NORMAL AND ABNORMAL INTERFERON ALPHA RECEPTORS

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The Type I IFN-R is formed by at least three different subunits: 1) the alpha subunit (110kDa) recognized by the IFNaR3 monoclonal antibody, 2) the beta subunit (100 kDa) recognized by the IFNaRbeta1 monoclonal antibody, and 3) the cloned receptor subunit with an approximate MW of 75 kDa. The cloned receptor subunit binds very low levels of IFNalpha8 only at 37 degrees C, although Daudi cell, the source of mRNA for the cloning, bind all IFNalpha subtypes at 4 degrees C. The MW of the cloned subunit expressed in Cos 7 cells (approx. 75 kDa) differs from the main IFNalpha-binding proteins with MWs 110 and 100 kDa (alpha and beta subunits,respectively) observed in affinity crosslinking experiments. Thus, there are many questions that have not been addressed by the cloning and expression of this receptor subunit. For instance, 1) what subunits of the receptor are necessary for IFNalpha binding? 2) What is the role of the cloned subunit?, and 3) How many receptor subunits are necessary to obtain IFNaplha binding? The knowledge of the Type I IFN-R structure at the protein and DNA level will be important in the understanding of the function of the IFN system. Over the last three years we have proved our hypotheses involving a multichain structure for the Type I IFN-R by developing monoclonal antibodies against the alpha and beta subunits. These antibodies ar essential tools for the cloning of these Type I IFN-R subunits and to further our knowledge about the Type I IFN-R in other areas such as signal transduction. Type I IFNs play a central role in viral infections and in the treatment of some types of cancer. Furthermore, some components of the IFN system such as the p68 kinase and IRF-1 may be tumor suppressor genes. Thus, the unraveling of the structure and function of the interferon receptor is essential to understand the role of the IFN system as a first defense against viral infections and in the development and treatment of cancer. In this application we propose to clone the alpha subunit of the Type I IFN-R. Our long term objectives involve the cloning of other component of the IFNalphaR, such as the beta subunit, and to determine the role of each receptor component in the IFNalpha signal transduction. In this application we propose to clone the alpha subunit of the Type I IFN-R. Different approaches for the cloning of the alpha subunit will be used: 1) purification of the alpha subunit using affinity chromatography on a wheat germ lectin column followed by affinity chromatography with specific anti-alpha subunit monoclonal antibody IFNaR3, microsequencing of the purified protein and screening of lambdagt11 and 3) eukaryotic expression libraries with the IFNaR3 monoclonal antibody. Once we clone the cDNA for the alpha subunit, we will express it in human and mouse cells alone and in association with the already cloned receptor subunit. This approach will allow us to determine the role of the alpha and cloned subunits in IFNalpha binding. Finally, we will characterize the structure of the gene encoding the alpha subunit at the genomic level. This part of the project will also conform the localization of the alpha subunit on human chromosome 21.