Activated macrophages play a pivotal role in promoting host defense and pathologic processes. Based on work from several laboratories, including my own, we now know that interferon-gamma (IFN gamma) is one of the major cytokines responsible for effecting macrophage activation both in vitro and in vivo. For the past 7 years, my laboratory has focused its efforts on elucidating IFN gamma's mechanism of action at the molecular level in vitro and in defining the physiologic role of IFN gamma-activated macrophages in vivo. One of our major contributions to this field has been the identification and characterization of the IFN gamma receptor of human and murine mono-nuclear phagocytes. During the past granting period we purified the human IFN gamma receptor, produced receptor-specific monoclonal antibodies, and cloned the human and murine IFN gamma receptor cDNAs. We now wish to use these unique reagents to define the structure of the functionally active human IFN gamma receptor and to elucidate its role in regulating the activity of macrophages and other cells. To achieve this goal, we intend to pursue three specific aims. First, we will continue to study the structure and cell biology of the 90 kDa glycoprotein (gp90) that is the universal IFN gamma binding protein on cell surfaces. Using biosynthetic labelling, immunoprecipitation, and electron microscopic techniques, we will elucidate the receptor's post-translational processing and define its life cycle and intracellular trafficking through cells. Second, we intend to pursue our unique observation that ligand induces the phosphorylation of gp90. We plan to characterize the specificity, kinetics, and reversibility of this reaction at the molecular level and determine its biologic consequences. We will put special emphasis on examining whether receptor phosphorylation accounts for the well documented but poorly understood desensitization of macrophages effected by ligand, immune complexes, and other biologic particles. Third, we will use state of the art molecular biology techniques to obtain high level expression of human gp90 in murine cells and investigate whether these cells become responsive to human IFN gamma. The latter experiments will also examine whether accessory polypeptides are needed to form a functionally active receptor. These studies should help to better define the molecular basis of macrophage activation and provide new insights into the roles of IFN gamma and activated macrophages in promoting physiologically important immune responses.
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