor subunits and specific pathway components. To accomplish this long range goal we intend to pursue the following three specific aims. I. We will elucidate the structure and function of the human IFNgamma receptor beta chain. Here we will use mutagenesis approaches to identify the structure-function relationships that are operative within the intracellular domain of the IFNgamma receptor beta chain and explore how this region of the molecule participates in the signal transduction process. We will also assess the mechanism by which the beta chain is recruited into the signaling process by ligand. II. We will define in detail the interaction of p9l with the phosphorylated IFNgamma receptor a chain. We intend to finish the description of the interaction by exploring whether p9l can be coprecipitated with the IFNgamma receptor a chain, define whether the interaction of p9l with the phosphorylated receptor a chain occurs directly or requires an adapter protein, and study the structures on p9l (and/or the adapter protein) needed to mediate the interaction. III. We will define the molecular basis of the IFNgamma inducible tyrosine kinase activity. This study will establish the identity of the tyrosine kinase(s) that associate with the IFNgamma receptor alpha chain, define the basis of the specificity of the receptor-kinase associations, and elucidate the mechanism of kinase activation. These studies are certain to provide new insights that will further elucidate the molecular mechanisms underlying IFNgamma's important immunomodulatory and proinflammatory activities.

干扰素-γ在促进宿主免疫中起着关键作用。 对多种胞内外微生物的防御 病原体和肿瘤细胞。在过去的12年里，我的实验室 其重点是阐明干扰素-γ的分子机制。 在体内的作用和生理作用。在本资金期限内，我们 人和小鼠配体结合链的克隆与鉴定 IFNGamma受体(现在被称为阿尔法链)展示了 对第二种匹配多肽(现在称为Beta)的需求 链)，证明了配体依赖的酪氨酸磷酸化 并确定了干扰素-γ受体的作用机制 受体α链酪氨酸磷酸化将受体偶联到其 信号转导系统。在此工作的基础上，以及其他人的工作 我们已经建立了一个IFNGamma信号转导的模型 这涉及到以下步骤：(L)配体诱导组装 激活的受体复合体(2)受体相关酪氨酸的激活 激酶(3)特定酪氨酸残基的磷酸化 受体a链的胞内区，(4)潜伏期的结合 胞浆/膜相关转录因子p9l到 磷酸化受体a链，(5)p9l的磷酸化/活化 受体相关活性酪氨酸激酶和(6)An的组装 激活了含有转录因子复合体的p9l，然后 转位到细胞核，在那里它转录激活IFNGamma 可诱导基因。本次更新中提出的研究重点 应用程序是对上述模型进行严格测试，并 在分子水平上阐明分子之间发生的相互作用 干扰素-γ受体亚基和特定的通路成分。要完成 这一长远目标，我们打算追求以下三个具体目标 目标。一、我们将阐明人类的结构和功能 干扰素-γ受体β链。在这里，我们将使用突变方法来 确定在内部运行的结构-功能关系 干扰素-γ受体β链胞内结构域的研究与探索 这个分子区域是如何参与信号转导的 进程。我们还将评估Beta链的作用机制 被配体招募到信号传递过程中。II.我们将在 详述p9l与磷酸化的干扰素受体a的相互作用 链条。我们打算通过探索来完成对交互的描述 P91是否可以与IFNGamma受体a链共沉淀， 确定p9l与磷酸化受体a的相互作用 链直接发生或需要适配蛋白，并研究 P9l上的结构(和/或接头蛋白)需要介导 互动。III.我们将定义IFNGamma的分子基础 可诱导的酪氨酸激酶活性。这项研究将确定身份 与干扰素-γ受体α相关的酪氨酸激酶(S) 链，定义受体-激酶特异性的基础 关联，并阐明了激酶激活的机制。这些 研究肯定会提供新的见解，进一步阐明 干扰素-γ重要免疫调节作用的分子机制 和促炎活动。