Ultimately, the work completed in this proposal will provide a tool to be used in additional experiments to aid in elucidating the role of MUC1 mucin in airway health and disease. More specifically, the work of this proposal will provide a better understanding of the role that the cytoplasmic domain of MUC1 mucin plays in the signal transduction pathway. This knowledge will be gained by the completion of three specific aims: 1) To establish a stable cell line expressing a chimeric receptor containing the extracellular and transmembrane domain of CD8 and the cytoplasmic domain of human MUC1 mucin; 2) To induce phosphorylation of the chimeric receptor and identify the amino acids phosphorylated in the cytoplasmic domain of MUC1 mucin; and 3) to determine in a preliminary manner whether some of the transcription factors involved in inflammation can be activated following the phosphorylation of MUC1 mucin. Phosphorylation of the chimeric receptor will be induced with an anti-CD8 antibody and monitored by immunoblotting with an anti- phosphotyrosine antibody. To identify the phosphorylated amino acids, PCR site directed mutagenesis and radiolabeled phosphoamino acid analysis will be carried out. Gel mobility shift assay will be performed with oligonucleotides corresponding to the transcription factors AP1 and NFkB. Modulation of these transcription factors is known to be involved in the pro-inflammatory and inflammatory response in the airway and may be linked to airway disease and its progression.
