FUNCTIONAL ANALYSIS OF CFTR

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As its long term objective, this project seeks to understand the function of CFTR (Cystic Fibrosis Transmembrane conductance Regulator), the protein whose defect causes cystic fibrosis. The specific aims required to achieve this objective include (1) the raising of polyclonal or monoclonal antibodies reactive to CFTR peptides; these can serve as immunoadsorbents to purify the intact molecule. Such purified (and likely denatured) material will then be used to obtain (2) a panel of monoclonal antibodies reactive against a very much wider range of specific epitopes. During the screening of these second generation antibodies, it should be possible to identify those that are high affinity and those that are low affinity. (3) Low affinity antibodies will be used in a second round of immunopurification, with specific precautions and mild conditions designed to promote retention of biological activity. (4) In parallel work, CFTR will also be purified by biochemical techniques, beginning with either a human cell line (T84) presumed to have high levels of CFTR, or with an overexpression system designed using full length cDNA. These biochemical steps will include use of triazine dye-coupled resins that interact with proteins (e.g. CFTR) having nucleotidebinding folds. Should the earlier work (above) prove difficult, this provides another opportunity to obtain antigens for production of a monoclonal antibody panel. With purified CFTR available, two fundamental issues will be addressed directly. (1) The more challenging experiments will identify endogenous materials that enhance or inhibit the ATP hydrolytic activity of isolated CFTR. It is likely (but not certain) that CFTR transports a molecule into or out of a membrane-bound compartment (the cell, a minor organelle, etc.), and that this substrate is key to the operation of CFTR. Arguing from other systems, it should be possible to identify this key molecule (and other effectors) by a stimulation of CFTR ATPase activity. This will immediately set the stage for an effective pharmacology for treatment of cystic fibrosis. (2) In the second set of experiments, the high affinity monoclonal antibodies will be used to affix CFTR to an affinity column. This will allow a characterization of proteins that interact with CFTR itself. Such proteins will become candidates members of a regulatory network that controls epithelial cell function. Together, these experiments have potential for revealing both small and large molecular targeted to CFTR. Such information should constitute the central core of knowledge about how membrane function is regulated in both normal and disease states.