The objectives of the next five years are to understand how expression of the receptor for mouse IFNgamma (MuIFN-gammaR) is controlled during activation of macrophages for tumor cell killing, and to obtain clues as to how it transduces signals. The rationale for proposing the work is that this receptor is the initiator of activation and a variety of other important host defensive and homeostatic pathways. Better understanding of how expression of the receptor is regulated, and how it initiates functions should lead to better understanding of the pathways that it serves. There will be four specific aims. First, monoclonal antibodies (MoAbs) will be used to map the external and internal domains of the receptor, which we are now characterizing, will be instrumental in the first of these studies. Analysis of the intracellular domain's role in signal transduction will be examined by introducing into macrophages (J774 cell line) MoAbs that bind to defined, spatially distinct epitopes. Blockage/augmentation of IFNgamma-inducible functions will be sought. Either cationic liposomes or electroporation will be the means of introducing the MoAbs. Second, expression of the receptor's mRNA, the amount of new protein that is synthesized from receptor-specific message, and expression of the receptor protein on the cell surface will be quantified in time course studies under conditions that activate macrophages for tumor cell killing. Preliminary studies suggest that activators down-regulate expression of receptor- specific message. Key reagents in these studies will be the riboprobe that we have synthesized from a 3' fragment of the receptor's cDNA and our MoAbs that bind to the receptor's external domain. The MoAbs will also be used to study receptor-specific protein synthesis by immunoprecipitating newly biosynthetically radiolabeled (35S-methionine) receptor protein. Third, we will clone the IFN-gammaR's gene from a mouse genomic cosmid library and sequence its exons, intronic boundaries and 5' flanking region. The rationale for doing so is that sequencing will provide the information that will be needed to identify the gene's transcriptional regulators. Fourth, we will functionally characterize the cis-acting sequences that regulate transcription of the gene that encodes the MuIFN-gammaR. We will concentrate initially on defining the organization of the promoter by transiently transfecting test DNA fragments into cells of the mouse macrophage line, J774. The luciferase gene will be used as a reporter element in these constructs. Enhancer/repressor sequences will be sought if either negative regulation of receptor mRNA expression by activators or differences in levels of receptor protein expression in different cell types is shown to have a transcriptional basis. The proposed research is important because the mechanisms responsible for regulating expression of the IFN-gammaR in macrophages could be important means of regulating activation for tumor cell killing and other inflammatory/immunologic functions that are attributable to IFN-gamma. Clues as to how the receptor transduces signals may also be obtained. The results should be of general interest, because virtually all other nucleated cell types express and use the receptor in homeostatic and/or defensive responses.
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