STRUCTURE AND FUNCTION OF THE MONOCYTE MCP-1 RECEPTOR

本提案的总体目标是研究 两个最近克隆的单核细胞趋化蛋白1受体 （MCP-1）。 MCP-1是一种有效的特异性单核细胞激动剂， 是趋化因子家族的一员 proteins. MCP-1被认为是单核细胞增殖的重要介质。 在多种炎症过程中的组织浸润 包括动脉粥样硬化。观察氧化低密度 脂蛋白刺激血管壁细胞产生MCP-1， 致动脉粥样硬化脂蛋白和募集 单核细胞到动脉壁。 为了阐明 单核细胞活化MCP-1，我们最近克隆和测序两个 人MCP-1受体仅胞内羧基不同 特米尼这两种形式的受体赋予强大的和显着的具体 当在非洲爪蟾中表达时对纳摩尔浓度MCP-1的反应 卵母细胞在这个提议中， 将检测MCP-1受体的两种形式。稳定转染的细胞 将用于比较MCP-1与两种形式的 受体，并表征和比较它们的信号通路， 哺乳动物细胞中的失活机制。为了确定结构 根据两种形式的受体，我们将克隆MCP的基因， 1受体，并确定内含子/外显子边界。第二个目标是 MCP-1受体的配体结合域的鉴定和 通过构建MIP-1 α/RANTES受体， 嵌合体该方法将创造和研究嵌合体，其中 氨基末端延伸和选择的细胞外环的两个 受体互换。这些混合受体将被评估为 它们结合并响应于加入的趋化因子而发出信号的能力， 识别决定配体特异性的受体区域。一 该基金的第三个目标是阐明 MCP-1受体。在COS-7细胞中的初步实验显示， MCP-1受体和单核细胞受体之间的信号传导存在显著差异。 与白细胞介素8（IL-8）受体密切相关。一系列混合动力 将构建受体，其中IL-8的细胞内环 和MCP-1受体互换以鉴定 与G蛋白相互作用并介导信号转导。实验 在这个补助金中提出的将提供有关相对的第一个信息 表达、结合亲和力和信号转导机制， MCP-1受体的两种形式的失活。

The overall goal of this proposal is to study the structure and function of two recently cloned receptors for monocyte chemoattractant protein 1 (MCP-1). MCP-1 is a potent and specific monocyte agonist and chemoattractant and is a member of the chemokine family of chemoattractant proteins. MCP-1 has been implicated as an important mediator of monocytic infiltration of tissues in a wide variety of inflammatory processes including atherosclerosis. The observation that oxidized low density lipoproteins stimulate MCP-1 production in vascular wall cells provides a possible link between atherogenic lipoproteins and the recruitment of monocytes to the artery wall. To elucidate the molecular mechanisms of monocyte activation by MCP-1 we have recently cloned and sequenced two human MCP-1 receptors that differ only in their intracellular carboxyl termini. Both forms of the receptor confer robust and remarkably specific responses to nanomolar concentrations of MCP-1 when expressed in Xenopus oocytes. In this proposal the expression, properties, and origin of the two forms of the MCP-1 receptor will be examined. Stably transfected cells will be used to compare the binding affinities of MCP-1 to both forms of the receptor, and to characterize and compare their signaling pathways and mechanisms of deactivation in mammalian cells. To determine the structural basis of the two forms of the receptor we will clone the gene for the MCP- 1 receptor and identify the intron/exon boundaries. A second goal will be the identification of ligand-binding domains of the MCP-1 receptor and the closely related MIP-1alpha/RANTES receptor by constructing receptor chimeras. The approach will be to create and study chimeras in which the amino-terminal extensions and selected extracellular loops of the two receptors are interchanged. These hybrid receptors will be evaluated for their ability to bind and signal in response to added chemokines to identify regions of the receptor that determine ligand specificity. A third goal of the grant will be to elucidate the signaling pathways of the MCP-1 receptor. Preliminary experiments in COS-7 cells have revealed significant differences in signaling between the MCP-1 receptor and the closely related interleukin 8 (IL-8) receptor. A series of hybrid receptors will be constructed in which the intracellular loops of the IL-8 and MCP-1 receptor are interchanged to identify receptor domains that interact with G-proteins and mediate signal transduction. Experiments proposed in this grant will provide the first information on the relative expression, binding affinities, and mechanisms of signal transduction and deactivation of the two forms of the MCP-1 receptor.